: cr ; CO i Lo i f^ CD o PROTOZOOTvOGY (^rd Edition) PROTOZOOLOGY By RICHARD R. KUDO, D.Sc. Professor of Zoology The University of Illinois Urbana, Illinois With three hundred and thirty-six illustrations CHARLES C THOMAS • PUBLISHER 301-327 EAST LAWRENCE AVENUE SPRINGFIELD • ILLINOIS 1946 Published by Charles C Thomas 301-327 East Lawrence Avenue, Springfield, Illinois Published simultaneously in Canada by The Ryerson Press, Toronto All rights in this book are reserved. No part may be repro- duced in any form w^hatsoever without permission in writing from the publisher, except by a reviewer who wishes to quote extremely brief passages in connection with a critical review. Reproduction in whole or in part in digests, in condensations of the literature, in lectures, or in films; or by multigraphing, lithoprinting, or by any other processes or devices, is reserved by the publisher. For information, address Charles C Thomas. Copyright, 19^6, by Charles C Thomas First Edition, January 1931 Second Edition, September 1939 Third Edition, January 1946 Printed in the United States of America 'The revelations of the Microscope are perhaps not excelled in importance by those of the telescope. While exciting our curiosity, our ivonder and admiration, they have proved of infinite service in advancing our knowledge of things around us." Leidy Preface to the third edition IN REVISING Protozoology for the second time, the author has maintained the original aim of the work for setting forth "in- troductory information on the common and representative genera of all groups of both free-living and parasitic Protozoa," and en- deavored to limit its expansion to minimum. Errors, typograph- ical and otherwise, which had appeared in the last edition have been corrected. In this he is much indebted to his former students, both graduates and undergraduates, who untiringly detected them and suggested improvement of many passages for clearer presentation. The criticisms offered by the reviewers of the second edition have also been considered. Published papers that had been overlooked before and those that have appeared in the last six years have been consulted and referred to in the present edition. Chapters 4 to 6 were largely rewritten and enlarged in the light of recent works. Alterations and additions have been made in all other chapters. Two new chapters have been added; they are chapter 7 (Major groups and phylogeny of Protozoa) and chapter 45 (Collection, cultivation, and observation of Protozoa). Since there is now a greater demand of the information on Proto- zoa that parasitize man than in the past, they have been more thoroughly treated in the present edition. The author continues to believe in the importance of adequate illustrations in this sort of work. Sixty-nine new figures have been added; of these forty-seven have been newly prepared or rearranged from former figures, while twenty-two have been taken from his Manual of human Protozoa (1944). The author once more expresses his indebtedness to numerous authors of published papers for the materials which have been in- corporated in the work. He is under special obligation to Doctor Harold Kirby, University of California; Doctor Reginald D. Man- well, Syracuse University; Doctor Tracy M. Sonneborn, Indiana University; Doctor David H. Wenrich, University of Pennsylvania; and Doctor Lorande L. Woodruff, Yale University, for their kind advices, sincere criticisms and suggestions. He further wishes to thank Mr. Charles C Thomas for the interest and care with which the present edition has been put in print. Richard R. Kudo Urbana, Illinois October, 1945 Preface to the second edition THE present work is similar in its primary aim to that of its predecessor, Handbook of Protozoology (1931), in presenting "introductory information on the common and representative genera of all groups of both free-living and parasitic Protozoa," to advanced undergraduate and graduate students in zoology in colleges and universities. With the expansion of courses in protozoology at the University of Illinois and elsewhere, it seemed advisable to incorpor- ate more material for lecture and discussion, in addition to the en- largement of the taxonomic section. The change of the text-contents has, therefore, been so extensive that a new title, Protozoologij , is now given. Chapters 1 to 6 deal with introduction, ecology, morphology, physiology, reproduction, and variation and heredity, of Proto- zoa. Each subject-matter has been considered in the light of more recent investigations as fully as the space permitted. Selection of material from so great a number of references has been a very diffi- cult task. If any important papers have been omitted, it was en- tirely through over-sight on the part of the author. The taxonomic portion (Chapters 7 to 43) has also been com- pletely rewritten and enlarged. Numerous genera and species, both old and new, have been added ; synonymy of genera and species has as far as possible been brought down to date; new taxonomic ar- rangement of major and minor subdivisions in each class has resulted in numerous changes. The class Ciliata has completely been reclassi- fied, following Kahl's admirable work on free-living ciliates (1930- 1935) ; however, unlike the latter, all parasitic ciliates have also been considered in the present work. The author continues to believe that good illustrations are in- dispensable in this kind of work, since they are far more easily comprehended than lengthy descriptions. Therefore, many old illustrations have been replaced by more suitable ones and numerous new illustrations have further been added. All illustrations were especially prepared for this work and in the case of those which have been redrawn from illustrations found in published papers, the indebtedness of the author is indicated by mentioning the names of the investigators from whose works the illustrations were taken. In order to increase the reference value, all figures are accompanied by scales of magnification which are uniformly somewhat greater than those of Handbook of Protozoology, since the microscope now used in the class-room has been improved upon in recent years. X PREFACE The list of references appended to the end of each chapter has been enlarged and is meant to aid those who wish to obtain fuller information than that which is given in this volume. Since com- prehensive monographs on various groups of Protozoa are widelj^ scattered and ordinarily not easily accessible, the author has en- deavored to provide for each group as complete an information as possible for general reference purpose within the limited space, and hopes that the present work has reference value for teachers of biology, field workers in pure and applied biological sciences, veterinarians, physicians, public health workers, laboratory tech- nicians, and others. The author is under obligation to numerous writers for their valuable contributions which have been incorporated in the text. Special thanks are due Professor L. R. Cleveland, Harvard Uni- versity; Professor R. P. Hall, New York University; Professor H. Kirby, Jr., University of California; Professor L. E. Noland, Uni- versity of Wisconsin; Professor H. J. Van Cleave, University of Illinois; Professor D. H. Wenrich, University of Pennsylvania; and Professor L. L. Woodruff, Yale University, for their valued criti- cisms and suggestions. The author further wishes to express his appreciation to Mr. Charles C Thomas, for his patient and kind cooperation which has aided greatly in the completion and appear- ance of the present work. R.R.K. Urbana, Illinois, U.S.A. July, 1939 CONTENTS Preface vii Part I: General biology 3 CHAPTER 1 Introduction 5 Relationship of protozoology to other fields of biological science, p. 6; the history of protozool- ogy, p. 10. 2 Ecology 17 The free-living Protozoa, p. 17; the parasitic Protozoa, p. 24. 3 Morphology 33 The nucleus, p. 34; the cytosome, p. 38; loco- motor organellae, p. 41; fibrillar structures, p. 52; protective or supportive organellae, p. 61; hold-fast organellae, p. 65; the parabasal appa- ratus, p. 66; the blepharoplast, p. 67; the Golgi apparatus, p. 68; the chondriosomes, p. 70; the contractile and other vacuoles, p. 73; the chro- matophore and associated organellae, p. 78. 4 Physiology 84 Nutrition, p. 84; the reserve food matter, p. 98; respiration, p. 101; excretion and secretion, p. 103; movements, p. 106; irritability, p. 113. 5 Reproduction 122 Nuclear division, p. 122; cytosomic division, p. 143; colony formation, p. 145; asexual repro- duction, p. 147; sexual reproduction and life- cycles, p. 149; regeneration, p. 170. 6 Variation and heredity 176 Part II: Taxonomy and special biology 191 CHAPTER 7 Major groups and phylogeny of Protozoa 193 8 Phylum Protozoa 198 Subphylum 1 Plasmodroma 198 Class 1 Mastigophora 198 Subclass 1 Phytomastigina 200 Order 1 Chrysomonadina 200 XI 59941 CONTENTS 9 Order 2 Cryptomonadina 213 10 Order 3 Phytomonadina 217 11 Order 4 Euglenoidina 232 Order 5 Chloromonadina 243 12 Order 6 Dinoflagellata 245 13 Subclass 2 Zoomastigina 263 Order 1 Rhizomastigina 263 14 Order 2 Protomonadina 268 15 Order 3 Polymastigina 293 16 Order 4 Hypermastigina 318 17 Class 2 Sarcodina 328 Subclass 1 Rhizopoda 329 Order 1 Proteomyxa 329 18 Order 2 Mycetozoa 335 19 Order 3 Amoebina 343 20 Order 4 Testacea 374 21 Order 5 Foraminifera 394 22 Subclass 2 Actinopoda 406 Order 1 Heliozoa 406 23 Order 2 Radiolaria 417 24 Class 3 Sporozoa 427 Subclass 1 Telosporidia 427 Order 1 Gregarinida 428 25 Order 2 Coccidia 464 26 Order 3 Haemosporidia 484 27 Subclass 2 Acnidosporidia 507 Order 1 Sarcosporidia 507 Order 2 Haplosporidia 510 28 Subclass 3 Cnidosporidia 515" Order 1 Myxosporidia 515 Order 2 Actinomyxidia 531 29 Order 3 Microsporidia 535 Order 4 Helicosporidia 542 30 Subphylum 2 Ciliophora 545 Class 1 Ciliata 545 Subclass 1 Protociliata 547 31 Subclass 2 Euciliata 551 Order 1 Holotricha 551 Suborder 1 Astomata 552 32 Suborder 2 Gymnostomata 560 Tribe 1 Prostomata 560 33 Tribe 2 Pleurostomata 580 CONTENTS xiii Tribe 3 Hypostomata 585 34 Suborder 3 Trichostomata 593 35 Suborder 4 Hymenostomata 608 36 Suborder 5 Thigmotricha 623 37 Suborder 6 Apostomea 630 38 Order 2 Spirotricha 636 Suborder 1 Heterotricha 636 39 Suborder 2 Oligotricha 652 40 Suborder 3 Ctenostomata 665 41 Suborder 4 Hypotricha 668 42 Order 3 Chonotricha 681 43 Order 4 Peritricha 683 44 Class Suctoria 695 45 Collection, cultivation, and observation of Protozoa 710 Author and subject index 731 PROTOZOOLOGY PROTOZOOLOGY PART I: GENERAL BIOLOGY Chapter 1 Introduction PROTOZOA are unicellular animals. The body of a protozoan is morphologically a single cell and manifests all characteristics common to the living thing. The various activities which make up the phenomena of life are carried on by parts within the body or cell. These parts are comparable with the organs of a metazoan which are composed of a large number of cells grouped into tissues and are called organellae or cell-organs. Thus one sees that the one-celled protozoan is a complete organism somewhat unlike the cell of a metazoan, each of which is dependent upon other cells and cannot live independently. From this viewpoint, certain students of proto- zoology maintain that the Protozoa are non-cellular, and not uni- cellular, organisms. Dobell (1911) for example, pointed out that the term "cell" is employed to designate (1) the whole protozoan body, (2) a part of a metazoan organism, and (3) a potential whole organ- ism (a fertilized egg) which consequently resulted in a confused state of knowledge regarding living things, and, therefore, proposed to define a cell as a mass of protoplasm composing part of an organ- ism, and further considered that the protozoan is a non-cellular but complete organism, differently organized as compared with cellular organisms, the Metazoa and Metaphyta. The great majority of protozoologists, however, continue to consider the Protozoa as uni- cellular animals. Through the processes of organic evolution, they have undergone cytological differentiation and the Metazoa histo- logical differentiation. In being unicellular, the Protozoa and the Protophyta are alike. The majority of the Protozoa are quite clearly distinguishable from the majority of the Protophyta on the basis of nuclear condition, method of nutrition, direction of division-plane, etc. While numerous Protophyta appear to possess scattered nuclear material or none at all, the Protozoa contain at least one nucleus. It is generally con- sidered that the binary fission of the Protozoa and of the Protophyta is longitudinal and transverse, respectively. A great majority of Ciliata, however, multiply by transverse division. In general the nutrition of Protozoa is holozoic and of Protophyta, holophytic; but there are large numbers of Protozoa which nourish themselves by holophytic method. Thus an absolute and clean-cut separation of the two groups of unicellular organisms is not possible. Haeckel 6 PROTOZOOLOGY coined the name Protista to include these organisms in a single group, but this is not generally adopted, since it includes undoubted animals and plants, thus creating an equal amount of confusion between it and the animal or the plant. Calkins (1933) excluded chromatophore-bearing Mastigophora from his treatment of Pro- tozoa, thus placing organisms similar in every way, except the presence or absence of chromatophores, in two different groups. This intermingling of characteristics between the two groups of microorganisms shows clearly their close interrelationship and sug- gests strongly their common ancestry. Although the majority of Protozoa are solitary and the body is composed of a single cell, there are several forms in which the organism is made up of more than one cell. These forms, which are called colonial Protozoa (p. 145), are well represented by the mem- bers of Phytomastigina, in which the individuals are either joined by cytoplasmic threads or embedded in a common matrix. These cells are alike both in structure and in function, although in a few forms there may be a differentiation of the individuals into repro- ductive and vegetative cells. Unlike the cells in a metazoan which form tissues, these vegetative cells of colonial Protozoa are not de- pendent upon other cells; therefore, they do not form any tissue. The reproductive cells produce zygotes through sexual fusion, which subsequently undergo repeated division and may produce a stage comparable with the blastula stage of a metazoan, but never reach- ing the gastrula stage. Thus colonial Protozoa are only cell-aggre- gates without histological differentiation and may thus be distin- guished from the Metazoa. Between 15,000 and 20,000 species of Protozoa are known to man. From comparatively simple forms such as Amoeba, up to highly complicated organisms as represented by numerous ciliates, the Protozoa vary exceedingly in their body organization, morphological characteristics, behavior, habitat, etc., which necessitates a tax- onomic arrangement for proper consideration as set forth in detail in chapters 8 to 44. Relationship of protozoology to other fields of biological science A brief consideration of the relationship of Protozoology to other fields of biology and its possible applications may not be out of place here. Since the Protozoa are single-celled animals manifesting the characteristics common to all living things, they have been studied by numerous investigators with a view to dis- INTRODUCTION 7 covering the nature and mechanism of various phenomena, the sum-total of which is known collectively as life. Though the in- vestigators generally have been disappointed in the results, in- asmuch as the assumed simplicity of unicellular organisms has proved to be offset by the complexity of their cell-structure, never- theless any discussion of biological principles today must take into account the information obtained from studies of Protozoa. It is now commonly recognized that adequate information on various types of Protozoa is a prerequisite to a thorough comprehension of biology and to proper application of biological principles. Practically all students agree in assuming that the higher types of animals have been derived from organisms which existed in the remote past and which probably were somewhat similar to the Protozoa of the present day. Since there is no sharp distinction between the Protozoa and the Protophyta or between the Protozoa and the Metazoa, and since there are intermediate forms between the major classes of the Protozoa themselves, progress in proto- zoology contributes toward the advancement of our knowledge on the probable steps by which living things in general evolved. Geneticists have undertaken studies on heredity and variation among Protozoa. "Unicellular animals," wrote Jennings (1909), "present all the problems of heredity and variation in miniature. The struggle for existence in a fauna of untold thousands showing as much variety of form and function as any higher group, works itself out, with ultimate survival of the fittest, in a few days under our eyes, in a finger bowl. For studying heredity and variation we get a generation a day, and we may keep unlimited numbers of pedigreed stock in a watch glass that can be placed under the micro- scope." Morphological variations are encountered commonly in all forms. Whether variation is due to germinal or environmental condi- tions, is often difficult to determine. The recent discovery of the mating types in Paramecium aurelia (Sonneborn; Kimbell) and in P. bursaria (Jennings) will probably assist in bringing to light many genetic problems of Protozoa which have remained obscure in the past. Parasitic Protozoa are limited to one or more specific hosts. Through studies of the forms belonging to one and the same genus or species, the phylogenetic relation among the host animals may be established or verified. The mosquitoes belonging to the genera Culex and Anopheles, for instance, are known to transmit avian and human Plasmodium respectively. They are further infected by specific microsporidian parasites. For instance, Thelohania 8 PROTOZOOLOGY has been found widely only in many species of anopheline mosqui- toes; T. opacita has, on the other hand, been found exclusively in culicine mosquitoes, although the larvae of the species belonging to these two genera live frequently in the same body of water. By ob- serving certain intestinal Protozoa in some monkeys, Hegner ob- tained evidence on the probable phylogenetic relationship between them and other higher mammals. The relation of various Protozoa of the wood-roach to those of the termite, as revealed by Cleveland and his associates, gives further proof that the Blattidae and the Isoptera are of the common origin. Study of a particular group of parasitic Protozoa and their hosts may throw light on the geographic condition of the earth in the remote past. The members of the genus Zelleriella are usually found in the colon of the frogs belonging to the family Leptodactylidae. Through an extensive study of these amphibians from South Amer- ica and Australia, Metcalf found that the species of Zelleriella occur- ring in the frogs of the two continents are almost identical. He finds it more difficult to conceive of convergent or parallel evolution of both the hosts and the parasites, than to assume that there once existed between Patagonia and Australia a land connection over which frogs, containing Zelleriella, migrated. Experimental studies of large Protozoa have thrown light on the relation between the nucleus and the cytoplasm, and have furnished a basis for an understanding of regeneration in animals. In Protozoa we find various types of nuclear divisions ranging from a simple amitotic division to a complex process comparable in every detail with the typical metazoan mitosis. A part of our knowledge in cytology is based upon studies of Protozoa. Through the efforts of various investigators in the past fifty years, it has now become known that numerous parasitic Protozoa occur in man (Kudo, 1944). Entamoeba histolytica, Balantidium coli, and three species of Plasmodium, all of which are pathogenic to man, are widely distributed throughout the world. In certain restricted areas are found other pathogenic forms, such as Trypanosoma and Leishmania. Since all parasitic Protozoa presumably have originated in free-living forms and since our knowledge of the morphology, physiology, and reproduction of the parasitic forms has largely been obtained in conjunction with the studies of the free-living organ- isms, a general knowledge of the entire phylum is necessary to under- stand the parasitic forms. Recent studies have further revealed that almost all domestic animals are hosts to numerous parasitic Protozoa, many of which INTRODUCTION 9 are responsible for serious infectious diseases. Many of the forms found in domestic animals are morphologically indistinguishable from those occurring in man. Balantidium coli is now generally considered as a parasite of swine, and man is its secondary host. Knowledge of protozoan parasites is useful to medical practitioners, just as it is essential to veterinarians inasmuch as certain diseases in animals, such as Texas fever, dourine, nagana, blackhead, coccidio- sis, etc., are caused by protozoans. Sanitary betterment and improvement are fundamental re- quirements in the modern civilized world. One of man's necessities is safe drinking water. The majority of Protozoa live freely in various bodies of water and some of them are responsible, if present in suffi- ciently large numbers, for giving certain odors to the waters of reservoirs or ponds (p. 100). But these Protozoa which are occasion- ally harmful are relatively small in number compared with those which are beneficial to man. It is generally understood that bacteria live on various waste materials present in polluted water, but that upon reaching a certain population, they would cease to multiply and would allow the excess organic substances to undergo decompo- sition. Numerous holozoic Protozoa, however, feed on the bacteria and prevent them from reaching the saturation population. Protozoa thus seem to help indirectly in the purification of the water. Protozo- ology therefore must be considered as part of modern sanitary science. Young fish feed extensively on small aquatic organisms, such as larvae of insects, small crustaceans, annelids, etc., all of which de- pend largely upon Protozoa and Protophyta as sources of food sup- ply. Thus the fish are indirectly dependent upon Protozoa as food material. On the other hand, there are numbers of Protozoa which live at the expense of fish. The Myxosporidia are almost exclusively parasites of fish and sometimes cause death to large numbers of com- mercially important fishes. Success in fish-culture, therefore, requires among other things a thorough knowledge of Protozoa. Since Russel and Hutchinson suggested some thirty years ago that Protozoa are probably a cause of limitation of the numbers, and therefore the activities of bacteria in the soil and thus tend to de- crease the amount of nitrogen which is given to the soil by the nitrifying bacteria, several investigators have brought out the fact that in the soils of temperate climates Protozoa are present com- monly and active throughout the year. The exact relation between specific protozoans and bacteria in the soil is a matter which still awaits future investigations, although numerous experiments and 10 PROTOZOOLOGY observations have already been made. All soil investigators should be acquainted with the biology and taxonomy of free-living pro- tozoans. It is a matter of common knowledge that the silkworm and the honey bee suffer from protozoan infection known as microsporidiosis. Sericulture in southern Europe suffered great damages in the middle of the nineteenth century because of the "pebrine" disease, caused by the microsporidian, Nosema hombycis. During the first decade of the present century, another microsporidian, Nosema apis, was found to infect a large number of honey bees. Methods of control have been developed and put into practice so that these micro- sporidian infections are at present not serious, even though they still occur. On the other hand, other Microsporidia are now known to in- fect certain insects, such as mosquitoes and lepidopterous pests, which, when heavily infected, die sooner or later. Methods of de- struction of these insects by means of chemicals are more and more used, but attention should also be given to utilization of the parasitic Protozoa and Protophyta for this purpose. While the majority of Protozoa lack permanent skeletal structures and their fossil forms are unknown, there are at least two large groups in the Sarcodina which possess conspicuous shells and which are found as fossils. They are Foraminifera and Radiolaria. From early palaeozoic era down to the present day, the carbonate of lime which makes up the skeletons of numerous Foraminifera has been left embedded in various rock strata. Although there is no dis- tinctive foraminiferan fauna characteristic of a given geologic pe- riod, there are certain peculiarities of fossil Foraminifera which dis- tinguish one formation from the other. From this fact one can un- derstand that knowledge of foraminiferous rocks is highly useful in checking up logs in well drilling. The skeletons of the Radiolaria are the main constituent of the ooze of littoral and deep-sea regions. They have been found abundantly in siliceous rocks of the palaeozoic and the mesozoic eras, and are also identified with the clays and other formations of the miocene period. Thus knowledge of these two orders of Sarcodina, at least, is essential for the student of geology and paleontology. The history of protozoology Aside from a comparatively small number of large forms. Protozoa are unobservable with the naked eye, so that we can easily under- stand why they were unknown prior to the invention of the micro- scope. Antony van Leeuwenhoek (1632-1723) is commonly recog- INTRODUCTION 11 nized as the father of protozoology. Grinding lenses himself, Leeuwenhoek made more than four hundred simple microscopes, in- cluding one which, it is said, had a magnification of 270 times (Harting). Among the many things he discovered were various Pro- tozoa. According to Dobell (1932), Leeuwenhoek saw in 1674 for the first time free-living Protozoa in fresh water. Among them, he observed bodies "green in the middle, and before and behind white," which Dobell interprets were Euglena. Between 1674 and 1716 he observed numerous microscopic organisms which he communicated to the Royal Society of London and which, as Dobell considered, were Vorticella, Stylonychia, Carchesium, Volvox, Haematococcus, Coleps, Kerona, Anthophysis, Elphidium, Polytoma, etc. According to Dobell, Huygens gave in 1678 "unmistakable descriptions of Chilodon(ella), Paramecium, Astasia and Vorticella, all found in in- fusions." Colpoda was seen by Bonanni (1691) and Harris (1696) rediscov- ered Euglena. In 1718 there appeared the first treatise on micro- scopic organisms, particularly of Protozoa, by Joblot who empha- sized the non-existence of abiogenesis by using boiled hay-infusions in which no Infusoria developed without exposure to the atmosphere. This experiment confirmed that of Redi who, some forty years be- fore, had made his well-known experiments by excluding flies from meat. Joblot illustrated, according to Woodruff (1937), Paramecium, the slipper animalcule, with the first identifiable figure. Trembley (1745) studied division in some ciliates, including probably Para- mecium, which generic name was coined by Hill in 1752. Noctiluca w^as first described by Baker (1753). Rosel von Rosenhoff (1755) observed an organism, possibly either Pelomyxa carolineyisis Wilson (Chaos chaos Linnaeus (Schaeffer, 1926)) or a mycetozoan (Mast and Johnson, 1931), which he called "der kleine Proteus," and also Vorticella, Stentor, and Volvox. Wris- berg (1764) coined the term "Infusoria" (Dujardin; Woodruff). By using the juice of geranium, Ellis (1770) caused the extrusion of the 'fins' (trichocysts) in Paramecium. Eichhorn (1783) observed the heliozoan, Actinosphaerium, which now bears his name. O. F. Miiller described Ceratium a little later and published two works on the In- fusoria (1773, 1786). Although he included unavoidably some Meta- zoa and Protophyta in his monographs, some of his descriptions and figures of Ciliata were so well done that they are of value even at the present time. At the beginning of the nineteenth century the cylcosis in Para- mecium was brought to light by Gruithuisen. Goldfuss (1817) coined 12 PROTOZOOLOGY the term Protozoa, including in it the coelenterates. Ten years later there appeared d'Orbigny's systematic study of the Foramini- fera, which he considered "microscopical cephalopods." In 1828 Ehrenberg began publishing his observations on Protozoa and in 1838 he summarized his contributions in Die Infusionsthierchen als Vollkommene Organismen, in which he diagnosed genera and species so well that many of them still hold good. Ehrenberg excluded Rota- toria and Cere aria from Infusoria. Through the studies of Ehrenberg the number of known Protozoa increased greatly; he, however, pro- posed the term "Polygastricha," under which he placed Mastigo- phora, Rhizopoda, Ciliata, Suctoria, desmids, etc., since he believed that the food vacuoles present in them were stomachs. This hypothe- sis became immediately the center of controversy, which incidentally, together with the then-propounded cell theory and improvements in microscopy, stimulated researches on Protozoa. Dujardin (1835) took pains in studying the protoplasm of various Protozoa and found it alike in all. He named it sarcode. In 1841 he published an extensive monograph of various Protozoa which came under his observations. The term Rhizopoda was coined by this investigator. The commonly used term protoplasm was coined by Purkinje in 1840. The Protozoa was given a distinct definition by Siebold in 1845, as follows: "Die Thiere, in welchen die verschied- enen Systeme der Organe nicht scharf ausgeschieden sind, und deren unregelmassige Form und einfache Organization sich auf eine Zelle reduzieren lassen." Siebold subdivided Protozoa into Infusoria and Rhizopoda. The sharp differentiation of Protozoa as a group cer- tainly inspired numerous microscopists. As a result, various stu- dents brought forward several group names, such as Radiolaria (J. Muller, 1858), Ciliata (Perty, 1852), Flagellata (Cohn, 1853), Suctoria (Claparede and Lachmann, 1858), Heliozoa, Protista (Haeckel, 1862, 1866), Mastigophora (Diesing, 1865), etc. Of Suc- toria, Stein failed to see the real nature (1849), but his two mono- graphs on Ciliata and Mastigophora (1854, 1859-1883) contain con- cise descriptions and excellent illustrations of numerous species. Haeckel (1873), who went a step further than Siebold by distinguish- ing between Protozoa and Metazoa, devoted ten years to his study of Radiolaria, especially those of the Challenger collection, and de- scribed in his celebrated monographs more than 4000 species. In 1879 the first comprehensive monograph on the Protozoa of North America was put forward by Leidy under the title of Fresh- water Rhizopods of North America, which showed the wide distribu- tion of many known forms of Europe and revealed a number of new INTRODUCTION 13 and interesting forms. This work was followed by Stokes' The Fresh- water Infusoria of the United States, which appeared in 1888. Butschli (1880-1889) established Sarcodina and made an excellent contribution to the taxonomy of the then-known species of Protozoa, which is still considered as one of the most important works on gen- eral protozoology. The painstaking researches by Maupas, on the conjugation of ciliates, corrected erroneous interpretation of the phenomenon observed by Balbiani some thirty years before and gave impetus to a renewed cytological study of Protozoa. The variety in form and structure of the protozoan nuclei became the subject of in- tensive studies by several cytologists. Weismann (1881) put into words the immortality of the Protozoa. Schaudinn contributed much toward the cytological and developmental studies of Protozoa. In the first year of the present century, Calkins in the United States and Doflein in Germany wrote modern textbooks on protozo- ology dealing with the biology as well as the taxonomy. Calkins initiated the so-called isolation pedigree culture of ciliates in order to study the physiology of conjugation and other phenomena connected with the life-history of the ciliates. Recently application of bacteria- free culture technique to certain free-living flagellates and ciliates has brought to light hitherto unknown facts regarding nutritional re- quirements of these organisms. Today the Protozoa are more and more intensively and exten- sively studied from both the biological and the parasitological sides, and important contributions appear continuously. Since all parasitic Protozoa appear to have originated in free-living forms, the com- prehension of the morphology, physiology, and development of the latter group is obviously fundamentally important for a thorough understanding of the former group. Compared with the advancement of our knowledge on free-living Protozoa, that on parasitic forms has been very slow. This is to be ex- pected, of course, since the vast majority of them are so minute that the discovery of their presence has been made possible only through improvements in the microscope and in technique. Here again Leeuwenhoek seems to have been the first to observe a parasitic protozoan, for he observed, according to Dobell, in the fall of 1674, the oocysts of the coccidian, Eimeria stiedae, in the con- tents of the gall bladder of an old rabbit; in 1681, Giardia intestinalis in his own diarrhceic stools; and in 1683, Opalina and Nyctotherus in the gut contents of frogs. The oral Trichomonas of man was ob- served by O. F. Miiller (1773) who named it Cercaria tenax (Dobell, 1939). There is no record of anyone having seen Protozoa living in 14 PROTOZOOLOGY other organisms, until 1828, when Dufour's account of the gregarine from the intestine of coleopterous insects appeared. Some ten years later. Hake rediscovered the oocysts of Eimeria stiedae. A flagellate was observed in the blood of salmon by Valentin in 1841, and the frog trypanosome was discovered by Gluge and Gruby (1842), the latter author creating the genus Trypanosoma for it. The gregarines were a little later given attention by Siebold (1839), KoUiker (1848) and Stein (1848). The year 1849 marks the first rec- ord of an amoeba being found in man, for Gros then observed Enta- moeba gingivalis in the human mouth. Five years later, Davaine found in the stools of cholera patients two flagellates (Trichomonas and Chilomastix). Kloss in 1855 observed the coccidian, Klossia heli- cina, in the excretory organ of Helix ; and Eimer (1870) made an ex- tensive study of Coccidia occurring in various animals. Balantidium coll was discovered by Malmsten in 1857. Lewis in 1870 observed Entamoeba coli in India, and Losch in 1875 found Entamoeba histo- lytica in Russia. At the beginning of the last century, an epidemic disease, pebrine, of the silkworm appeared in Italy and France, and a number of biologists became engaged in its investigation. Foremost of all, Pasteur (1870) made an extensive report on the nature of the causative organism, now known as Nosema bombycis, and also on the method of control and prevention. Perhaps this is the first scientific study of a parasitic protozoan to result in an effective practical method of control of its infection. Lewis observed in 1878 an organism which is since known as Trypanosoma lewisi in the blood of rats. In 1879 Leuckart created the group "Sporozoa," including in it the gregarines and coccidians. Other groups under Sporozoa were soon definitely designated. They are Myxosporidia (Biitschli, 1881), Microsporidia and Sarcosporidia (Balbiani, 1882). Parasitic protozoology received a far-reaching stimulus when Laveran,(1880) discovered the malarial parasite in the human blood. Smith and Kilbourne (1893) demonstrated that the Babesia of the Texas fever of cattle in the southern United States was transmitted by the cattle tick from host to host, and thus brought to light for the first time the close relationship which exists between an arthropod and a parasitic protozoan. Two years later, Bruce discovered Try- panosoma brucei in the blood of horses and cattle suffering from "nagana" disease in Africa, and in the following year he showed by experiments that the tsetse fly transmits the trypanosome from host to host. Studies of malarial diseases continued and several important contributions appeared. Golgi (1886, 1889) studied the schizogony INTRODUCTION 15 and its relation to the occurrence of fever and was able to distinguish two types of fever. MacCallum (1897-1898) found in the United States the union of a microgamete and a macrogamete of Haemopro- teus of birds. Almost at the same time, Schaudinn and Siedlecki (1897) showed that anisogamy results in the production of zygotes in Coccidia. The latter author published later further observations on the life-cycle of Coccidia (1898, 1899). Ross (1898) showed how Plasmodium relictum (P. praecox) was carried by Culex fatigans and described its life-cycle. Since that time several investigators have brought to light important observations concerning the biology and development of malarial organisms and their relation to man. In the present centur}'^, Forde and Button (1901) observed that the sleeping sickness in equatorial Africa was due to an infection by Trypanosoma gambiense. In 1903 Leishman and Donovan recognized Leishmania of "kala-azar." Artificial cultivation of bacteria had contributed toward a very rapid advancement in bacteriology, and it was natural, as the num- ber of known parasitic Protozoa rapidly increased, that attempts to cultivate them in vitro should be made. Musgrave and Clegg (1904) cultivated, on bouillon-agar, small free-living amoebae from old faecal matter. In 1905 Novy and McNeal cultivated successfully the trypanosome of birds in blood-agar medium, which remained free from bacterial contamination and in which the organisms underwent multiplication. Almost all species of Trypanosoma and Leishmania have since been cultivated in a similar manner. This serves for de- tection of a mild infection and also identification of the species in- volved. It was found, further, that the changes which these organ- isms underwent in the culture media were imitative of those that took place in the invertebrate host, thus contributing toward the life-cycle studies of them, Bass (1911), and Bass and Johns (1912) demonstrated that Plas- modium of man could be cultivated in vitro for a few generations. During and since the World War I, it became known that numerous intestinal Protozoa of man are widely present throughout the tropi- cal, subtropical and temperate zones. Taxonomic, morphological and developmental studies on these forms have therefore appeared in an enormous number. Cutler (1918) seems to have succeeded in cultivating Entamoeba histolytica, though his experiment was not repeated by others. Barrett and Yarborough (1921) cultivated Balantidium coli and Boeck (1921) cultivated Chilomastix mesnili. Boeck and Drbohlav (1925) succeeded in cultivating Entamoeba histolytica, and their work was repeated and improved upon by sev- 16 PROTOZOOLOGY eral investigators. While the cultivation has not yet thrown much light on this and similar amoebae, it has revealed certain evidences that there is no sexual reproduction in these amoebae. References BtJTSCHLi, O. 1887-1889 Bronn's Klassen und Ordnungen des Thier-reichs. Vol. 1, Part 3. Calkins, G. N. 1933 The biology of the Protozoa. 2 ed. Philadelphia. Cole, F. J. 1926 The history of protozoology . London. DoBELL, C. 1911 The principles of protistology. Arch. f. Protis- tenk., Vol. 23. 1932 Antony van Leeuivenhoek and his "little animals.'' New York. 1939 The common flagellate of the human mouth, Tri- chomonas tenax (O.F.M.): its discovery and its nomenclature. Parasitology, Vol. 31. DoFLEiN, F. and E. Reichenow. 1929 Lehrhuch der Protozoen- kunde. 5 ed. Jena. DujARDiN, F. 1841 Histoire natiirelle des Zoophytes. Paris. Kudo, R. R. 1944 Manual of Human Protozoa. Springfield, Illinois. Nordenskiold, E. 1928 The history of biology. New York. Woodruff, L. L. 1937 Louis Joblot and the Protozoa. Sci. Monthly, Vol. 94. 1939 Some pioneers in microscopy, with special reference to protozoology. Tr. N. Y. Acad. Sci., Ser. 2, Vol. 1. Chapter 2 Ecology WITH regard to their habitats, the Protozoa may into free-living forms and those Hving on or in other organisms. Mastigophora, Sarcodina, Cihata, and Suctoria include both free- living and parasitic Protozoa, but Sporozoa are exclusively parasi- tic. The free-living Protozoa The vegetative or trophic stages of free-living Protozoa have been found in every type of fresh and salt water, soil and decaying or- ganic matter. Even in the circumpolar regions or at extremely high altitudes, certain protozoa occur at times in fairly large numbers. The factors, which influence their distribution in a given body of wa- ter, are temperature, light, chemical composition, acidity, kind and amount of food, and degree of adaptability of the individual proto- zoans to various environmental changes. Their early appearance as living organisms, their adaptability to various habitats, and their capacity to remain viable in encysted condition, probably account for the wide distribution of the Protozoa throughout the world. The common free-living amoebae, numerous testaceans and others, to mention a few, of fresh waters, have been observed in innumerable places of the world. Temperature. The majority of Protozoa are able to live only within a small range of temperature variation, although in the en- cysted state they can withstand a far greater temperature fluctua- tion. The lower limit of the temperature is marked by the freezing of the protoplasm, and the upper limit by the destructive chemical change within the body protoplasm. The temperature toleration seems to vary among different species of Protozoa; and even in the same species under different conditions. For example, Chalkley (1930) placed Paramecium caudatum in 4 culture media (balanced saline, saline with potassium excess, saline with calcium excess, and saline with sodium excess), all with pH from 5.8 or 6 to 8.4 or 8.6, at 40°C. for 2-16 minutes and found that (1) the resistance varies with the hydrogen-ion concentration, maxima appearing in the alkaline and acid ranges, and a minimum at or near about 7.0; (2) in a bal- anced saline, and in saline with an excess of sodium or potassium, the alkaline maximum is the higher, while in saline with an excess of calcium, the acid maximum is the higher; (3) in general^ acidity de- creases and alkalinity increases resistance; and (4) between pH 6.6 17 18 PROTOZOOLOGY and 7.6, excess of potassium decreases resistance and excess of cal- cium increases resistance. Glaser and Coria (1933) cultivated Para- mecium caudatum on dead yeast free from living organisms at 20-28°C. (optimum 25°C.) and noted that at 30°C. the organisms were killed. Doudoroff (1936), on the other hand, found that in P. multimicronucleatum its resistance to raised temperature was low in the presence of food, but rose to a maximum when the food was exhausted, and there was no appreciable difference in the resistance between single and conjugating individuals. The thermal waters of hot springs have been known to contain liv- ing organisms including Protozoa. Glaser and Coria obtained from the thermal springs of Virginia, several species of Mastigophora, Ciliata, and an amoeba which were living in the water, the tempera- ture of which was 34-36°C., but did not notice any protozoan in the water which showed 39-41°C. Uyemura and his co-workers made a series of studies on Protozoa living in various thermal waters of Ja- pan, and reported that many species lived at unexpectedly high temperatures. Some of the Protozoa observed and the temperatures of the water in which they were found are as follows: Amoeba sp., Vahlkampfia Umax, A. radiosa, 30-51°C.; Amoeba verrucosa, Chilo- donella sp., Lionotus fasciola, Paraynecium caudatum, 36-40°C.; Oxytricha fallax, 30-56°C. Under experimental conditions, it has been shown repeatedly that many protozoans become accustomed to a very high temperature if the change be made gradually. Dallinger (1887) showed a long time ago that Tetramitus rostratus and two other species of flagellates became gradually acclimatized up to 70°C. in several years. In na- ture, however, the thermal death point of most of the free-living Protozoa appears to lie between 36° and 40°C. and the optimum temperature, between 16° and 25°C. On the other hand, the low temperature seems to be less detri- mental to Protozoa than the higher one. Many protozoans have been found to live in water under ice, and several haematochrome- bearing Phytomastigina undergo vigorous multiplication on snow in high altitudes, producing the so-called "red snow." Klebs (1893) sub- jected the trophozoites of Euglena to repeated freezing without ap- parent injury and Jahn (1933) found no harmful effect when Euglena cultures were kept without freezing at — 0.2°C. for one hour, but when kept at — 4°C. for one hour the majority were killed. Gaylord (1908) exposed Trypanosoma gambiense to liquid air for 20 minutes without apparent injury, but the organisms were killed after 40 min- utes' immersion. ECOLOGY 19 Kiihn (1864) observed that Amoeba and Actinophrys suffered no ill effects when kept at 0°C. for several hours as long as the culture medium did not freeze, but were killed when the latter froze. Molisch (1897) likewise noticed that Amoeba dies as soon as the ice forms in its interior or immediate vicinity. Chambers and Hale (1932) dem- onstrated that internal freezing could be induced in an amoeba by inserting an ice-tipped pipette at — 0.6°C., the ice spreading in the form of fine featherly crystals from the point touched by the pipette. They found that the internal freezing kills the amoebae, although if the ice is prevented from forming, a temperature as low as — 5°C. brings about no visible damage to the organism. At 0°C., Deschiens (1934) found the trophozoites of Entamoeba histolytica remained alive, though immobile, for 56 hours, but were destroyed in a short time when the medium froze at — 5°C. According to Greeley, when Stentor coeruleus was slowly sub- jected to low temperatures, the cilia kept on beating at 0°C. for 1-3 hours, then cilia and gullet were absorbed, the ectoplasm was thrown off, and the body became spherical. When the temperature was raised, this spherical body is said to have undergone a reverse proc- ess and resumed its normal activity. If the lowering of temperature is rapid and the medium becomes solidly frozen, Stentor perishes. Efimoff (1924) observed that Paramecium multiplied once in about 13 days at 0°C., withstood freezing at — 1°C. for 30 minutes, but died when kept for 50-60 minutes at the same temperature. He further stated that Paramecium caudatum, Colpidium colpoda, and Spiro- stomum amhiguum, perished in less than 30 minutes, when ex- posed below — 4°C., and that quick and short cooling (not lower than — 9°C.) produced no injury, but if it is prolonged, Paramecium be- came spherical and swollen to- 4-5 times normal size, while Colpid- ium and Spirostomum shrunk. Wolfson (1935) studied Paramecium sp. in gradually descending subzero-temperature, and observed that as the temperature decreases the organism often swims backward, its bodily movements cease at — 14.2°C., but the cilia continue to beat for some time. While Paramecium recover completely from a momentary exposure to — 16°C., long cooling at this temperature brings about degeneration. When the water in which the organisms are kept freezes, no survival was noted. Plasmodium knowlesi and P. inui in the blood of Macacus rhesus remain viable, according to Coggeshall (1939), for as long as 70 days at — 76°C., if frozen and thawed rapidly. Light. In the Phytomastigina which include chromatophore-bear- ing flagellates, the sun light is essential to photosynthesis (p. 92). The 20 PROTOZOOLOGY sun light further plays an important role in those protozoans which are dependent upon chromatophore-possessing organisms as chief source of food supply. Hence the light is another factor concerned with the distribution of free-living protozoans in the water. Chemical composition of water. The chemical nature of the water is another important factor which influences the very existence of Protozoa in a given body of water. Protozoa differ from one another in morphological as well as physiological characteristics. Individual protozoan species requires a certain chemical composition of the wa- ter in which it can be cultivated under experimental conditions, al- though this may be more or less variable among different forms (Needham et al., 1937). In their "biological analysis of water" Kolkwitz and Marsson (1908, 1909) distinguished four types of habitats for many aquatic plant, and a few animal, organisms, which were based upon the kind and amount of inorganic and organic matter and amount of oxygen present in the water: namely, katharobic, oligosaprobic, mesosapro- bic, and polysaprobic. Katharobic protozoans are those which live in mountain springs, brooks, or ponds, the water of which is rich in oxygen, but free from organic matter. Oligosaprobic forms are those that inhabit waters which are rich in mineral matter, but in which no purification processes are taking place. Many Phytomastigina, various testaceans and many ciliates, such as Frontonia, Lacrymaria, Oxytricha, Stylonychia, Vorticella, etc. inhabit such waters. Meso- saprobic protozoans live in waters in which active oxidation and de- composition of organic matter are taking place. The majority of freshwater protozoans belong to this group: namely, numerous Phytomastigina, Heliozoa, Zoomastigina, and all orders of Ciliata. Finally polysaprobic forms are capable of living in waters which, because of dominance of reduction and cleavage processes of organic matter, contain at most a very small amount of oxygen and are rich in carbonic acid gas and nitrogenous decomposition products. The black bottom slime contains usually an abundance of ferrous sul- phide and other sulphurous substances. Lauterborn (1901) called this sapropelic. Examples of polysaprobic protozoans are Pelomyxa palustris, Euglypha alveolata, Pamphagus armatus, Mastigamoeba, Trepomo7ias agilis, Hexamita inflata, Rhynchomonas nasuta, Hetero- nema acus, Bodo, Cercomonas, Dactylochlamys, Ctenostomata, etc. The so-called "sewage organisms" abound in such habitat (Lackey). Certain free-living Protozoa which inhabit waters rich in decom- posing organic matter are frequently found in the faecal matter of various animals. Their cysts either pass through the alimentary ECOLOGY 21 canal of the animal unharmed or are introduced after the faeces are voided, and undergo development and multiplication in the faecal infusion. Such forms are collectively called coprozoic Protozoa. The coprozoic protozoans grow easily in suspension of old faecal matter which are rich in decomposed organic matter and thus show a strik- ingly strong capacity of adapting themselves to conditions different from those of the water in which they normally live. Some of the Protozoa which have been referred to as coprozoic and which are mentioned in the present work are, as follows: Scytomonas pusilla, Rhynchomonas nasuta, Cercomonas longicauda, C. crassicauda, Tre- yomonas agilis, Dimastig amoeba gruheri, Acanthamoeba hyalina, Chlamydophrys stercorea and Tillina magna. As a rule, the presence of sodium chloride in the sea water prevents the occurrence of the large number of fresh-water inhabitants. Cer- tain species, however, have been known to live in both fresh and brackish or salt water. Among the species mentioned in the present work, the following species have been reported to occur in both fresh and salt waters: Mastigophora: Amphidinium lacustris, Cerat- ium hirundinella; Sarcodina: Lieberkuhnia wagneri; Ciliata: Meso- dinium pulex, Prorodon discolor, Lacrymaria olor, Amphileptus claparedei, Lionotus fasciola, Nassula aurea, Trochilioides recta, Chilodonella cucullulus, Trimyema compressum, Paramecium cal- kinsi, Colpidium campylum, Platynematum sociale, Cinetochilum margaritaceum, Pleuronema coronatum, Caenomorpha medusula, Spirostomum minus, S. teres, Climacostomum virens, and Thuricola follicidata; Snctoria: Metacineta mystacina, Endosphaera engelmanni. It seems probable that many other protozoans are able to live in both fresh and salt water, judging from the observations such as that made by Finley (1930) who subjected some fifty species of freshwater Protozoa of Wisconsin to various concentrations of sea water, either by direct transfer or by gradual addition of the sea water. He found that Bodo uncinatus, Uronema marina, Pleuron- ema jaculans and Colpoda aspera are able to live and reproduce even when directly transferred to sea water, that Amoeba verrucosa, Euglena, Phacus, Monas, Cyclidium, Euplotes, Lionotus, Para- mecium, Styl onychia, etc., tolerate only a low salinity when directly transferred, but, if the salinity is gradually increased, they live in 100 per cent sea water, and that Arcella, Cyphoderia, Aspidisca, Ble- pharisma, Colpoda cucullus, Halteria, etc. could not tolerate 10 per cent sea water even when the change was gradual. Finley noted no morphological changes in the experimental protozoans which might be attributed to the presence of the salt in the water, except Amoeba 22 PROTOZOOLOGY verrucosa, in which certain structural and physiological changes were observed as follows: as the salinity increased, the pulsation of the contractile vacuole became slower. The body activity continued up to 44 per cent sea water and the vacuole pulsated only once in 40 minutes, and after systole, it did not reappear for 10-15 minutes. The organism became less active above this concentration and in 84 per cent sea water the vacuole disappeared, but there was still a tendency to form the characteristic ridges, even in 91 per cent sea water, in which the organism was less fan-shaped and the cytoplasm seemed to be more viscous. Yocom (1934) found that Eiiplotes pa- tella was able to live normally and multiply up to 66 per cent of sea water; above that concentration no division was noticed, though the organism lived for a few da3^s in up to 100 per cent salt water, and Paramecium caudatum and Spirostomum ambiguum were less adaptive to salt water, rarely living in 60 per cent sea water. Frisch (1935) found that no freshwater Protozoa lived above 40 per cent sea water and that Paramecium caudatum and P. multimicronucle- atum died in 33-52 per cent sea water. Hardin (1942) reports that Oikomonas termo will grow when transferred directly to a glycerol- peptone culture medium, in up to 45 per cent sea water, and cultures contaminated with bacteria and growing in a dilute glycerol-peptone medium will grow in 100 per cent sea water. Hydrogen-ion concentration. Closely related to the chemical com- position is the hydrogen-ion concentration (pH) of the water which influences the distribution of Protozoa. The hydrogen-ion concentra- tion of freshwater bodies vary a great deal between highly acid bog waters in which various testaceans may frequently be present, to highly alkaline water in which such forms as Acanthocystis, Hyalo- bryon, etc., occur. In standing deep fresh water, the bottom region is often acid because of the decomposing organic matter, while the surface water is less acid or slightly alkaline due to the photosyn- thesis of green plants which utilize carbon dioxide. In some cases different pH may bring about morphological differences. For exam- ple, in bacteria-free cultures of Paramecium hursaria in a tryptone medium, Loefer (1938) found that at pH 7.6-8.0 the length averaged 86 or 87m, but at 6.0-6.3 the length was about 129^. The greatest variation took place at pH 4.6 in which no growth occurred. The shortest animals at the acid and alkaline extremes of growth, were the widest, while the narrowest forms (about 44ju wide) were found in culture at pH 5.7-7.4. Several workers have made observations on the pll range of the water or medium in which certain proto- zoans live, grow, and multiply, which data are collected in Table 1. ECOLOGY 23 Table 1. — Protozoa and hydrogen-ion concentration pH range of Protozoa medium in which Optimum Observers growth occurs range A. In bacteria-free cultures Euglena gracilis 3.5-9.0 — Dusi 3.0-7.7 6.7 Alexander 3.9-9.9 6.6 Jahn E. deses 6.5-8.0 7.0 Dusi 5.3-8.0 7.0 Hall E. pisciformis 6.0-8.0 6.5-7.5 Dusi 5.4-7.5 6.8 Hall Chilomonas Paramecium 4.8-8.0 6.8 Mast and Pace 4.1-8.4 4.9;7.0 Loefer Chlorogoniiim euchlorum 4.8-8.7 7.1-7.5 Loefer C. elongatum 4.8-8.7 7.1-7.5 Loefer C. teragamum 4.2-8.6 6.7-8.3 Loefer Colpidium campylum — 5.4 Kidder Glaucoma scintillans — 5.6-6.8 Kidder G. ficaria 4.0-9.5 5.1;6.7 Johnson Tetrahymena geleii — 5.6-8.0 Kidder T. vorax — 6.2-7.6 Kidder Paramecirim bursaria 5.3-8.0 6.7-6.8 Loefer B. In cultures containing bacteria Carteria ohtusa — 3.5-4.5 Wermel Acanthocystis aculeata 7.4 or above 8.1 Stern Paramecium caudatum 5.3-8.2 7.0 Darby 6.0-9.5 7.0 Morea P. aurelia 5.7-7.8 6.7 Morea 5.9-8.2 5.9-7.7 Phelps P. mullimicronucleatum 4.8-8.3 7.0 Jones P. sp. 7.8-8.0 Saunders 7.0-8.5 7.8-8.0 Pruthi Colpidium sp. 6.0-8.5 — Pruthi Colpoda cucullus 5.5-9.5 6.5;7.5 Morea Holophyra sp. 6.5-7.4 — Pruthi Plagiopyla sp. 6.9-7.5 — Pruthi Amphileptus sp. 6.8-7.5 7.1-7.3 Pruthi Spirostomum amhiguum 6.8-7.5 7.4 Saunders S. sp. 6.5-8.0 7.5 Morea Stentor coeruleus 7.8-8.0 — Hetherington Blepharisma undulans — 6.5 Moore Gastrostyla sp. 6.0-8.5 — Pruthi Stylonychia pustidata 6.0-8.0 6.7;8.0 Darby Seemingly various Protozoa require a definite pH value in order to carry on maximum metabolic activities. As a matter of fact, 24 PROTOZOOLOGY Pringsheim, Hall, Loefer, Johnson, and others, found that sodium acetate may increase or decrease the growth rate of various Phyto- mastigina subject to the hydrogen-ion concentration of the culture media. Food. The kind and amount of food available in a given body of water also controls the distribution of Protozoa. The food is ordinarily one of the deciding factors of the number of Protozoa in a natural habitat. Species of Paramecium and many other holo- zoic protozoans cannot live in waters in which bacteria or minute protozoans do not occur. If other conditions are favorable, then the greater the number of food bacteria, the greater the number of these protozoans. Didinium nasutum feeds almost exclusively on Para- mecium, hence it cannot live in the absence of the latter ciliate. As a rule, euryphagous protozoans are widely distributed and stenophagous forms are limited in their distribution. Some protozoans inhabit soil of various types and localities. Un- der ordinary circumstances, they occur near the surface, their maxi- mum abundance being found at a depth of about 10-12 cm. (Sandon, 1927). It is said that a very few protozoans occur in the subsoil. Here also one notices a very wide geographical distribution of ap- parently one and the same species. For example, Sandon found Amoeba proteus in samples of soil collected from Greenland, Tristan da Cunha, Gough Island, England, Mauritius, Africa, India, and Argentina. This amoeba is known to occur in various parts of North America, Europe, Japan, and Australia. The majority of Testacea inhabit moist soil in abundance. Sandon observed Trinema enchelys in the soils of Spitzbergen, Greenland, England, Japan, Australia, St. Helena, Barbados, Mauritius, Africa, and Argentina. The parasitic Protozoa ' Some Protozoa belonging to all groups live on or in other organ- isms. The Sporozoa are made up exclusively of parasites. The rela- tionships between the host and the protozoan differ in various ways, which make the basis for distinguishing the associations into three types as follows: commensalism, symbiosis, and parasitism. Commensalism is an association in which an organism, the com- mensal, is benefited, while the host is neither injured nor benefited. Depending upon the location of the commensal in the host body, the term ectocommensalism or endocommensalism is used. Ecto- commensalism is often represented by Protozoa which may attach themselves to any aquatic animals that inhabit the same body of water, as shown by various species of Chonotricha, Peritricha, and ECOLOGY 25 Suctoria. In other cases, there is a definite relationship between the commensal and the host. For example, Kerona polyporum is found on various species of Hydra, and many ciliates placed in Thigmo- tricha (p. 623) are inseparably associated with certain species of the mussels. Endocommensalism is often difficult to distinguish from endo- parasitism, since the effect of the presence of a commensal upon the host cannot be easily understood. On the whole, the protozoans which live in the lumen of the alimentar}^ canal may be looked upon as endocommensals. These protozoans undoubtedly use part of the food material which could be used by the host, but they do not in- vade the host tissue. As examples of endocommensals may be men- tioned: Endamoeha hlattae, Lophomonas blattarum, L. striata, Nyciotherus ovalis, etc., of the cockroach; Entamoeba coli, lodamoeba biitschlii, Endolimax nana, Dientamoeba fragilis, Chilomasiix mes- nili, etc., of the human intestine; numerous species of Protociliata of Anura, etc. Because of the difficulties mentioned above, the term parasitic Protozoa, in its broad sense, includes the commenals also. Symbiosis on the other hand is an association of two species of organisms, which is of mutual benefit. The cryptomonads belonging to Chrysidella ("Zooxanthellae") containing yellow or brown chrom- atophores, which live in Foraminifera and Radiolaria, and certain algae belonging to Chlorella ("Zoochlorellae") containing green chromatophores, which occur in some freshwater protozoans, such as Paramecium bursaria, Stentor amethystinus, etc., are looked upon as holding symbiotic relationship with the respective protozoan host. Several species of the highly interesting Hypermastigina, which are present commonly and abundantly in various species of termites and the woodroach Cryptocercus, have been demonstrated by Cleveland to digest the cellulose material which makes up the bulk of wood- chips the host animals take in and to transform it into glycogenous substances that are used partly by the host insects. If deprived of these flagellates by being subjected to oxygen under pressure or to a high temperature, the termites die, even though the intestine is filled with wood-chips. If removed from the gut of the termite, the flagellates die. Thus the association here may be said to be an abso- lute symbiosis. Parasitism is an association in which one organism (the parasite) lives at the expense of the other (the host). Here also ectoparasitism and endoparasitism occur, although the former is not commonly found. Hydramoeba hydroxena (p. 370) feeds on the body cells of Hydra which, according to Reynolds and Looper, die on an average 26 PROTOZOOLOGY in 6.8 days as a result of the infection and the amoebae disappear in from 4 to 10 days if removed from a host Hydrsi. Costia necatrix (p. 297) often occurs in an enormous number, attached to various freshwater fishes especially in an aquarium, by piercing through the epidermal cells and appears to disturb the normal functions of the host tissue. Ichthyophthirius muUifiliis (p. 568), another ectoparasite of freshwater fishes, goes further by completely burying themselves in the epidermis and feeds on the host's tissue cells and, not infre- quently, contributes toward the cause of the death of the host fishes. The endoparasites absorb by osmosis the vital body fluid, feed on the host cells or cell-fragments by pseudopodia or cytostome, or enter the host tissues or cells themselves, living on the cj^oplasm or in some cases on the nucleus. Consequently they bring about abnor- mal or pathological conditions upon the host which often succumbs to the infection. Endoparasitic Protozoa of man are Entamoeba histolytica, Balantidium coli, species of Plasmodium and Leishmania, Trypanosoma gamhiense, etc. The Sporozoa, as was stated before, are without exception coelozoic, histozoic, or cytozoic parasites. Because of their modes of living, the endoparasitic Protozoa cause certain morphological changes in the cells, tissues, or organs of the host. The active growth of Entamoeba histolytica in the glands of the colon of the victim, produces slightly raised nodules first which de- velop into abscesses and the ulcers formed by the rupture of ab- scesses, may reach 2 cm. or more in diameter, completely destroying the tissues of the colon wall. Similar pathological changes are also noticed in the case of infection by Balantidium coli. In Leishmania donovani, the victim shows an increase in number of the large macro- phages and mononuclears and also an extreme enlargement of the spleen. Trypanosoma cruzi brings about the degeneration of the in- fected host cells and an abundance of leucocytes in the infected tissues, followed by an increase of fibrous tissue. T. gambiense, the causative organism of African sleeping sickness, causes enlargement of lymphatic glands and spleen, followed by changes in meninges and an increase of cerebro-spinal fluid. Its most characteristic changes are the thickening of the arterial coat and the round-celled infiltration around the blood vessels of the central nervous system. Brand's (1938) summary of the carbohydrate metabolism of the pathogenic trj^panosomes tends to show that the sugar is only par- tially oxidized in the presence of oxygen and that the carbohydrate metabolism of the infected host is disturbed, as shown mainly by the unbalanced condition of the blood sugar, by lowering of the glycogen reserves, and by reduced ability to build glycogen from ECOLOGY 27 sugar. Malarial infection is invariably accompanied by an enormous enlargement of the spleen ("spleen index"); the blood becomes watery; the erythrocytes decrease in number; the leucocytes, sub- normal; but mononuclear cells increase in number; pigment granules which are set free in the blood plasma at the time of merozoite- liberation are engulfed by leucocytes; and enlarged spleen contains large amount of pigments which are lodged in leucocytes and endo- thelial cells. In Plasmodium falciparum, the blood capillaries of brain, spleen and other viscera may completely be blocked by in- fected erythrocytes. pfiKppillTf Fig. 1. Histological changes in host fish caused by myxosporidian in- fection, X1920 (Kudo), a, portion of a cyst of Myxobolus intestinalis, sur- rounded by peri-intestinal muscle of the black crappie; b, part of a cyst of Thelohanellus notatus, enveloped by the connective tissue of the blunt- nosed minnow. In Myxosporidia which are either histozoic or coelozoic parasites of fishes, the tissue cells that are in direct contact with highly en- larging parasites, undergo various morphological changes. For exam- ple, the circular muscle fibers of the small intestine of Pomoxis sparoides, which surround Myxobolus intestinalis, a myxosporidian, become modified a great deal and turn about 90° from the original direction, due undoubtedly to the stimulation exercised by the myxosporidian parasite (Fig. 1, a). In the case of another myxo- sporidian, Thelohanellus notatus, the connective tissue cells of the host fish surrounding the protozoan body, transform themselves into 28 PROTOZOOLOGY "epithelial cells" (Fig. 1, h), a state comparable to the formation of the ciliated epithelium from a layer of fibroblasts lining a cyst formed around a piece of ovary inplanted into the adductor muscle of Pecten as observed by Drew (1911). Practically all Microsporidia are cytozoic, and the infected cells become hypertrophied enormously, producing in one genus the so- called Glugea cysts (Fig. 257). In many cases, the hypertrophy of the nucleus of the infected cell is far more conspicuous than that of the cytoplasm (Fig. 255). Information concerning toxic substances produced by parasitic Protozoa is meager. Sarcosporidia appear to produce a certain toxic substance which, when injected into the blood vessel, is highly toxic to experimental animals. This was named sarcocystine (Laveran and Mesnil) or sarcosporidio toxin (Teichmann and Braun). For the great majority of parasitic Protozoa, there exists a de- finite host-parasite relationship and animals other than the specific hosts possess a natural immunity against an infection by a particular parasitic protozoan. Immunity involved in diseases caused by Pro- tozoa has been most intensively studied on haemozoic forms, es- pecially Plasmodium and Trypanosoma, since they are the causative organisms of important diseases. Development of these organisms in hosts depends on various factors such as the species and strains of the parasites, the species and strains of vectors, and immunity of the host. Boyd and co-workers showed that reinoculation of persons who have recovered from an infection with Plasmodium vivax or P. falciparum with the same strain of the parasites, will not result in a second clinical attack, because of the development of homologous immunity, but with a different strain of the same species or different species, a definite clinical attack occurs, thus there being no hetero- logous tolerance. The homologous immunity was found to continue for at least three years and in one case for about seven years in P. vivax, and for at least four months in P. falciparum after apparent eradication of the infection. In the case of leishmaniasis, recovery from a natural or induced infection apparently develops a lasting immunity against reinfection with the same species of Leishmania. It has been shown that in infections with avian, monkey and hu- man Plasmodium or Trypanosoma lewisi, a considerable number of the parasites are destroyed during the developmental phase of the infection and that after a variable length of time, resistance to the parasites often develops in the host, as the parasites disappear from the peripheral blood and symptoms subside, though the host still harbors the organisms. In malarious countries, the adults and chil- ECOLOGY 29 dren show usually a low and a high rate of malaria infection respect- ively, but the latter frequently do not show symptoms of infection, even though the parasites are detectable in the blood. Apparently repeated infection produces tolerance which can keep, as long as the host remains healthy, the parasites under control. There seems to be also racial difference in the degree of immunity against Plasmodium and Trypanosoma, as shown by James, Milam and Kusch, etc. As to the mechanism of immunity, the destruction of the parasites by phagocytosis of the endothelial cells of the spleen, bone marrow and liver and continued regenerative process to replace the de- stroyed blood cells, are the two important phases in the cellular de- fense mechanism. Besides, there are indications that humoral de- fense mechanism through the production of antibodies is in active operation in infections by Plasmodium knowlesi (Coggeshall and Kumm, Eaton, etc.) and trypanosomes (Taliaferro), With regard to the origin of parasitic Protozoa, it is generally agreed among biologists that the parasite in general evolved from the free-living form. The protozoan association with other organ- isms was begun when various protozoans which lived attached to, or by crawling on, submerged objects happened to transfer them- selves to various invertebrates which occur in the same water. These Protozoa benefit by change in location as the host animal moves about, and thus enlarging the opportunity to obtain a con- tinued supply of food material. Such ectocommensals are found abundantly; for example, the peritrichous ciliates attached to the body and appendages of various aquatic animals such as larval in- sects and microcrustaceans. Ectocommensalism may next lead to ectoparasitism as in the case of Costia or Hydramoeba, and then again instead of confining themselves to the body surface, the Pro- tozoa may bore into the body wall from outside and actually acquire the habit of feeding on tissue cells of the attached animals as in the case of Ichthyophthirius. The next step in the evolution of parasitism must have been reached when Protozoa, accidentally or passively, were taken into the digestive system of the Metazoa. Such a sudden change in habitat appears to be fatal to most protozoans. But certain others possess extraordinary capacity to adapt themselves to an entirely different environment. For example, Dobell (1918) observed in the tadpole gut, a typical free-living limax amoeba, with characteristic nucleus, contractile vacuoles, etc., which was found in numbers in the water containing the faecal matter of the tadpole. Glaucoma pyriformis (Tetrahymena geleii), a free-living ciliate, was found to 30 PROTOZOOLOGY occur in the body cavity of the larvae of Theohaldia annulata (after MacArthur) and in the larvae of Chironomus -plumosus (after Treil- lard and Lwoff). Lwoff successfully inoculated this ciliate into the larvae of Galleria mellonella which died later from the infection. Recently Janda and Jirovec (1937) injected bacteria-free culture of this ciliate into annelids, molluscs, crustaceans, insects, fishes, and amphibians, and found that only insects — all of 14 species (both larvae and adults) — became infected by this ciliate. In a few days after injection the haemocoele became filled with the ciliates. Of various organs, the ciliates were most abundantly found in the adipose tissue. The organisms were much larger than those present in the original culture. The insects, into which the ciliates were in- jected, died from' the infection in a few days. The course of develop- ment of the ciliate within an experimental insect depended not only on the amount of the culture injected, but also on the temperature. At 1-4°C. the development was much slower than at 26°C.; but if an infected insect was kept at 32-36°C. for 0.5-3 hours, the ciliates were apparently killed and the insect continued to live. When Glaucoma taken from Dixippus morosus were placed in ordinary water, they continued to live and underwent multiplication. The ciliate showed a remarkable power of withstanding the artificial digestion; namely, at 18°C. they lived 4 days in artificial gastric juice with pH 4.2; 2-3 days in a juice with pH 3.6; and a few hours in a juice with pH 1.0. Cleveland (1928) observed Tritrichomonas fecalis in faeces of a single human subject for three years which grew well in faeces diluted with tap water, in hay infusions with or with- out free-living protozoans or in tap water with tissues at —3° to 37°C., and which, when fed per os, was able to live indefinitely in the gut of frogs and tadpoles. Reynolds (1936) found that Colpoda steini, a free-living ciliate of fresh water, occurs naturally in the intestine and other viscera of the land slug, Agriolimax agrestis, the slug forms being much larger than the free-living individuals. It may be further speculated that Vahlkampfia, Hydramoeba, Schizamoeba, and Endamoeba, are the different stages of the course the intestinal amoebae might have taken during their evolution. Obviously endocommensalism in the alimentary canal was the initial phase of endoparasitism. When these endocommensals began to consume an excessive amount of food or to feed on the tissue cells of the host gut, they became the true endoparasites. Destroying or penetrating through the intestinal wall, they became first established in the body or organ cavities and then invaded tissues, cells or even nuclei, thus developing into pathogenic Protozoa. The endoparasites ECOLOGY 31 developing in invertebrates which feed upon the blood of vertebrates as source of food supply, will have opportunities to establish them- selves in the higher animals. References Calkins, G. N. and F. M. Summers (editors). 1941 Protozoa in biological research. New York. Chalkley, H. W. 1930 Resistance of Paramecium to heat as af- fected by changes in hydrogen-ion concentration and in inor- ganic salt balance in surrounding medium. U. S. Publ. Health. Rep., Vol. 45. Cleveland, L. R. 1926 Symbiosis among animals with special reference to termites and their intestinal flagellates. Quart. Rev. Biol., Vol. 1. 1928 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. Jour. Hyg., Vol. 8. Dallinger, W. H. 1887 The president's address. Jour. Roy. Micr. Soc. for 1887. DoBELL, C. 1918 Are Entamoeba histolytica and Entamoeba ranarum the same species? Parasitology, Vol. 10. DouDOROFF, M. 1936 Studies in thermal death in Paramecium. Jour. Exp. Zool., Vol. 72. Efimoff, W. W. 1924 Ueber Ausfrieren und Ueberkaltung der Protozoen. Arch. f. Protistenk., Vol. 49. Finley, H. E. 1930 Toleration of freshwater Protozoa to increased salinity. Ecology, Vol. 11. Glaser, R. W. and ISF. A. Coria. 1933 The culture of Paramecium caudatum free from living microorganisms. Jour. Parasit. Vol. 20. Janda, V. and 0. Jirovec 1937 Ueber kiinstlich hervorgerufenen Parasitismus eines f reilebenden Ciliaten Glaucoma piriformis und Infektionsversuche mit Euglena gracilis und Spirochaeta hiflexa. Mem. soc. zool tehee, de Prague, Vol. 5. Kidder, G. W. 1941 Growth studies on cihates. VII. Biol. Bull., Vol. 80. Kolkwitz, R. and M. Marsson 1909 Oekologie der tierischen Saprobien. Intern. Rev. Ges. Hydrobiol. u. Hydrogr., Vol. 2. Kudo, R. R. 1929 Histozoic Myxosporidia found in freshwater fishes of Illinois, U.S.A. Arch. f. Protistenk., Vol. 65. Lackey, J. B. 1925 The fauna of Imhof tanks. Bull. New Jersey Agr. Exp. Stat., No. 417. Lauterborn, R. 1901 Die "sapropelische" Lebewelt. Zool. Anz., Vol. 24. Needhum, J. G., P. S. Galtsoff, F. E. Lutz and P. S. Welch. 1937 Culture methods for invertebrate animals. Ithaca, N.Y. Noland, L. E. 1925 Factors influencing the distribution of fresh- water ciliates. Ecology, Vol. 6. 32 PROTOZOOLOGY Reynolds, B. D. 1936 Colpoda steini, a facultative parasite of the land slug, Agriolimax agrestis. Jour. Parasit., Vol. 22. and J. B. Looper 1928 Infection experiments with Hydra- moeha hydroxena nov. gen. Ibid., Vol. 15. Sandon, H. 1927 The composition and distribution of the protozoan fauna of the soil. Edinburgh. Taliaferro, W. H. 1926 Host resistance and types of infections in trypanosomiasis and malaria. Quart. Rev. Biol., Vol. 1. VON Brand, T. 1938 The metabolism of pathogenic trypanosomes and the carbohydrate metabolism of their hosts. Ibid., Vol. 13. Wenyon, C. M. 1926 Protozoology. 2 vols. London and New York. WoLFSON, C. 1935 Observations on Paramecium during exposure to sub-zero temperatures. Ecology, Vol. 16. YocoM, H. B. 1934 Observations on the experimental adaptation of certain freshwater ciliates to sea water. Biol. Bull., Vol. 67. Chapter 3 Morphology PROTOZOA range in size from ultramicroscopic to macroscopic, though they are on the whole minute microscopic animals. The parasitic forms, especially cytozoic parasites, are often extremely small, while free-living protozoans are usually of much larger dimen- sions. Noctiluca, Foraminifera, Radiolaria, many ciliates such as Stentor, Bursaria, etc., represent larger forms. Colonial proto- zoans such as Carchesium, Zoothamnium, Ophrj^dium, etc., are even greater than the solitary forms. On the other hand, Plasmodium, Leishmania, and microsporidian spores may be mentioned as exam- ples of the smallest forms. The unit of measurement employed in protozoology is, as in general microscopy, 1 micron (ju) which is equal to 0.001 mm. The body form of Protozoa is even more varied, and because of its extreme plasticity it frequently does not remain constant. Fur- thermore the form and size of a given species may vary according to the kind and amount of food as is discussed elsewhere (p. 94). From a small simple spheroidal mass up to large highly complex forms, all possible body forms occur. Although the great majority are without symmetry, there are some which possess a definite symmetry. Thus bilateral symmetry is noted in all members of Diplomonadina (p. 311); radial symmetry in Gonium, Cyclonexis, etc.; and universal symmetry, in certain Heliozoa, Volvox, etc. The fundamental component of the protozoan body is the pro- toplasm which is without exception differentiated into the nucleus and the cytosome. Haeckel's monera are now considered as non- existent, since improved microscopic technique has failed in recent years to reveal any anucleated protozoans. The nucleus and the cyto- some are inseparably important to the well-being of a protozoan, as has been shown by numerous investigators since Verworn's pioneer work. In all cases, successful regeneration of the body is accomplished only by the nucleus-bearing portions and enucleate parts degenerate sooner or later. On the other hand, when the nucleus is taken out of a protozoan, both the nucleus and cytosome degenerate, which indi- cates their intimate association in carrying on the activities of the body. It appears certain that the nucleus controls the assimilative phase of metabclism which takes place in the cytosome in normal animals, while the cytosome is capable of carrying on the catabolic 33 34 PROTOZOOLOGY phase of the metabolism. Aside from the importance as the control- ling center of metabolism, evidences point to the conclusion that the nucleus contains the genes or hereditary factors which characterize each species of protozoans from generation to generation, as in the cells of multicellular animals and plants. The nucleus Because of a great variety of the body form and organization, the protozoan nuclei are of various forms, sizes and structures. At one extreme there is a small nucleus and, at the other, a large voluminous one and, between these extremes, is found almost every conceivable variety of form and structure. The majority of Protozoa contain a single nucleus, though many may possess two or more throughout the greater part of their life-cycle. In several species, each individual possesses two similar nuclei, as in Diplomonadina, Protoopalina and Zelleriella. In Euciliata and Suctoria, two dissimilar nuclei, a macronucleus and a micronucleus, are typically present. The macro- nucleus is always larger than the micronucleus, and controls the trophic activities of the organism, while the micronucleus is con- cerned with the reproductive activity. Certain Protozoa possess numerous nuclei of similar structure, as for example, in Pelomyxa, Mycetozoa, Actinosphaerium, Opalina, Cepedea, Myxosporidia, Microsporidia, etc. The essential components of the protozoan nucleus are the nuclear membrane, chromatin, plastin and nucleoplasm. Their interrela- tionship varies sometimes from one developmental stage to an- other, and vastly among different species. Structurally, they fall in general into one of the two types: vesicular and compact. The vesicular nucleus (Fig. 2, a) consists of a nuclear membrane which is sometimes very delicate but distinct, nucleoplasm and chromatin. Besides there is an intranuclear body which is, as a rule, more or less spherical and which appears to be of different make-ups as judged by its staining reactions among different nuclei. It may be composed of chromatin, of plastin, or of a mixture of both. The first type is sometimes called karyosome and the second, nucleolus or plasmosome. Absolute distinction between these two terms cannot be made as they are based upon the difference in affinity to nuclear stains which cannot be standardized and hence do not give uni- formly the same result. Following Minchm and others, the term endosome is advocated here to designate one or more conspicuous bodies other than the chromatin granules, present within the nuclear membrane. MORPHOLOGY 35 When viewed in life, the nucleoplasm is ordinarily homogeneous and structureless. But, upon fixation, there appear invariably plastin strands or networks which seem to connect the endosome and the nuclear membrane. Some investigators hold that these strands or networks exist naturally in life, but due to the similarity of refractive indices of the strands and of the nucleoplasm, they are not visible and that, when fixed, they become readily recognizable because of a change in these indices. In some nuclei, however, certain strands have been observed in life, as for example in the nucleus of the species of Barbulanympha (Fig. 152, c), according to Cleveland and his associates (1934). Others maintain that the achromatic structures prominent in fixed vesicular nuclei are mere artifacts brought about Nuclear membrane Endosome Achromatic strand Chromatin granules a b Fig. 2. a, vesicular nucleus; b, compact nucleus (diagrams). by fixation and do not exist in life and that the nucleoplasm is a homogeneous liquid matrix of the nucleus. The chromatin substance is ordinarily present as small granules although at times they may be in block forms. Precise knowledge of chromatin is still lacking. At present the determination of the chromatin depends upon the following tests: (1) artificial digestion which does not destroy this substance, while non-chromatinic parts of the nucleus are completely dissolved; (2) acidified methyl green which stains the chromatin bright green; (3) 10 per cent sodium chloride solution which dissolves, or causes swelling of, chromatin granules, while nuclear membrane and achromatic sub- stances remain unattacked; and (4) in the fixed condition Feulgen's nucleal reaction. The vesicular nucleus is most commonly present in various orders of the Sarcodina and Mastigophora. The compact nucleus (Fig. 2, b), on the other hand, contains a large amount of chromatin substance and a comparatively small amount of nucleoplasm, and is thus massive. The macronucleus of the Cihophora is almost always of this kind. The variety of forms of the compact nuclei is indeed remarkable. It may be spherical, ovate, cylindrical, club-shaped, band-form, moniliform, horseshoe- form, filamentous, or dendritic. The nuclear membrane is always 36 PROTOZOOLOGY distinct, and the chromatin substance is usually of spheroidal form, varying in size among different species and often even in the same nucleus. In the majority of species, the chromatin granules are small and compact, though in some forms, such as Nydotherus ovalis (Fig. 3), they may reach 20^ or more in diameter in some individuals. Fig. 3. Parts of four macronuclei of Nydotherus ovalis, showing chromatin spherules of different sizes, X650 (Kudo). and while the smaller chromatin granules seem to be solid, larger forms contain alveoli of different sizes in which smaller chromatin granules are suspended (Kudo, 1936). There is no sharp demarcation between the vesicular and compact nuclei, since there are numerous nuclei the structures of which are intermediate between the two. Moreover what appears to be a vesicular nucleus in life, may approach a compact nucleus when fixed and stained as in the case of Euglenoidina. Several experimental observations show that the number, size, and structure of the endo- MORPHOLOGY 37 some in the vesicular nucleus, and the amount and arrangement of the chromatin in the compact nucleus, vary according to the physio- logical state of the whole organism. The macronucleus may be divided into two or more parts with or without connections among them and in Dileptus anser into more than 200 small nuclei, each of which is "composed of a plastin core and a chromatin cortex" (Cal- kins; Hayes). In general, the chromatin granules or spherules fill the intra- nuclear space compactly, in which one or more endosomes may occur. In many nuclei these chromatin granules appear to be sus- pended freely, while in others a reticulum appears to make the background. The chromatin of compact nuclei gives a strong posi- tive Feulgen's nucleal reaction. The macronuclear and micronuclear chromatin substances respond differently to Feulgen's nucleal re- action or to the so-called nuclear stains, as judged by the difference in the intensity or tone of color. In Paramecium caudatum, P. aurelia, Chilodonella, Nyctotherus ovalis, etc., the macronuclear chromatin is colored more deeply than the micronuclear chromatin, while in Colpoda, Urostyla, Euplotes, Stylonychia, and others, the reverse seems to be the case, which may support the validity of the assumption by Heidenhain that the two types of the nuclei of Euciliata and Suctoria are made up of different chromatin sub- stances — idiochromatin in the micronucleus and trophochromatin in the macronucleus — and in other classes of Protozoa, the two kinds of chromatin are present together in a single nucleus. Chromidia. Since the detection of chromatin had solely depended on its affinity to certain nuclear stains, several investigators found extranuclear chromatin granules in many protozoans. Finding such granules in the cytosome of Actinosphaeriu7n eichhorni, Arcella vul- garis, and others, Hertwig (1902) called them chromidia, and main- tained that under certain circumstances, such as lack of food ma- terial, the nuclei disappear and the chromatin granules become scat- tered throughout the cytosome. In the case of Arcella vulgaris, the two nuclei break down completely to produce a chromidial-net which later reforms into smaller secondary nuclei. It has, however, been found by Belar that the lack of food caused the encystment rather than chromidia-formation in Actinosphaerium and, according to Reichenow, Jollos observed that in Arcella the nuclei persisted, but were thickly covered by chromidial-net which could be cleared away by artificial digestion to reveal the two nuclei. In Difflugia, the chromidial-net is vacuolated or alveolated in the fall and in each alveolus appear glycogen granules which seem to serve as reserve 38 PROTOZOOLOGY food material for the reproduction that takes place during that season (Zuelzer), and the chromidia occurring in Actinosphaerium appear to be of a combination of a carbohydrate and a protein (Rumjantzew and Wermel). Apparently the widely distributed volutin (p. 101), and many inclusions or cytozoic parasites, such as Sphaerita, which occur occasionally in different Sarcodina, have in some cases been called chromidia. Bj^ using Feulgen's nucleal reac- tion, Reichenow (1928) obtained a diffused violet-stained zone in Chlamydomonas and held them to be dissolved volutin. Calkins (1933) found the chromidia of Arcella vulgaris negative to the nucleal reaction, but by omitting acid-hydrolysis and treating with fuchsin- sulphurous acid for 8-14 hours, the chromidia and the secondary nuclei were found to show a typical positive reaction and believed that the chromidia were chromatin. Thus at present the real nature of chromidia is still not clearly known, although many protozoolo- gists are inclined to think that the substance is not chromatinic, but, in some way, is connected with the metabolism of the protozoan. The cytosome The extranuclear part of the protozoan body is the cytosome. It is composed of the cytoplasm, a colloidal system, which may be homogenous, granulated, vacuolated, reticulated, or fibrillar in optical texture, aud is almost always colorless. The chromatophore- bearing Protozoa are variously colored, and those with symbiotic algae or cryptomonads are also greenish or brownish in color. Fur- thermore, pigment or crystals which are produced in the body, may give protozoans various colorations. In several forms pigments are diffused throughout the cytoplasm. For example, many dinoflagel- lates are beautifully colored, which, according to Kofoid and Swezy, is due to a thorough diffusion of pigment in the cytoplasm. Stentor coeruleus is ordinarily blue-colored, the pigment stentorin (Lankester) is lodged as granules between the surface striae; and rose- or purple- coloration of several species of Blepharisma appears to be due to a special pigment, zoopurpurin (Arcichovskij) which is said to be lodged in the ectoplasmic granules often called protrichocysts (p. 65). The development of zoopurpurin is definitely correlated with the sun-light, as shown by Giese. Deeply pink specimens will lose color completely in a few hours when exposed to strong sun-light and the recoloration takes place in darkness very slowly. The extent and nature of the cytosomic differentiation differ greatly among various groups. In the majority of Protozoa, the cytoplasm is differentiated into the ectoplasm and the endoplasm. MORPHOLOGY 39 The ectoplasm is the cortical zone which is hyaline and homogene- ous. In the Ciliophora, it is a permanent and distinct part of the body and contains several organellae; in the Sarcodina and the Sporozoa, it is more or less a temporarily differentiated zone and hence varies greatly at different times and, in the Mastigophora, it seems to be more or less permanent. The endoplasm is more voluminous and fluid. It is granulated or alveolated and contains various organellae. While the alveolated cytoplasm is normal in forms such as the members of Heliozoa and Radiolaria, in other cases the alveolation of normally granulated or vacuolated cytoplasm indicates invariably the degeneration of the protozoan body. In Amoeba and other Sarcodina, the "hyaline cap" and "layer" (Mast) make up the ectoplasm, and the "plasmasol" and "plamagel" (Mast) compose the endoplasm (Fig. 44). In numerous Sarcodina and certain Mastigophora, the body surface is naked and not protected by any form-giving organella. According to the observations by Kite, Rowland, and others, the surface layer is not only elastic, but solid, and therefore the name plasma-membrane may be applied to it. Such forms are capable of undergoing amoeboid movement by formation of pseudopodia and by continuous change of form due to the movement of the cytoplasm which is more fluid. However, the majority of Protozoa possess a characteristic and constant body form due to the development of a special envelope, the pellicle. In Amoeba striata and A. verrucosa, there is a distinct pellicle. The same is true with some flagellates, such as certain species of Euglena, Peranema, and Astasia, in which it is elastic and expansible so that the organisms show a great deal of plasticity. The pellicle of a ciliate is much thicker and more definite, and often variously ridged or sculptured. In many, linear furrows and ridges run longitudinally, obliquely, or spirally; and, in others, the ridges are combined with hexagonal or rectangular depressed areas. Still in others, such as Coleps, elevated platelets are arranged paral- lel to the longitudinal axis of the bodJ^ In certain peritrichous ciliates, such as Vorticella monilata, Carchesiiim granulatum, etc., the pellicle may possess nodular thickenings arranged in more or less parallel rows at right angles to the body axis. While the pellicle always covers the protozoan body closely, there are other kinds of protective envelopes produced by Protozoa which may cover the body rather loosely. These are the shell, test, lorica or envelope. The shell of various Phytomastigina is usually made up of cellulose, a carbohydrate, which is widely distributed 40 PROTOZOOLOGY in the plant kingdom. It may be composed of a single or several layers, and may possess ridges or markings of various patterns on it. In addition to the shell, gelatinous substance may in many forms be produced to surround the shelled body or in the members of Volvo- cidae to form the matrix of the entire colony in which the individuals are embedded. In the dinoflagellates, the shell is highly developed and often composed of numerous plates which are variously sculp- tured. In other Protozoa, the shell is made up of chitin or pseudo-chitin (tectin). Common examples are found in the testaceans; for example, in Arcella and allied forms, the shell is made up of chitinous material constructed in particular ways which characterize the different gen- era. Newly formed shell is colorless, but older ones become brownish, because of the presence of iron oxide. Difflugia and related genera form shells by gluing together small sand-grains, diatom-shells, debris, etc., with chitinous or pseudochitinous substances which they secrete. Many foraminiferans seem to possess a remarkable selective power in the use of foreign materials, for the construction of their shells. According to Cushman, Psammosphaera fusca uses sand- grains of uniform color but of different sizes, while P. parva uses grains of more or less uniform size but adds, as a rule, a single large acerose sponge spicule which is built into the test and which extends out both ways considerably. Cushman thinks that this is not acci- dental, since the specimens without the spicules are few and those with a short or broken spicules are not found. P. howmanni, on the other hand, uses only mica flakes which are found in a comparatively small amount, and P. rustica uses acerose sponge spicules for the framework of the shell, skilfully fitting smaller broken pieces into polygonal areas. Other foraminiferans combine chitinous secretion with calcium carbonate and produce beautifully complicated shells (Fig. 4) with one or numerous pores. In the Coccolithidae, variously shaped platelets of calcium carbonate ornament the shell. The silica is present in the shells of various Protozoa. In Euglypha and related testaceans, siliceous scales or platelets are produced in the endoplasm and compose a new shell at the time of fission or of encystment together with the chitinous secretion. In many helio- zoans, siliceous substance forms spicules, platelets, or combination of both which are embedded in the mucilaginous envelope that surrounds the body and, in some cases, a special clathrate shell com- posed of silica, is to be found. In some Radiolaria, isolated siliceous spicules occur as in Heliozoa, while in others the lateral development of the spines results in production of highly complex and the most MORPHOLOGY 41 beautiful shells with various ornamentations or incorporation of foreign materials. Many pelagic radiolarians possess numerous con- spicuous radiating spines in connection with the skeleton, which ap- parently aid the organisms in maintaining their existence in the open sea. Fig. 4. Diagram of the shell of Fetieroplis pertusus, X about 35 (Carpenter), ep, external pore; s, septum; sc, stolon canal. Certain Protomonadina possess a funnel-like collar in the flagel- lated end and in some in addition a chitinous lorica surrounds the body. The lorica found in the Ciliophora is mostly composed of chitinous substance alone, especially in Peritricha, although others produce a house made up of gelatinous secretion containing foreign materials as in Stentor (p. 645). In the Tintinnidae, the loricae are either solely chitinous in numerous marine forms not mentioned in the present work or composed of sand-grains or coccoliths ce- mented together by chitinous secretion, which are found in fresh- water forms. Locomotor organellae Closely associated with the body surface are the organellae of locomotion : pseudopodia, flagella, and cilia. These organellae are not confined to Protozoa alone and occur in various cells of Metazoa. All protoplasmic masses are capable of movement which may result in change of their forms. Pseudopodia. A pseudopodium is a temporary projection of part of the cytoplasm of those protozoans which do not possess a definite pellicle. Pseudopodia are therefore a characteristic organella of 42 PROTOZOOLOGY Sarcodina, though many Mastigophora and certain Sporozoa, which lack a pellicle, are also able to produce them. According to their form and structure, four kinds of pseudopodia are distinguished. 1). Lobopodium is formed by an extension of the ectoplasm, accompanied by a flow of endoplasm as is commonly found in Amoeba proteus (Figs. 44; 161). It is finger- or tongue-like, sometimes branched, and its distal end is typically rounded. It is quickly formed and equally quickly retracted. In many cases, there are many pseudopodia formed from the entire body surface, in which the largest one will counteract the smaller ones and the organism will move in one direction; while in others, there may be a single pseudopodium formed, as in Amoeba striata, A. guttula, Vahlkampfia Umax, Pelomyxa caroUnensis, etc., in which case it is a broadly tongue-like extension of the body in one direction and the progressive movement of the organisms is comparatively rapid. The lobopodia may occasionally be conical in general shape, as in Amoeba spumosa. Although ordinarily the formation of lobopodia is by a general flow of the cytoplasm, in some it is sudden and "eruptive," as in End- amoeba blattae or Entamoeba histolytica in which the flow of the endo- plasm presses against the inner zone of the ectoplasm and the ac- cumulated pressure finally causes breaks through the zone, resulting in a sudden extension of the endoplasmic flow at that point. 2). Filopodium is a more or less filamentous projection com- posed almost exclusively of the ectoplasm. It may be sometimes branched, but the branches do not anastomose. Many testaceans, such as Lecythium, Boderia, Plagiophrys, Pamphagus, Euglypha, etc., form this type of pseudopodia. The pseudopodia of Amoeba radiosa may be considered as approaching this type rather than the lobopodia. 3). Rhizopodium is also filamentous, but branching and anastomosing. It is found in numerous Foraminifera, such as Elphidium, Peneroplis (Fig. 5), etc., and in certain testaceans, such as Lieberkiihnia, Myxotheca, etc. The abundantly branching and anastomosing rhizopodia often produce a large network which serve almost exclusively for capturing prey. 4). Axopodium is, unlike the other three types, a more or less semi-permanent structure and composed of axial rod and cytoplas- mic envelope. Axopodia are found in many Heliozoa, such as Actino- phrys, Actinosphaerium, Camptonema, Sphaerastrum, and Acan- thocystis. The axial rod, which is composed of fibrils (Doflein; Roskin), arises from the central body or the nucleus located in the approximate center of the body, from each of the nuclei in multi- MORPHOLOGY 43 nucleate forms, or from the zone between the ectoplasm and endo- plasm (Fig. 6). Although semipermanent in structure, the axial rod is easily absorbed and reformed. In the genera of Heliozoa, not . \ M 1 A ." 'K I'' ' '7' r- Fig. 5. Pseudopodia of Elphidiuni strigilata, X about 50 (Schulze from Kiihn). mentioned above and in numerous radiolarians, the radiating fila- mentous pseudopodia are so extremely delicate that it is difficult to determine whether an axial rod exists in each or not, although they resemble axopodia in general appearance. There is no sharp demarcation between the four types of pseudo- 44 PROTOZOOLOGY podia, as there are transitional pseudopodia between any two of them. For example, the pseudopodia formed by Arcella, Lesquer- eusia, Hyalosphaenia, etc., resemble more lobopodia than filopodia, though composed of the ectoplasm only. The pseudopodia of Actino- monas, Elaeorhanis, Clathrulina, etc., may be looked upon as transitional between rhizopodia and axopodia. Fig. 6. Portion of Adinosphaerium eichhorni, X800 (Kiihn), ar, axial rod; cv, contractile vacuole; ec, ectoplasm; en, endoplasm; n, nucleus. While the pseudopodia formed by an individual are usually of characteristic form and appearance, they may show an entirely different appearance under different circumstances. According to the often-quoted experiment of Verworn, a limax amoeba changed into a radiosa amoeba upon addition of potassium hydrate to the water (Fig. 7). Mast has recently shown that when Amoeba proteus or A. duhia was transferred from a salt medium into pure water, the amoeba produced radiating pseudopodia, and when transferred back to a salt medium, it changed into monopodial form, which change, he was inclined to attribute to the difference in the water contents of the amoeba. In some cases during and after certain in- ternal changes, an amoeba may show conspicuous differences in pseudopodia (Neresheimer). As was stated before, pseudopodia occur widely in forms which are placed under classes other than Sarcodina during a part of their life-cycle. Care, therefore, should be exer- MORPHOLOGY 45 cised in using them for taxonomic consideration of the Protozoa. Flagella. The flagellum is a filamentous extension of the cyto- plasm and is ordinarily extremely fine and highly vibratile, so that it is difficult to recognize it in life under the microscope with a moderate magnification. It is most clearly observed over a darkfield condenser. Lugol's solution (p. 721) stains it, though the organ- ism is killed. In a number of species, the flagellum, however, can be seen in life as a long filament, as for example in Peranema. As a rule, the number of flagella present in a single individual is small, varying from one to eight, but in Hypermastigina there are numerous fla- 5^®ID Fig forms dition of KOH solution to the water 7. Form-change in a limax-amoeba (Verworn). a, b, contracted c, individual showing typical form; d-f, radiosa-forms, after ad- gella. A flagellum appears to be composed of at least two parts (Fig. 8, a, b). An elastic axial filament takes its origin in the basal granule. Surrounding this filament there is a sheath of contractile cytoplasm which varies in thickness alternately on the opposite sides of the filament. The flagellum ordinarily tapers toward its distal end where the axial filament is said to be frequently exposed. Vlk (1938) found, besides the kind mentioned above which he called the whip-flagellum, another form named by him ciliary flagel- lum. The latter is said to be uniformly thick, but possesses dense ciliary projections which are arranged on it in one or two spiral rows 46 PROTOZOOLOGY b Fig. S. Diagrams of flagella. a, flagellum of Euglena (Biitschli) ; b, flagel- lum of Trachelomonas (Plenge); c, ciliary flagellum w'ith one row of cilia; d, a ciliary flagellum with two rows of cilia; e, whip-flagella of Polytoma uvella; f, ciliary flagellum of Urceolus cyclostomus; g, the flagella of Monas socialis (Vlk). (Fig. 8, c, d). The whip-flagellum occurs in Chlamydomonas, Poly- toma uvella (e), Cercomonas crassicaiida, Trepomonas rotans, T. agilis, Hexamita inflata, Urophagus rostratus, etc.; the ciliary MORPHOLOGY 47 flagellum, in Mallomonas, Chromulina, Trachelomonas, Urceolus (/), Phaciis, Euglena, Astasia, Distigma, etc.; and both kinds in Synura, Uroglena, Dinobryon, Monas (g), etc. (Vik). The flagellum is most frequently inserted near the anterior end of the body and directed forward, its movement pulling the organ- ism forward. Combined with this, there may be a trailing flagellum which is directed posteriorly and serves to steer the course of move- ment or to push the body forward to a certain extent. In a compara- tively small number of flagellates, the flagellum is inserted near the Flagellum Undulating membrane Nucleus Anterior flagellum Basal granule Blepharoplast Rhizoplast Nucleus Parabasal body Basal granule m Blepharoplast f^7^ Posterior flagellum Fig. 9. Diagrams of two flagellates, showing their structures (Kiihn). a, Trypanosoma brucei; b, Proteromonas lacertae. posterior end of the body and would push the body forward by its vibration. Lankester coined the terms tractella and pulsella for pulling and pushing flagella respectively. In certain parasitic Mastigophora, such as Trypanosoma (Fig. 9, a). Trichomonas, etc., there is a very delicate membrane extending out from the side of the body, a flagellum bordering its outer margin. When this membrane vibrates, it shows a characteristic undulating movement, as will easily be seen in Trypanosoma rotatorium of the frog, and is called the undulating membrane. In many of the dino- flagellates, the transverse flagellum seems to be similarly constructed (Kofoid and Swezy) (Fig. 107, d,f). Cilia. The cilia are the organella of locomotion found in the Cilio- phora. They aid in the ingestion of food and serve often as a tactile organella. The cilia are fine and more or less short processes of ecto- plasm and occur in large numbers in the majority of the Holotricha. 48 PROTOZOOLOGY They may be uniformly long, as in Protociliata, or may be of differ- ent lengths, being Ipnger at the extremities, on certain areas, in peristome or in circumoral areas. Ordinarily the cilia are arranged in longitudinal, oblique, or spiral rows, being inserted either on the ridges or in the furrows. Again the cilia may be confined to certain parts or zones of the body. Fig. 10. Diagrams of cilia (Klein), a, Coleps; b, Cyclidium glaucoma; c, Colpidi'uni colpoda. af, axial filament; bg, basal granule; cf, circular fibril; cs, cross-striation; sg, secondary granule. Each cilium originates in a basal granule situated in the deeper part of the ectoplasm and, in a few species, a cilium is found to be made up of an elastic axial filament arising from the basal granule, and the contractile sheath. Gelei observed in flagella and cilia, lipoid substance in granular or rod-like forms which differed even among different individuals of the same species; and Klein found in many cilia of Colpidium colpoda, an argentophilous substance in granular form much resembling the lipoid structure of Gelei and called them "cross-striation" of the contractile component (Fig. 10). MORPHOLOGY 49 Cirrus fiber Ectoplasmic granules Basal plate of the cirrus Basal granules of component cilia Undulating membrane Fig. 11. a, five anal cirri of Ewplotes eurystomus (Taylor); b, schematic ventral view of Stylonychia to show the distribution of the cirri. 50 PROTOZOOLOGY The cilia are often present more densely in a certain area than in other parts of body and, consequently, such an area stands out conspicuously, and is sometimes referred to as a ciliary field. If this area is in the form of a zone, it may be called a ciliary zone. Some authors use pectinellae for short longitudinal rows or transverse bands of close-set cilia. In a number of forms, such as Coleps, Sten- tor, etc., there occur, mingled among the vibratile cilia, immobile stiff cilia which are apparently solely tactile in function. cpg Fig. 12. Diagrams of cirrus and membranella of Euplotes eurystomus, X1450 (Taylor), a, an anal cirrus in side view; b, a membranella; bg, basal granule; cpg, coagulated protoplasmic granules; cr, ciliary root; fp, fiber plate. In the Hypotricha, the cilia are largely replaced by cirri, although in some species both may occur. A cirrus is composed of a number of cilia arranged in 2 to 3 rows that fused into one structure com- pletely (Figs. 11, 12), which was demonstrated by Taylor. Klein also showed by desiccation that each marginal cirrus of Stylonychia was composed of 7 to 8 cilia. In some instances, the distal portion of a cirrus may show two or more branches. The cirri are confined to the ventral surface in Hypotricha, and called frontal, ventral, anal, caudal, and marginal cirri, according to their location (Fig. 11). Un- like the cilia, the cirri may move in any direction so that the organ- isms bearing them, show various types of locomotion. Oxytricha, MORPHOLOGY 51 Stylonychia, etc., walk on f rentals, ventrals, and anals, while swim- ming movement by other species is of different types. In all euciliates except Holotricha, there are adoral membranellae. A membranella is composed of a double ciliary lamella, fused com- pletely into a plate (Fig. 12). A number of these membranellae occur on a margin of the peristome, forming the adoral zone of membranel- lae, which serves for bringing the food particles to the cytostome. The frontal portion of the zone, the so-called frontal membrane appears to serve for locomotion and Kahl considers that it is prob- ably made up of three lamellae. The oral membranes which are often found in Holotricha and Heterotricha, are transparent thin mem- branous structures composed of one or two rows of cilia, which are i^^ n jHOl' Fig. 13. Diagrams showing the possible development of a suctorian tentacle from a cytostome and cytopharynx of a ciliate (Collin). more or less strongly fused. The membranes, located in the lower end of the peristome, are sometimes called perioral membranes, and those in the cytopharynx, undulating membranes. In Suctoria, cilia are present only during the developmental stages, and, as the organisms become mature, tentacles develop in their stead. The tentacles are concerned with food-capturing, and are either prehensile or usually suctorial. In some instances the tentacles are tubular and this type is interpreted by Collin as possibly derived from a cytostome and cytopharynx of the ciliate (Fig. 13). Although the vast majority of Protozoa possess only one of the three organellae of locomotion mentioned above, a few may possess pseudopodia in one phase and flagella in another phase during their life-cycle. Among many examples, may be mentioned Dimastig- amoebidae (Fig. 160), Tetramitus rostratus (Fig. 134), etc. Further- more, there are some protozoans which possess two types of organ- ellae at the same time. Flagellum or flagella and pseudopodia occur 62 PROTOZOOLOGY in many Phytomastigina and Rhizomastigina, and a flagellum and cilia are present in Ileonema (Fig. 273, b, c). In the cytosome of Protozoa there occur various organellae, each of which will be considered briefly here. Fibrillar structures One of the fundamental characteristics of the protoplasm is its contractility. If a fully expanded Amoeba proteus is subjected to a mc i*^:*/ ^^- bg 1 1® - f 1 ; z -i;VVt'; \ = E \ f:! glS Fig. 14. Myonemes in Stentor coeruleus (Schroder), a, cross-section of ectoplasm; b, surface view of three myonemes; c, two isolated myonemes; bg, basal granules; cl, cilium; gis, granules between striae; m, myonemes; mc, myoneme canal. mechanical pressure, it retracts its pseudopodia and contracts into a more or less spherical form. In this response there is no special or- ganella, and the whole body reacts. But in certain other Protozoa, there are special organellae of contraction. Many Ciliophora are able to contract instantaneously when subjected to mechanical pressure, as will easily be noticed by following the movement of Stentor, Spirostomum, Trachelocerca, Vorticella, etc., under a dissecting microscope. The earliest observer of the contractile elements of MORPHOLOGY 53 Protozoa appears to be Lieberkiihn (1857) who noted the "muscle fibers" in the ectoplasm of Stentor which were later named myonemes (Haeckel) or neurophanes (Neresheimer). The myonemes of Stentor have been studied by several in- vestigators. According to Schroder (1906), there is a canal between each two longitudinal striae and in it occurs a long banded myoneme which measures in cross-section 3-7m high by about l/x wide and which appears cross-striated (Fig. 14). Roskin (1923) considers that, oe " 0^^< Fig. 15. a, b, fibrillar structures of the stalk of Zoothamnium (Kolt- zoff); c, m}''onemes in Gregarina (Schneider), ef, elastic fiber; ie, inner envelope; k, kinoplasm; oe, outer envelope; t, thecoplasm. the myoneme is a homogeneous cytoplasm (kinoplasm) and the wall of the canal is highly elastic and counteracts the contraction of the myonemes. All observers agree that the myoneme is a highly con- tractile organella. Many stalked peritrichous ciliates have well-developed myonemes not only in the body proper, but also in the stalk. Koltzoff 's studies show that the stalk is a pseudochitinous tube, enclosing an inner tube filled with granulated thecoplasm, which surrounds a central 54 PROTOZOOLOGY rod, composed of kinoplasm, on the surface of which are arranged skeletal fibrils (Fig. 15). The contraction of the stalk is brought about by the action of kinoplasm and walls, while elastic rods will lead to extension of the stalk. Myonemes present in the ciliates aid in the contraction of body, but those which occur in many Gre- garinida aid apparently in locomotion, being arranged longitudi- nally, transversely and probably spirally (Fig. 15,c). In certain Radio- laria, such as Acanthometron elasticum (Fig. 195, c), etc., each axial spine is connected with 10-30 myonemes (mj^ophrisks) originating in the body surface. When these myonemes contract, the body vol- ume is increased, thus in this case functioning as a hj^drostatic organella. In Isotricha prostoma and I. intestinalis, Schuberg (1888) observed that the nucleus is suspended by ectoplasmic fibrils and called the apparatus karyophore. In some forms these fibrils are replaced by ectoplasmic membranes as in Nydotherus ovalis (Zuluta; Kudo), ten Kate (1927) studied fibrillar systems in Opalina, Nyctotherus, Ichthyophthirius, Didinium, and Balantidium, and found that there are numerous fibrils, each of which originates in a basal granule of a cilium and takes a transverse or oblique course through the endoplasm, ending in a basal granule located on the other side of the body. He further noted that the cytopharynx and nucleus are also connected with these fibrils, ten Kate suggested morphonemes for them, since he believed that the majority were form-retaining fibrils. The well-coordinated movement of cilia in the ciliate has long been recognized, but it was Sharp (1914) who definitely showed that this ciliary coordination is made possible by a certain fibrillar system which he discovered in Epidinium (Diplodinium) ecaudatum (Fig. 16). Sharp recognized in this ciliate a complicated fibrillar system connecting all the motor organellae of the cytostomal region, and thinking that it was "probably nervous in function," as its size, ar- rangement and location did not suggest supporting or contractile function, he gave the name neuromotor apparatus to the whole system. This apparatus consists of a central motor mass, the motorium (which is stained red with Zenker fixation and modified Mallory's connective tissue staining), located in the ectoplasm just above the base of the left skeletal area, from which definite strands radiate: namely, one to the roots of the dorsal membranellae (a dorsal motor strand) ; one to the roots of the adoral membranellae (a ventral motor strand); one to the cytopharynx (a circum-oeso- phageal ring and oesophageal fibers); and several strands into the MORPHOLOGY 55 Fig. 16. A composite drawing from three median sagittal sections of Epidinium ecaudatum, fixed in Zenker and stained with Mallory's connec- tive tissue stain, X1200 (Sharp), am, adoral membranellae; c, cytostome; cp, cytopharynx; cpg, cytopyge; cpr, circumpharyngeal ring; dd, dorsal disk; dm, dorsal membrane; ec, ectoplasm; en, endoplasm; m, motorium; DC, oral cilia; od, oral disk; oef, oesophageal fibers; of, opercular fibers J p, pelhcle; prs, pharyngeal retractor strands; si, skeletal laminae; vs, ven- tral skeletal area. 56 PROTOZOOLOGY ectoplasm of the operculum (opercular fibers). A similar apparatus has since been observed in many other ciliates: Euplotes CYocom; Taylor), Balantiduum (McDonald), Paramecium (Rees; Brown; Lund), Tintinnopsis (Campbell), Boveria (Pickard), Dileptus (Visscher), Chlamydodon (MacDougall), Entorhipidium and Le- chriopyla (Lynch), Eupoterion (MacLennan and Connell), Metopus (Lucas), Troglodytella (Robertson), Oxytricha (Lund), Ancistruma and Conchophthirus (Kidder), etc. Fig. 17. Diagrams showing the neuromotor apparatus of Euplotes pa- tella (Taylor), a, diagrammatic dorsal view of the entire apparatus, X1600; b, dissected portion of disintegrating membranella fiber plates attached to the membranella fiber; c, a dissociated fiber plate of a frontal cirrus with its attached fibers, X1450. acf, anal cirrus fiber; afp, anal fiber plate; eg, small and large ectoplasmic granules; m, motorium; mf, mem- branella fiber; mfp, membranella fiber plate. Euplotes patella, a common free-living hypotrichous ciliate, has been known for nearly 50 years to possess definite fibrils connecting the anal cirri with the anterior part of the body. Engelmann sug- gested that their function was more or less nervelike, while others maintained that they were supporting or contracting in function. Yocom (1918) traced the fibrils to the motorium, a very small bilobed body (about 8/i by 2^) located close to the right anterior corner of the triangular cytostome (Fig. 17, a). Joining with its left end are five long fibers from the anal cirri which converge and appear to unite with the motorium as a single strand. From the right end of the motorium extends the membranella-fiber anteriorly and then to left MORPHOLOGY 57 along the proximal border of the oral lip and the bases of all mem- branellae. Yocom further noticed that within the lip there is a latticework structure whose bases very closely approximate the cyto- stomal fiber. Taylor (1920) recognized two additional groups of fibrils in the same organism: (1) membranella fiber plates, each of which is contiguous with a membranella basal plate, and is attached at one end to the membranella fiber; (2) dissociated fiber plates con- tiguous with the basal plates of the frontal, ventral and marginal cirri, to each of which are attached the dissociated fibers (c). By means of microdissection needles, Tajdor demonstrated that these fibers have nothing to do with the maintenance of the body form, since there results no deformity when Euplotes is cut fully two- thirds its width, thus cutting the fibers, and that when the motorium is destroyed or its attached fibers are cut, there is no coordination in the movements of the adoral membranellae and anal cirri. A striking feature common to all neuromotor systems, is that there seems to be a central motorium from which radiate fibers to different ciliary structures and that, at the bases of such motor or- ganellae, are found the basal granules or plates to which the "nerve" fibers from the motorium are attached. Independent of the studies on the neuromotor system of American investigators, Klein (1926) introduced the silver-impregnation method which had first been used by Golgi in 1873 to demonstrate various fibrillar structures of metazoan cells, to Protozoa in order to demonstrate the cortical fibers present in ciliates, by dry-fixation and impregnating with silver nitrate. Klein (1926) subjected the ciliates of numerous genera and species to this method, and observed that there was a fibrillar system in the ectoplasm at the level of the basal granules which could not be demonstrated by other methods. Klein (1927) named the fibers silver lines and the whole complex, the silverline system, which vary among different species (Fig. 18). Gelei, Chatton and Lwoff, Jirovec, Lynch, Jacobson, Kidder, Lund, and others, applied the silver-impregnation method to many other ciliates and confirmed Klein's observations. Chatton and Lwoff (1935) found in Apostomea, the system remains even after the embryonic cilia have entirely disappeared and considered it in- fraciliature. The question whether the neuromotor apparatus and the silver- line system are independent structures or different aspects of the same structure has been raised frequently. Turner (1933) found that in Euplotes patella (E. eurystomus) the silverline system is a regular latticework on the dorsal surface and a more irregular network on 58 PROTOZOOLOGY the ventral surface. These lines are associated with rows of rosettes from which bristles extend. These bristles are held to be sensory in function and the network, a sensory conductor system, which is connected with the neuromotor system. Turner maintains that the neuromotor apparatus in Euplotes patella is augmented by a distinct but connected external network of sensory fibrils. He however finds no motorium in this protozoan. Lund (1933) also made a comparative study of the two systems in Paramecium multimicronucleatum, and observed that the silverline system of this ciliate consists of two parts. One portion is made up Fig. 18. The silverline system of Ancistruma imjlili, XlOOO (Kidder), a, ventral view; b, dorsal view. of a series of closely-set polygons, usually hexagons, but flattened into rhomboids or other quadrilaterals in the regions of the cyto- stome, cytopyge, and suture. This system of lines stains if the or- ganisms are well dried. Usually the lines appear solid, but fre- quently they are interrupted to appear double at the vertices of the polygons which Klein called "indirectly connected" (pellicular) conductile system. In the middle of the anterior and posterior sides of the hexagons is found one granule or a cluster of 2-4 granules. MORPHOLOGY 59 which marks the outer end of the trichocyst. The second part which Klein called "directly connected" (subpellicular) conductile system consists essentially of the longitudinal lines connecting all basal granules in a longitudinal row of hexagons and of delicate transverse fibrils connecting granules of adjacent rows especially in the cyto- stomal region CFig. 19). Fig. 19. Diagram of the cortical region of Paramecium mtdtimicromi- cleatum, showing various organellas, X7300 (Lund), bg, basal granule; c, cilia; et, tip of trichocyst; If, longitudinal fibril; p, pellicle; t, trichocyst; tf, transverse fibril. By using Sharp's technique, Lund found the neuromotor system of Paramecium multimicronucleatum constructed as follows: The subpellicular portion of the system is the longitudinal fibrils which connect the basal granules. In the cytostomal region, the fibrils of right and left sides curve inward forming complete circuits (the circular cytosomal fibrils) (Fig. 20). The postoral suture is separated at the point where the cytopyge is situated. Usually 40-50 fibrils radiate outward from the cytostome (the radial cytostomal fibrils). The pharyngeal portion is more complex and consists of (1) the oesophageal network, (2) the motorium and associated fibrils, (3) penniculus which is composed of 8 rows of basal granules, thus form- ing a heavy band of cilia in the cytopharynx, (4) oesophageal process, (5) paraoesophageal fibrils, (6) posterior neuromotor chain, and (7) postoesophageal fibrils. Lund concludes that the so-called silverline 60 PROTOZOOLOGY Fig. 20. The neuromotor system of Paramecium multimicromicleatum (Lund), a, oral network; b, motorium, X1670. aep, anterior end of pen- niculus; c, cytopyge; ccf, circular cytostomal fibril; cof, circular oesopha- geal fibril; cpf, circular pharyngeal fibril; ef, endoplasmic fibrils; Ibf, longitudinal body fibril; lof, longitudinal oesophageal fibrils; Ipf, longi- tudinal pharyngeal fibril; m, motorium; oo, opening of oesophagus; op, oesophageal process; paf, paraoesophageal fibrils; pep, posterior end of penniculus; pnc, posterior neuromotor chain; pof, postoesophageal fibrils; rcf, radial cytostomal fibril; s, suture. MORPHOLOGY 61 system includes three structures: namely, the peculiarly ridged pellicle; trichocysts which have no fibrillar connections among them or with fibrils, hence not conductile; and the subpellicular sys- tem, the last of which is that part of the neuromotor system that concerns with the body cilia, ten Kate (1927) suggested that senso- motor apparatus is a better term than the neuromotor apparatus. Protective or supportive organellae The external structures as found among various Protozoa which serve for body protection, have already been considered (p. 39). Here certain internal structures will be discussed. The greater part of the shell of Foraminifera is to be looked upon as endoskeleton and thus supportive in function. In Radiolaria, there is a mem- branous structure, the central capsule, which divides the body into a central region and a peripheral zone. The intracapsular portion contains the nucleus or nuclei, and is the seat of reproductive proc- esses, and thus the capsule is to be considered as a protective or- ganella. The endoskeletal structures of Radiolaria vary in chemical composition and forms, and are arranged with a remarkable regular- ity (pp. 418-425). In some of the astomatous euciliates, there are certain structures which seem to serve for attaching the body to the host's organ, but which seem to be supportive to a certain extent also. The peculiar organella /wrcwZa, observed by Lynch in Lechriopyla (p. 597) is said to be concerned with either the neuromotor system or protection. The members of the family Ophryoscolecidae (p. 654), which are common commensals in the stomach of ruminants, have conspicuous endoskeletal plates which arise in the oral region and extend posteri- orly. Dogiel (1923) believed that the skeletal plates of Cycloposthium and Ophryoscolecidae are made up of hemicellulose, "ophryoscole- cin," which was also observed by Strelkow (1929). MacLennan found that the skeletal plates of Polyplastron multivesiculatum were composed of small, roughly prismatic blocks of paraglycogen, each possessing a central granule. In certain Polymastigina and Hypermastigina, there occurs a flexible structure known as the axostyle, which varies from a fila- mentous structure as in several Trichomonas, to a very conspicuous rod-like structure occurring in Parajoenia, Gigantomonas, etc. The anterior end of the axostyle is very close to the anterior tip of the body, and it extends lengthwise through the cytoplasm, ending near the posterior end or extending beyond the body surface. In other cases, the axostyle is replaced by a bundle of axostylar filaments 62 PROTOZOOLOGY which have connections with the fiagella as seen in certain Hyper- mastigina such as Lophomonas. In trichomonad flagellates there is often present along the line of attachment of the undulating membrane, a rod-like structure which has been known as costa (Kunstler) and which, according to Kirby's extensive study, appears to be most highly developed in Pseudo- trypanosoma and Trichomonas. The staining reaction indicates that its chemical composition is different from that of fiagella, blepharo- plast, parabasal body, or chromatin. Fig. 21. a, trichites in Spathidium spathula, X300 (Woodruff and Spencer); b, trichites in Pseudoprorodon fardus, X400 (Roux). In the gymnostomatous ciliates, the cytopharynx is often sur- rounded by rod-like bodies, and the entire apparatus is often called oral or pharyngeal basket, which is considered as supportive in function. The rod-like bodies appear in most cases to be trichites which may have been derived from the trichocysts, but which do not explode as do the latter. For example, in Chilodonella cucuUulus the oral basket is composed of 12 trichites which are so completely fused in part that the lower portion appears as a smooth tube and in Pseudoprorodon farctus (Fig. 21, h) much longer trichites form the basket, with reserve structures scattered throughout the cytoplasm (Engelmann). In Spathidium spathida (Fig. 21, a), trichites are imbedded like a paling in the thickened rim of the anterior end. They are also distributed throughout the endoplasm and, according to Woodruff and Spencer, ''some of these are apparently newly formed and being transported to the oral region, while others may well be MORPHOLOGY 63 thr •i ;: J) extr W%s^ t > trb trg trb rt Fig. 22. a, b, cortical region of Frontonia leucas, with embedded and extruded trichocysts (Tonniges); c, d, embedded and discharged tricho- cysts of Dileptiis anser, X4200 (Hayes); e, two extruded trichocysts of Paramecium caudatum, X1530 (original), ci, cilium; ec, ectoplasm; en, en- doplasm; extr, extruded trichocyst; p, pellicle; rt, root of trichocyst; th", thread of trichocyst; tr, trichocyst; trb, bulb of trichocyst; trg, trichocyst granule. 64 PROTOZOOLOGY trichites which have been torn away during the process of prey in- gestion." Whether the numerous 12-20/i long needle-like endoskele- tal structures which Kahl observed in Remanella (p. 584) are modi- fied trichites or not, is not known. In numerous ciliates, there is another ectoplasmic organella, the trichocyst, which is much shorter, though somewhat similar in general form. As seen in a Paramecium, the refractile fusiform trichocysts are embedded in the ectoplasm and arranged regularly at right angles to the body surface, while in forms, such as Cyclo- gramma they are situated obliquely (Fig. 278, c). In Frontonia leucas (Fig. 22), Tonniges found that the trichocysts originate in the chromatinic endosomes of the macronucleus and development takes place during their migration to the ectoplasm; on the other hand, Brodsky believes that the trichocysts are composed of col- loidal excretory substances and are first formed in the vicinity of the macronucleus, becoming fully formed during the course of their migration toward the periphery of the body. In species of Prorodon, Kriiger recently observed that the rod-like trichocysts of these ciliates are composed of a cylindrical sac containing a long filament which is arranged in a manner somewhat similar to the polar capsule of cnidosporidian spores. The end facing the body surface is fila- mentous and connected with the pellicle. The extrusion of the trichocysts is easily induced by means of mechanical pressure or chemical (acid or alkaline) stimulation, though the mechanism of extrusion is not well understood in all forms. Brodsky maintains that the fundamental force is not the mechanical pressure, but that under the influence of certain stimuli the expansion of the colloidal substances results in the extrusion of the trichocysts through the pellicle. The fully extruded tricho- cysts are needle-like in general form. The trichocysts of Frontonia leucas are about 6m long, but when extruded, measure 50-60m in length, and those of Paramecium caudatum may reach 40m in length. Dileptus anser feeds on various ciliates through the cytostome, located at the base of the proboscis, which possesses a band of long trichocysts on its ventral side. When food organisms come in contact with the ventral side of the proboscis, they give a violent jerk, and remain motionless. Visscher saw no formed elements discharged from the trichocysts, and, therefore, considered that these tricho- cysts contained a toxic fluid and named them toxicytes. Recently Hayes found that the exploded trichocysts (Fig. 22) could be dis- tinctly seen and suggested that these trichocysts themselves may be toxic. MORPHOLOGY 65 Although the trichocyst was first discovered by Elhs (1769) and so named by Allman (1855), nothing concrete is yet known as to their function. Ordinarily the trichocysts are considered as a de- fensive organella as in the case of the oft-quoted example Parame- cium, but, as Mast demonstrated, the extruded trichocysts of this ciliate do not have any effect upon Didinium other than forming a viscid mass about the former to hamper the latter. Penard con- siders that some trichocysts may be secretory organellae to produce material for loricae or envelope, with which view Kahl concurs, as granular to rod-shaped trichocysts occur in Metopus, Amphileptus, etc. Klein has called these ectoplasmic granules protrichocysts, and in Prorodon, Kriiger observed, besides typical tubular trichocysts, torpedo-like forms to which he applied the same name. To this group may belong the trichocysts recognized by Kidder in Con- chophthirus mijtili. The trichocysts present in certain Cryptomonad- ina (Chilomonas and Cyathomonas) are probably homologous with the protrichocysts. The pigments, which give a beautiful coloration to certain ciliates such as Stentor and Blepharisma, are said to be lodged in the protrichocysts. Hold-fast organellae In the Mastigophora, Ciliophora, and a few Sarcodina, there are forms which possess a stalk supporting the body or the lorica. With the stalk the organism is attached to a solid surface. In some cases, as in Anthophysis, Maryna, etc., the dendritic stalks are made up of gelatinous substances rich in iron, which gives to them a reddish brown color. In parasitic Protozoa, there are special or- ganellae developed for attachment. Many genera of cephaline gregarines are provided with an epimerite of different structures (Figs, 208-210), by which the organisms are able to attach them- selves to the gut epithelium of the host. In Astomata, such as Into- shellina, Maupasella, Lachmannella, etc., simple or complex pro- trusible chitinous structures are often present in the anterior region; or a certain area of the body may be concave and serves for ad- hesion to the host, as in Rhizocaryum, Perezella, etc.; or, again, there may be a distinctive sucker-like organella near the anterior extremity of the body, as in Haptophyra, Steinella, etc. A sucker is also present on the antero-ventral part of Giardia intestinalis. In the Myxosporidia and Actinomyxidia, there appear, during the development of spore, 1-4 special cells which develop into polar capsules, each, when fully formed, enclosing a more or less long spirally coiled delicate thread, the polar filament (Fig. 259). 66 PROTOZOOLOGY The polar filament is considered as a temporary anchoring organella of the spore at the time of its germination after it gained entrance into the alimentary canal of a suitable host. In the Microsporidia, the filament may or may not be enclosed within a capsule. The ne- matocysts (Fig. 110, h) of certain dinoflagellates belonging to Nema- toidium and Polykrikos, are almost identical in structure with those Fig. 23. Parabasal apparatus in: a, Lojihomonas blattarum (Kudo); b, Metadevescovina debilis; c, Devescovina sp. (KirbjO- af, axostylar fila- ments; bl, blepharoplasts; f, food particles; fl, flagella;n, nucleus; pa, para- basal apparatus. found in the coelenterates. They are distributed through the cyto- plasm, and various developmental stages were noticed by Chatton, and Kofoid and Swezy, which indicates that they are characteristic structures of these dinoflagellates and not foreign in origin as had been held by some. The function of the nematocysts in these proto- zoans is not understood. The parabasal apparatus In the cytosome of many parasitic flagellates, there is frequently present a conspicuous structure known as the parabasal apparatus (Janicki), consisting of the parabasal body and often thread (Cleve- land), which latter may be absent in some cases. This structure MORPHOLOGY 67 varies greatly among different genera and species in appearance, structure and position within the body. It is usually connected with the blepharoplast and located very close to the nucleus, though not directly connected with it. It may be single, double, or multiple, and may be pyriform, straight or curved rod-like, bandform, spirally coiled or collar-like (Fig. 23). Kofoid and Swezy considered that the parabasal body is derived from the nuclear chromatin, varies in size according to the metabolic demands of the organism, and is a "kinetic reservoir." On the other hand, Duboscq and Grasse main- tain that this body is the Golgi apparatus, since (1) acetic acid destroys both the parabasal body and the Golgi apparatus; (2) both are demonstrable with the same technique; (3) the parabasal body is made up of chromophile and chromophobe parts as is the Golgi apparatus; and (4) there is a strong evidence that the parabasal body is secretory in function. According to Kirby, who has made an extensive study of this organella, the parabasal body could be stained with Delafield's haematoxylin or Mallory's triple stain after fixation with acetic acid-containing fixatives and the body does not show any evidence to indicate that it is a secretory organella. More- over the parabasal body is discarded or absorbed at the time of divi- sion of the body and two new ones are formed. The parabasal body of Lophomonas hlattarum to which the name was originally applied, is discarded when the organism divides and two new ones are reformed from the centriole or blepharoplast (Fig. 62), and its function appears to be supportive. Possibly not all so- called parabasal bodies are homologous or analogous. A fuller com- prehension of the structure and function of the organella rests on further investigations. The blepharoplast In the Mastigophora or in other groups in which flagellate stages occur, the flagellum ends internally in a basal granule, which, in turn, is sometimes connected by a much larger bod}^ This latter organella has been called the blepharoplast. In many instances they appear to be combined in one. The blepharoplast is further connected by a fibril, the rhizoplast, with the nucleus (Fig. 24). The blepharoplast and centriole are considered synonymous by Minchin, Cleveland, and others, since they give rise to the kinetic organella. Woodcock and Minchin held, on the other hand, that the blepharoplast was a nucleus holding a special relation with loco- motor organellae, and called it kinetonucleus. In recent years it has become known that the blepharoplast of many flagellates re- 68 PROTOZOOLOGY spends positively to Feulgen's nucleal reaction which may indicate the presence of thymonucleic acid or chromatin in this structure. The Golgi apparatus With the discovery of a wide distribution of the so-called Golgi apparatus in metazoan cells, a number of protozoologists also re- tm Fig. 24. Flagellar attachment in Euglenoidina (Hall and Jahn"). a, Euglena deses, X2025; b, E. actis, X750; c, E. spirogyra, X720; d, Menoiclium incurvum, X1550. ported a homologous structure from many protozoans. It seems im- possible at present to indicate just exactly what the Golgi appara- tus is, since the so-called Golgi techniques, the important ones of which are based upon the assumption that the Golgi material is MORPHOLOGY 69 osmiophile and argentophile, and possesses a strong affinity to neutral red, are not specific and the results obtained by using the same method often vary a great deal. Some of the examples of the Golgi apparatus reported from Protozoa are summarized in Table 2, It appears thus that the Golgi bodies occurring in Protozoa are small osmiophilic granules or larger spherules which are composed of osmiophile cortical and osmiophobe central substances. Fre- 93.0 o Fig. 25. The Golgi bodies in Amoeba yroteus (Brown). quently the cortical layer is of unequal thickness, and, therefore, crescentic forms appear. Ringform apparatus was noted in Chilo- donella and Dogielella by Nassonov and network-like forms were ob- served by Brown in Pyrsonympha and Dinenympha. The Golgi ap- paratus of Protozoa as well as of Metazoa, appears to be composed of a lipoidal material in combination with protein substance. In line with the suggestion made for the metazoan cell, the Golgi apparatus of Protozoa is considered as having something to do with secretion or excretion. Nassonov considers that osmiophilic lipoidal substance, which he observed in the vicinity of the walls of the contractile vacuole and its collecting canals in many ciliates and flagellates, is homologous with the metazoan Golgi apparatus and secretes the fluid waste material into the vacuole from which it is excreted to the exterior. According to Brown, there is no blackening by osmic impregnation of the contractile vacuole in Amoeba proteus, 70 PROTOZOOLOGY but fusion of minute vacuoles associated with crescentic Golgi bodies produces the vacuole. Duboscq and Grasse who hold that the parabasal body is the Golgi apparatus, maintain that this body is a source of energy which is utilized by the motor organellae. Joyet-Lavergne pointed out that Table 2. — Golgi apparatus in Protozoa Protozoa Golgi apparatus Observers Monocystis, Gregarina Spheres, rings, crescents Hirschler Endamoeha blattae Spheres, rings, crescents Hirschler Adelea Crescents, beaded grains King and Gatenby Entamoeba gingivalis Rings, crescents to network Causey Vorticella, Lionotus, The membrane of contrac- Nassonov Paramecium, Dogiel- tile vacuole and collecting ella, Nassula, Chilo- canals monas, Chilodonella Holomastigotes, Pyr- Parabasal bodies Dubocsq and sonympha, etc. Grass6 Aggregata, gregarines Crescents, rings Joyet-Lavergne Euglenoidina Stigma Grass6 Chilomonas Granules, vacuoles Hall Peranema Rings, globules, granules Hall Chromulina, Astasia Rings, spherules with a dark Hall Amoeba proteus (Fig. rim Rings, crescents, globules, Brown 25) granules Pyrsonympha, Di- Rings, crescents, spherules; Brown nenympha granules break down to form network near pos- terior end Euglena gracilis Spherical, discoidal with dark rim; tend to group around or near nucleus Brown Blepharisma undulans Rings in the cytoplasm Moore in certain sporozoans the Golgi apparatus is granular and may be the center of enzyme production. The exact morphological and physiological information of the Golgi apparatus must be looked for in future observations. The chondriosomes Widely distributed in many metazoan cells, the chondriosomes have also been recognized in various Protozoa. The chondriosomes possess a low refractive index, and are composed of substances easily MORPHOLOGY 71 soluble in alcohol, acetic acid, etc. Osmium tetroxide blackens the chondriosomes, but the color bleaches faster than in the Golgi bodies. Janus green B stains them even in 1 : 500,000 solution, but stains also other inclusions, such as the Golgi bodies (in some cases) and certain bacteria. According to Horning (1926), janus red is said to be a more exclusive chondriosome stain, as it does not stain bacteria. The chemical composition of the chondriosome seems to be somewhat similar to that of the Golgi body; namely, it is a protein compounded with a lipoidal substance. If the protein is small in amount, it is Fig. 26. The chrondiosomes in Peranema trichophorum, X1750 (Hall) a, b, surface views and c, optical section of a single individual. said to be unstable and easily attacked by reagents; on the other hand, if the protein is relatively abundant, it is more stable and resistant to reagents. The chondriosomes occur as small spherical to oval granules, rod- like or filamentous bodies, and show a tendency to adhere to or re- main near protoplasmic surfaces. In many cases they are distributed without any definite order; in others, as in Paramecium or Opalina, they are regularly arranged between the basal granules of cilia (Horning). In Peranema trichophorum (Fig. 26), according to Hall, the chondriosomes are said to be located along the spiral striae of the pellicle. Causey (1925) noticed in Leishmania hrasiliensis usu- ally eight spherical chondriosomes in each individual, which become rod-shaped when the organism divides. He further observed spher- ical and rod-like chondriosomes in Notiluca scintillans. 72 PROTOZOOLOGY In certain Protozoa, the chondriosomes are not always demon- strable. For example, Horning states in Monocystis the chondrio- somes present throughout the asexual life-cycle as rod-shaped bodies, but at the beginning of the spore formation they decrease in size and number, and in the spore none exists. The chondriosomes appear as soon as the sporozoites are set free. Thus it would appear that the chondriosomes are reformed de novo. On the other hand, Faure- Fremiet, the first student of the chondriosomes in Protozoa, main- tained that they reproduce by division, which has since been con- firmed by many observers. As a matter of fact. Horning found in Opalina, the chondriosomes are twisted filamentous structures and undergo multiple longitudinal fission in asexual division phase. Be- fore encystment, the chondriosomes divide repeatedly transversely and become spherical bodies which persist during encystment and in the gametes. In zygotes, these spherical bodies fuse to produce longer forms which break up into elongate filamentous structures. Richardson and Horning further succeeded in bringing about divi- sion of the chondriosomes in Opalina by changing pH of the medium. As to the function of chondriosomes, opinions vary. A number of observers hold that they are concerned with the digestive process. After studying the relationship between the chondriosomes and food vacuoles of Amoeba and Paramecium, Horning suggested that the chondriosomes are the seat of enzyme activity and it is even probable that they actually give up their own substance for this purpose. Mast and Doyle hold that the "excretory granules" (chon- driosomes) in Amoeba proteus contribute to the formation of the contractile vacuole. The view that the chondriosomes may have something to do with the cell-respiration expressed by Kingsbury was further elaborated by Joyet-Lavergne through his studies on certain Sporozoa. That the chondriosomes are actively concerned with the development of the gametes of the Metazoa is well known. Zweibaum's observation, showing an increase in the amount of fatty acid in Paramecium just prior to conjugation, appears to suggest this function. On the othr hand. Calkins found that in Uroleptus, the chondriosomes became abundant in exconjugants, due to trans- formation of the macronuclear material into the chondriosomes. It may be stated that the chondriosomes appear to be associated with the formation of enzymes which participate actively in the processes of catalysis or synthesis in the protozoan body. The author agrees with McBride and Hewer who wrote: "it is a remarkable thing that so little is known positively about one of the 'best known' proto- plasmic inclusions." MORPHOLOGY 73 The contractile and other vacuoles The majority of Protozoa possess one or more vacuoles known as pulsating or contractile vacuoles. They occur regularly in all freshwater inhabiting Sarcodina and Mastigophora, and in Cilio- phora regardless of habitat. In the Sporozoa, which are all parasitic, and the Sarcodina and Mastigophora, which live either in salt water or in the body of other animals, there is no contractile vacuole. In various species of free-living amoebae, the contractile vacuole is formed by accumulation of wate-r in one or more droplets which finally fuse into one. It enlarges itself continuously until it reaches a maximum size (diastole) and suddenly bursts through the thin cytoplasmic layer above it (systole), discharging its content to out- %' % '•^'.*.. V Fig. 27. Diagrams showing the contractile vacuole, the accessory vacu- oles and the aperture, during diastole and systole in Conchophthirus (Kidder). side. The location of the vacuole is not definite in such forms and, therefore, it moves about with the cytoplasmic movements; and, as a rule, it is confined to the temporary posterior region of the body. Although almost spherical in form, it may occasionally be irregular in shape, as in Amoeba striata (Fig. 161, /). In many testaceans and heliozoans, the contractile vacuoles which are variable in number, are formed in the ectoplasm and the body surface bulges out above the vacuoles at diastole. In the Mastigophora, the contractile vacuole appears to be more or less constant in position. In Phytomastigina, they are usually located in the anterior region and, in Zoomastigina, as a rule, in the posterior half of the body. The number of the vacuoles present in an individual varies from one to several. In the Ciliophora, except Protociliata, there occur one to many contractile vacuoles, which seem to be located in the deepest part of the ectoplasm and therefore constant in position. Directly above each vacuole is found a pore in the pellicle, through which the con- tent of the vacuole is discharged to outside. In the species of Con- 74 PROTOZOOLOGY chophthirus, Kidder (1934) observed a narrow slit in the pellicle just posterior to the vacuole on the dorsal surface (Fig. 27). The margin of the slit is thickened and highly refractile. During diastole, the slit is nearly closed and, at systole, the wall of the contractile vacuole appears to break and the slit opens suddenly, the vacuolar content pouring out slowly. When there is only one contractile vacuole, it is usually located either near the cytopharynx or, more often, in the posterior part of the body. When several to many vacuoles are present, they may be distributed without apparent order, in linear series, or along the body outline. When the contrac- tile vacuoles are deeply seated, there is a delicate duct which con- nects the vacuole with the pore on the pellicle as in Paramecium Fig. 28. Diagrams showing the successive stages in the formation of the contractile vacuole in Paramecium muliimicromicleatum (King) ; up- per figures are side views; lower figures front views; solid lines indicate permanent structures; dotted lines temporary structures, a, full diastole; b-d, stages of systole; e, content of ampulla passing into injection canal; f, formation of vesicles from injection canals; g, fusion of vesicles to form contractile vacuole; h, full diastole. woodruffi, or in Ophryoscolecidae. In Balantidium, Nyctotherus, etc., the contractile vacuole is formed very close to the permanent cyto- pyge located at the posterior extremity, through which it empties its content. In a number of ciliates there occur radiating or collecting canals besides the main contractile vacuole. These canals radiate from the central vacuole in Paramecium, Frontonia, Disematostoma, etc. But when the vacuole is terminal, the collecting canals of course do not MORPHOLOGY 75 radiate, in which case the number of the canals varies among different species: one in Spirostomum, Stentor, etc., 2 in Clima- costomum, Eschaneustyla, etc., and several in Tillina. In Peritricha, Fig. 29. Contractile vacuoles of Paramecium muUimicronucleatum, X1200 (King), a, early systole, side view; b, diastole, front view; c, com- plete systole, front view; d, systole, side view. 76 PROTOZOOLOGY the contractile vacuole occurs near the posterior region of the peri- stome and its content is discharged through a canal into the vesti- bule, and in Ophrydium ectaium, the contractile vacuole empties its content into the cytopharynx through a long duct (Mast). Of numerous observations concerning the operation of the con- tractile vacuole, that of King (1935) on Paramecium multimicro- nucleatum (Figs. 28, 29) may be quoted here. In this ciliate, there are 2 to 7 contractile vacuoles which are located below the ecto- plasm on the aboral side. There is a permanent pore above each vacuole. Leading to the pore is a short tube-like invagination of the pellicle, with inner end of which the temporary membrane of the vacuole is in contact (Fig. 28, a). Each vacuole has 5-10 long col- lecting canals with strongly osmiophilic walls (Fig. 29), and each canal is made up of terminal portion, a proximal injection canal, and an ampulla between them. Surrounding the distal portion, there is osmiophilic cytoplasm which may be granulated or finely reticu- lated, and which Nassonov interpreted as homologous to the Golgi apparatus of the metazoan cell. The injection canal extends up to the pore. The ampulla becomes distended first with fluid transported discontinuously down the canal and the fluid next moves into the injection canal. The fluid now is expelled into the cytoplasm just beneath the pore as a vesicle, the membrane of which is derived from a membrane which closed the end of the injection canal. These fluid vesicles coalesce presently to form the contractile vacuole in full diastole and the fluid is discharged to exterior through the pore, which becomes closed by the remains of the membrane of the dis- charged vacuole. In Haptophrya michiganensis, MacLennan (1944) observed that accessory vacuoles appear in the wall of the contractile canal which extends along the dorsal side from the sucker to the posterior end, as the canal contracts. The canal wall expands and enlarging acces- sory vacuoles fuse with one another, followed by a full expansion of the canal. Through several excretory f)ores with short ducts the con- tent of the contractile canal is excreted to the exterior. The function of the contractile vacuole is considered in the following chapter (p. 103). Various other vacuoles or vesicles occur in different Protozoa. In the ciliates belonging to Loxodidae, there are variable numbers of MuUer's vesicles or bodies, arranged in 1-2 rows along the aboral sur- face. These vesicles (Fig. 30, a-c) vary in diameter from 5 to 8.5/i and contain a clear fluid in which one large spherule or several small highly refractile spherules are suspended. In some, there is a fila- MORPHOLOGY 77 mentous connection between the spherules and the wall of the vesicle. Penard maintains that these bodies are balancing cell-organs and called the vesicle, the statocyst, and the spherules, the stato- liths. Another vacuole, known as concrement vacuole, is a character- istic organella in Biitschliidae and Paraisotrichidae. As a rule, there is a single vacuole present in an individual in the anterior third of body. It is spherical to oval and its structure appears to be highly complex. According to Dogiel, the vacuole is composed of a pellicu- lar cap, a permanent vacuolar wall, concrement grains and two Fig. 30.a-c, Miiller's vesicles in Loxodes (a, b) and in Remanella (c) (a, Penard; b, c, Kahl); d, concrement vacuole of Blepharoprosthium (Dogiel). cf, centripetal fibril; eg, concrement grains; cp, cap; fw, fibrils of wall; p, pellicle; vp, vacuolar pore; w, wall. fibrillar systems (Fig. 30, d). When the organism divides, the an- terior daughter individual retains it, and the posterior individual de- velopes a new one from the pellicle into which concrement grains enter after first appearing in the endoplasm. This vacuole shows no external pore. Dogiel believes that its function is sensory and has named the vacuole, the statocyst, and the enclosed grains, the statoliths. Food vacuoles are conspicuously present in the holozoic Protozoa which take in whole or parts of other organisms as food. The food 78 PROTOZOOLOGY vacuole is a space in the cytoplasm, containing the fluid medium which surrounds the protozoans and in which are suspended the food matter, such as various Protophyta, other Protozoa or small Metazoa. In the Sarcodina, the Mastigophora and the Suctoria, which do not possess a cytostome, the food vacuoles assume the shape of the food materials and, when these particles are large, it is difficult to make out the thin film of water which surrounds them. When minute food particles are taken through a cytostome, as is the case with the majority of euciliates, the food vacuoles are usually spherical and of approximately the same size within a single proto- zoan. In the saprozoic Protozoa, which absorb fluid substances through the body surface, food vacuoles containing solid food, of course, do not occur. The chromatophore and associated organellae In the Phytomastigina and certain other forms which are green- colored, one to many chromatophore s (Fig. 31) or chloroplasts con- taining chlorophyll occur in the cytosome. The chromatophores vary in form among different species; namely, discoidal, ovoid, band- form, rod-hke, cup-like, fusiform, network or irregularly diffused. The color of the chromatophore depends upon the amount and kinds of pigment which envelops the underlying chlorophyll substance. Thus the chromatophores of Chrysomonadina are brown or orange, as they contain one or more accessory pigments, including phyco- chrysin, and those of Cryptomonadina are of various types of brown with very diverse pigmentation. In Chloromonadina, the chromato- phores are bright green, containing an excess of xanthophyfl. In dinoflagellates, they are dark yellow or brown, because of the pres- ence of pigments: carotin, phylloxanthin, and peridinin (Kylin), the last of which is said to give the brown coloration. A few species of Gymnodinium contain blue-green chromatophores for which phyco- cj^anin is held to be responsible. The chromatophores of Phytomon- adina and Euglenoidina are free from any pigmentation, and there- fore green. Aside from various pigments associated with the chro- matophores, there are carotinoid pigments which occur often outside the chromatophores, and are collectively known as haematochrome. The haematochrome occurs in Haematococcus pluvialis, Euglena sanguinea, E. rubra, Chlamydomonas, etc. In Haematococcus, it in- creases in volume and in intensity when there is a deficiency in phos- phorus and especially in nitrogen; and when nitrogen and phos- phorus are present sufficiently in the culture medium, the haemato- chrome loses its color completely (Reichenow; Pringsheim). Steinecke MORPHOLOGY 79 also noticed that the frequent yellow coloration of phytomonads in moorland pools is due to a development of carotin in the chro- matophores as a result of deficiency in nitrogen. Johnson (1939) noted that the haematochrome granules of Euglena rubra become collected in the central portion instead of being scattered through- out the body when sunlight becomes weaker. Thus this Euglena appears green in a weak light and red in a strong light. Flagella Stigma Pyrenoids Chromotophores — Nucleus — Shell Chromatophores Pyrenoids Fig. 31. a, Trachelomonas hispida, X530 (Doflein); b, c, living and stained reproductive cells of Pleodorina illinoisensis, XlOOO (Merton); d-f, terminal cells of Hydrurus foetidus, showing division of chromato- phore and pyrenoid (Geitler); g-i, Chlavujdomonas sp., showing the di- vision of pyrenoid (Geitler). In association with the chromatophores are found the pyrenoids (Fig. 31) which are usually embedded in them. The pyrenoid is a viscous structureless mass of protein (Czurda), and may or may not be covered by tightly fitting starch-envelope, composed of several pieces or grains which appear to grow by apposition of new material on the external surface. A pyrenoid divides when it reaches a certain size, and also at the time of the division of the organism in which it occurs. As to its function, it is generally agreed that the pyrenoid is concerned with the formation of the starch and allied anabolic prod- ucts of photosynthesis. 80 PROTOZOOLOGY Chromatophore-bearing Protozoa usually possess also a stigma (Fig. 31) or eye-spot. The stigma may occur in exceptional cases in colorless forms, as in Khawkinea, Polytomella, etc. It is ordi- narily situated in the anterior region and appears as a reddish or brownish red dot or short rod, embedded in the cortical layer of the cytoplasm. The color of the stigma is due to the presence of droplets of haematochrome in a cytoplasmic network. The stigma is incapable of division and a new one is formed de novo at the time of cell divi- sion. In many species, the stigma possesses no accessory parts, but, according to Mast, the pigment mass in Chlamydomonas, Pando- rina, Eudorina, Euglena, Trachelomonas, etc., is in cup-form, the concavity being deeper in the colonial than in solitary forms. There is a colorless mass in the concavity, which appears to function as a lens. In certain dinoflagellates, there is an ocellus (Fig. 107, c, d, g, h) which is composed of amyloid lens and a dark pigment mass (melan- osome) that is sometimes capable of amoeboid change of form. The stigma is, in general, regarded as an organella for the perception of light intensity. Mast (1926) considers that the stigma in the Volvo- cidae is an organella which determines the direction of the move- ment. References Belar, K. 1926 Der Formwechsel der Protistenkerne. Ergebn. u. Fortschr. Zool., Vol. 6. Brodsky, a. 1924 Die Trichocysten der Infusorien. Arch. rus. protist., Vol. 3. Brown, V. E. 1930 The Golgi apparatus of Amoeba proteus. Biol. Bull., Vol. 59. 1930 The Golgi apparatus of Pyrsonympha and Dine- nympha. Arch. f. Protistenk., Vol. 71. Calkins, G. N. and F. M. Summers (editors). 1941 Protozoa in biological research. New York. Causey, D. 1925-1926 Mitochondria and Golgi bodies in Enda- moeba gingivalis. Mitochondria in Leishniania brasiliensis. Mito- chondria in Noctiluca scintillans. Univ. Calif. Publ. Zool., Vol. 28. Chatton, E. and A. Lwoff 1935 Les cili^s apostomes. Arch. zool. exp. et gen., Vol. 77. 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., Vol. 17. CusHMAN, J. A. 1933 Foraminifera: their classification and economic use. Second edition. Sharon, Mass. DoFLEiN, F. 1916 Studien zur Naturgeschichte der Protozoen. VII. Zool. Jahrb. Abt. Anat., Vol. 39. MORPHOLOGY 81 DoGiEL, V. 1923 Cellulose als Bestandteil des Skellettes bei einigen Infusorien. Biol. Zentralbl., Vol. 43. 1929 Die sog. "Konkrementenvakuole" der Infusorien als eine Statocyste betrachtet. Arch. f. Protistenk., Vol. 68. DuBOSCQ, O. and P. P. Grasse 1933 L'appareil parabasal des flagelles. Arch. zool. exp. et gen., Vol. 63. Gelei, J. VON 1926 Zur Kenntnis des Wimperapparates. Zeitschr. f. ges. Anat., Abt. I, Vol. 81. GiESE, A. C. 1938 Reversible bleaching of Blepharisma. Trans. Amer. Micr. Soc, Vol. 57. Hall, R. P. 1929 Reaction of certain cytoplasmic inclusions to vital dyes and their relation to mitochondria and Golgi appara- tus in the flagellate Peranema trichophorum. Jour. Morph. Physiol., Vol. 48. and T. L. Jahn 1929 On the comparative cytology of cer- tain euglenoid flagellates and the systematic position of the families Euglenidae and Astasiidae. Trans. Amer. Micro. Soc, Vol. 48. Hayes, M. L. 1938 Cytological studies on Dilepius anscr. Ibid., Vol. 57. Hertwig, R. 1902 Die Protozoen und die Zelltheorie. Arch. f. Protistenk., Vol. 1. Horning, E. S. 1926 Observations on mitochondria. Austral. Jour. Exp. Biol. Med. Sci., Vol. 3. 1927 On the orientation of mitochondria on the surface cytoplasm of infusorians. Ibid., Vol. 4. 1929 Mitochondrial behavior during the life cycle of a sporozoan (Monocystis). Quart. Jour. Micr. Sci., Vol. 73. Janicki, C. v. 1911 Zur Kenntnis des Parabasalapparates bei parasitischen Flagellaten. Biol. Zentralbl., Vol. 31. Kidder, G. W. 1933 On the genus Ancistruma Strand (Ancistrum Maupas). Biol. Bull, Vol. 64. 1933 ConcJwphthirus caryoclada sp. nov. Ibid., Vol. 65. 1934 Studies on the ciliates from freshwater mussels. Ibid., Vol. 66. King, R. L. 1935 The contractile vacuole of Paramecium multi- micronucleatum. Jour. Morph., Vol. 58. KiRBY, Jr., H. 1931 The parabasal body in trichomonad flagel- lates. Trans. Amer. Micr. Soc, Vol. 50. Klein, B. M. 1926 Ergebnisse mit einer Silbermethode bei Cilia- ten. Arch. f. Protistenk., Vol. 56. 1927 Die Silverliniensystem der Ciliaten. Ibid., Vol. 58. 1929 Weitere Beitrage zur Kenntnis des Silberliniensys- tems der Ciliaten. Ibid., Vol. 65. 1930 Das Silberliniensystem der Ciliaten. Ibid., Vol. 69. KoFOiD, C. A. and Olive Swezy 1921 The free-living unarmored Dinoflagellata. Mem. Univ. California. Vol. 5. Koltzoff, N. K. 1911 Untersuchung iiber die Kontraktilitat des Stieles von Zoothamnium alternans. Biol. Zeitschr. Moskau., Vol. 2. 82 PROTOZOOLOGY Kruger, F. 1934 Untersuchungen iiber die Trichocysten einiger Prorodon-Arten. Arch. f. Protistenk., Vol. 83. Kudo, R. R. 1924 A biologic and taxonomic study of the Micro- sporidia. Illinois Biol. Monogr., Vol. 9. ■ — ■ 1926 Observations on Lophomonas blattarum, a flagellate inhabiting the colon of the cockroach, Blatta orientalis. Arch. f. Protistenk., Vol. 53. 1936 Studies on Nyctotherus ovalis Leidy, with special refer- ence to its nuclear structure. Ibid., Vol. 87. Lund, E. E. 1933 A correlation of the silverline and neuromotor systems of Paramecium. Univ. Calif. Publ. Zool., Vol. 39. Lynch, J. E. 1930 Studies on the ciliates from the intestine of Strongylocentrotus. II Lechriopyla mystax, gen. nov., sp. nov. Ibid. Vol. 33. MacLennan, R. F. 1944 The pulsatory cycle of the contractile canal in the ciliate Haptophrya. Trans. Amer. Micro. Soc, Vol. 63. Mast, S. O. 1928 Structure and function of the eye-spot in unicel- lular and colonial organisms. Arch. f. Protistenk., Vol. 60. 1944 A new peritrich belonging to the genus Ophrydium. Trans. Amer. Micro. Soc, VoK 63. Nassonov, D. 1924 Der Exkretionsapparat (kontraktile Vakuole) der Protozoen als Homologen des Golgischen Apparatus der Metazoenzelle. Arch. mikr. Anat., Vol. 103. Penard, E. 1922 Etudes sw les infusoires d'eau douce. Geneva, Piney, a. 1931 Recent advances in microscopy. London. Pringsheim, E. 1914 Die Ernahrung von Haematococcus pluvialis. Beitr. Biol. Pfianzen, Vol. 12. Reichenow, E. 1909 Untersuchungen an Haematococcus pluvialis nebst Bemerkungen iiber andere Flagellaten. Arch, kaiserl. Ge- sundheitsamt.. Vol. 33. 1928 Ergebnisse mit der Nuklealfarbung bei Protozoen. Arch. f. Protistenk., Vol. 61. ' Richardson, K. C. and E. S. Horning 1931 Cytoplasmic struc- tures in binucleate opalinids with special reference to the Golgi apparatus. Jour. Morph. Physiol., Vol. 52. RosKiN, G. 1923 La structure des Myonemes des infusoires. Bull. biol. France et Belg., Vol. 57. 1925 Ueber de Axopodien der Heliozoa und die Greif- tentakel der Ephelotidae. Arch. f. Protistenk., Vol. 52. RuMJANTZEW, A. and E. Wermel 1925 Untersuchungen ueber den Protoplasmabau von Actinosphaerium eichhorni. Ibid., Vol. 52. Schroder, O. 1906 Beitrage zur Kenntnis von Stentor coeruleus und St. roeselii. Ibid., Vol. 8. Schuberg, a. 1888 Die Protozoen des Wiederkauermagens. I. Zool. Jahrb. Abt. System., Vol. 3. Sharp, R. 1914 Diplodiniiim ecaudatum with an account of its neuromotor apparatus. Univ. Calif. Publ. Zool., Vol. 13. Strelkow, a. 1929 Morphologische Studien iiber oligotriche In- fusorien aus dem Darme des Pferdes. I. Arch. f. Protistenk., Vol. 68. MORPHOLOGY 83 Taylor, C. V. 1920 Demonstration of the function of the neuro- motor apparatus in Euplotes by the method of micro-dissection. Univ. Calif- Publ. ZooL, Vol. 19. TEN Kate, C. G. B. 1927 Ueber das Fibrillensystem der Ciliaten. Arch. f. Protistenk., Vol. 57. ToNNiGES, C. 1914 Die Trichocysten von Frontonia leucas und ihr chromidialer Ursprung. Ibid., Vol. 32. Turner, J. P. 1933 The external fibrillar system of Euplotes with notes on the neuromotor apparatus. Biol. Bull. Vol. 64. Verworn, M. 1903 Allgemeine Physiologic. Fourth edition. Jena. VisscHER, J. P. 1926 Feeding reactions in the ciliate Dileptus gigas, with special reference to the trichocysts. Biol. Bull., Vol. 45. Vlk, W. 1938 Ueber den Bau der Geissel. Arch. f. Protistenk., Vol. 90. Woodruff, L. L. and H. Spencer 1922 Studies on Spathidium spathula. I. Jour. Exp. Zool., Vol. 35. YocoM, H. B. 1918 The neuromotor apparatus of Euplotes patella. Univ. Calif. Publ. Zool, Vol. 18. Chapter 4 Physiology THE morphological consideration which has been given in the last chapter, is, though necessarily brief, indicative of the occur- rence of various and often complex organellae in Protozoa. The physiological activity of the whole protozoan is the sum-total of all the functions which are carried on by numerous minute parts or organellae of the cell body, unlike the condition found in a metazoan. Indeed, as Calkins (1933) stated, "physiological problems (of Protozoa) for the most part begin where similar problems of the Metazoa leave off, namely the ultimate processes of the single cell. Here the functional activities have to do with the action and inter- action of different substances which enter into the make-up of protoplasm and, for the most part, these are beyond our powers of analysis." A full discussion of various physiological problems per- taining to Protozoa is out of question in the present work and, there- fore, a general consideration on protozoan physiology will suffice for our purpose. Nutrition Protozoa obtain nourishment in manifold ways. Information on the nutrition of the Protozoa is undergoing an accelerated progress through improvements in technique in experimental cultivation of these organisms. Doyle (1943) has given an excellent review on the subject. It will be here briefly considered under three types: holozoic, holophytic, and saprozoic. Holozoic (zootrophic, heterotrophic) nutrition. This is the method by which all higher animals obtain their nourishment; namely, the protozoan uses other animals or plants as sources of food. It involves the food-capture and ingestion, digestion and assimilation, and re- jection of indigestible portions. The methods of food-capture vary among different forms. In the Sarcodina, the food organisms are captured and taken into the body at any point. The methods however vary. According to Rhumbler's oft-quoted observations, four methods of food-ingestion occur in amoebae (Fig. 32); namely, (1) by ''import," in which the food is taken into the body upon contact, with very little movement on the part of the amoeba (a); (2) by "circumfluence," in which the cytoplasm flows around the food organism as soon as it comes in contact with it on all sides and engulfs it (b) ; (3) by "circumvalla- tion," in which the amoeba without contact with the food, forms 84 PHYSIOLOGY 85 pseudopodia which surround the food on all sides and ingest it (c) ; (4) by "invagination," in which the amoeba touches and adheres to the food, and the ectoplasm in contact with it is invaginated into the endoplasm as a tube, the cytoplasmic membrane later disappears (d-h). Jennings, Kepner, Schaeffer and others, have made studies with reference to the food-ingestion in amoebae. Fig. 32. Various ways b^^ which amoebae capture food organisms, a, A moeba verrucosa feeding on Oscillatoria by 'import' (Rhumbler) ; b, ^ . proteus feeding on bacterial glea by 'circumfluence'; c, on Paramecium by 'circumvallation' (Kepner and Whitlock); d-h, A. verrucosa ingesting a food particle by 'invagination' (Gross- Allermann). In certain testaceans, such as Gromia, several rhizopodia cooper- ate in engulfing the prey and, in Lieberkiihnia (Fig. 33), Verworn noted ciliates are captured by and digested in rhizopodia. Similar observation was made by Schaudinn in the heliozoan Camptonema in which several axopodia anastomose to capture a prey (Fig. 109, d). In the holozoic Mastigophora, such as Hypermastigina, which do not possess cytostome, the food-ingestion is by pseudopodia also. The food particles become attached to the pseudopodium and are 86 PROTOZOOLOGY held there on account of the viscid nature of the pseudopodium. The sudden immobihty of active organisms upon coming in contact with pseudopodia of certain forms, such as Actinophrys, Actinosphaer- ium, Gromia, Elphidium, etc., suggests, however, probable discharge of poisonous substances. In the Suctoria which lack a cytostome, the tentacles serve as food-capturing organellae. The suctorial tentacle bears on its distal end a rounded knob which, when it comes in con- tact with an actively swimming ciliate, stops the latter immediately (Parapodophrya typha, Fig. 329, a). The prehensile tentacles of Ephelotidae are said to be similar in structure to the axopodia, in Fig. 33. Rhizopodia of Lieberkiihnia, capturing and digesting Colpiditun colpoda (Verworn). that each possesses a bundle of axial filaments around a cytoplasmic core (Roskin). These tentacles are capable of piercing through the body of a prey. In some suctorians, such as Choanophrya (Fig. 334, a), the tentacles are said to be tubular, and both solid and liquid food materials are sucked in through the cavity. The rapidity with which tentacles of a suctorian stop a very actively swimming ciliate is attributed to a certain substance secreted by the tentacles, which paralyses the prey. In the cytostome-bearing Mastigophora, the lashing of flagella will aid in bringing about the food particles to the cytostome, where it is taken into the endoplasm. In the ciliates there are numerous types of cytostomes and associated organellae. But food-capturing seems to be in general of two kinds. When the cytostome is perma- nently open, the organism ingests food particles which are small enough to pass the cytostome and cytopharynx, as in the case of Paramecium. Another type is one, such as noted in Coleps, Didi- PHYSIOLOGY 87 nium, etc., where the ciliate attacks other organism and sucks in the body substance of the latter through the enlarged cytostome. The ingested food particles are always surrounded by a film of fluid which envelops the organism and the whole is known as the food vacuole (p. 77). The quantity of fluid taken in with the food varies greatly and, generally speaking, it seems to be inversely pro- portional to the size, but proportional to the activity, of the food organisms. Food vacuoles composed entirely of surrounding liquid medium have occasionally been observed. Edwards (1925) observed ingestion of fluid medium by an amoeba by forming food-cups under changed chemical composition. Brug (1928) reports seeing Ent- amoeba histolytica engulf liquid culture medium by formation of lip- /T^ ^^ n n g; I Fig. 34. Ingestion of brine by Rhopalophrya salina (Kirby). like elevation of the ectoplasm and Kirby (1932) figures ingestion of the brine containing no visible organisms by the cytostome of Rhopalophrya salina (Fig. 34). Mast and Doyle (1934) state that if Amoeha protcus, A. duhia, A. dofleini, or A. radiosa is placed in an albumin solution, a hypertonic balanced salt solution, or a hyper- tonic solution of calcium gluconate it rapidly decreases in volume, and forms numerous tubes filled with fluid, which disintegrate sooner or later and release their fluid content in the cytoplasm. At times 50 or more such tubes may be present, which indicates that the organism ingests considerable quantities of fluid in this way. The two authors consider that it is *'a biological adaptation which serves to compen- sate for the rapid loss of water." The food vacuoles finally reach the endoplasm and in forms such as Amoebina the vacuoles are carried about by the moving endo- plasm. In the ciliates, the fluid endoplasm shows often a definite rotation movement. In Paramecium, the general direction is along the aboral side to the anterior region and down the other side, with PROTOZOOLOGY a short cyclosis in the posterior half of the body. In Carchesium, ac- cording to Greenwood, the food-vacuoles pass down to one end of the macronucleus and then move close along its concave surface to the anterior end of the nucleus where defecation to the vestibule takes place (Fig. 35). As stated above, in a number of species the food organisms are paralyzed or killed upon contact with pseudopodia, tentacles or ex- ploded trichocysts. In numerous other cases, the captured organism is taken into the food vacuole alive, as will easily be noted by ob- serving Chilomonas taken in by Amoeba proteus or actively moving bacteria ingested by Paramecium. But the prey ceases to move in a Fig. 35. Diagram showing the digestion within the food vacuoles in Carchesium polypinurn (Greenwood), a, digestion area; b, region of little change; c, region of acid reaction; d, region of neutral reaction; e, defeca- tion area. very short time. It is generally believed that some substances are se- creted into the food vacuole by the protoplasm of the organisms, to stop the activity of the prey within the food vacuole. Engelmann (1878) demonstrated that the granules of blue litmus, when ingested by Paramecium or Amoeba, became red in a few minutes. Brandt PHYSIOLOGY 89 (1881) examined the staining reactions of amoebae by means of haematoxylin, and found that the watery vacuoles contained an acid. Metschnikoff (1889) also showed that there appears an acid secretion around the ingested litmus grains in Mycetozoa. Green- wood and Saunders (1884) found in Carchesium that ingestion of food particles stimulated the cytoplasm to secrete a mineral acid (Fig. 35). According to Nirenstein (1925), the food vacuole in Paramecium undergoes change in reaction which can be grouped in two periods. The first is acid reaction and the second alkaline reaction, in which albumin digestion takes place. On the other hand, Khainsky (1910) observed that the food vacuole of ciliates, such as Paramecium, is cv Fig. 36. Diagram showing changes in reactions in food-vacuoles of Paramecium caudatum, after ingesting litmus (Shapiro), b, blue; cv, con- tractile vacuole; lb, light blue; Ir, light red; r, red. acid during the entire period of protein digestion, and becomes neu- tral to finally alkaline when the solution of the food substance is ended. Metalnikoff (1912) found that in the food vacuoles of Para- mecium, besides acid-alkaline reaction change, some vacuoles never show acid reaction and others occasionally show sustained acid reac- tion. Shapiro (1927) studied the reaction change of the food vacuoles 90 PROTOZOOLOGY in Paramecium caudatum (Fig. 36) by using phenol red, neutral red, Congo red, and litmus, and found that when the organism is kept in a medium with pH 7, its food vacuoles are first alkaline (pH 7.6), soon reach a maximum acidity (pH 4.0), while still in the posterior half of the body. Later, the vacuoles show a decreased acidity, finally reaching pH 7.0. In Vorticella sp. and Stylonychia pustulata, the range of pH observed in the food vacuoles was said to be 4.5- 7.0 and 4.8-7.0 respectively. The food vacuoles of Actinosphaer- ium, according to Howland (1928), possess at the beginning pH 6.0-7.0 for 5 to 10 minutes, but this soon changes to acid (pH 4.3) in which digestion appears to be carried on. In older food vacuoles which are of less acid (pH 5.4-5.6), the digestion appears to be at an end. In the species of Bresslaua, Claff, Dewey and Kidder (1941) noted that a Colpoda taken into the food vacuole is instantly killed with a sudden release of an acid which shows pH 3.0-4.2. During digestion the protoplasm of the prey becomes alkaline and the un- digested residue becomes acid before extrusion. Mast's recent obser- vations (1942) on the food vacuoles in Amoeba proteus and A. duhia containing Chilomonas or Colpidium indicate: (1) the fluid in the vacuoles becomes first acid and then alkaline; (2) the increase in the acidity of the fluid in the vacuole is not due to cytoplasmic secre- tion, but is probably due to respiration in the ingested organisms, chemical changes associated with their death, etc. ; and (3) the death of the organisms taken in the food vacuoles is probably caused by the decrease in oxygen in the vacuoles, owing to the respiration of the organisms in them. Just exactly what processes take place in the food vacuole have been observed only in a few cases. Nirenstein noticed the appear- ance of numerous neutral red-stainable granules around the food vacuole which pass into the interior of the vacuole, and regarded them as carriers of a tryptic ferment, while Roskin and Levinsohn demonstrated the oxidase reaction in these granules. A number of enzymes have been reported in the Protozoa, some of which are mentioned in Table 3. These findings suffice to indicate that the digestion in Protozoa is carried on also by enzymes and its course appears to vary among different Protozoa. The albuminous substances are digested and de- composed into simpler compounds by enzymes and absorbed by the surrounding cytoplasm. The power to digest starch into soluble sugars is widely found among various Protozoa. It has been re- ported in Mycetozoa, Foraminifera, Pelomyxa, Amoeba, Enta- moeba, Ophryoscolecidae and other ciliates by several investigators. PHYSIOLOGY 91 In Pelomyxa, Stole (1900) found that the so-called refringent bodies are intimately associated with the carbohydrate metabolism in that they are filled with glycogen which amount is proportionate to the food matter the organism obtains. The members of Vampyrella (p. 330) are known to dissolve the cellulose wall of algae, especially Spirogyra in order to feed on their contents. Pelomyxa (Stole), Foraminifera (Schaudinn), Amoeba (Rhumbler), Hypermastigina, Polymastigina (Cleveland), etc., have also been known for possessing the power of cellulose digestion. Many of the Hypermastigina and Polymastigina which lead symbi- Table 3. — Enzymes in Protozoa Protozoa Enzymes Observers Aethalium septicujn Pelomyxa palustris Soil amoebae Balantidium coli Euglena gracilis Glaucoma pyriformis Colpidium striatum Poly- and Hyper- mastigina in wood roach Pepsin-like enzyme, dissolving albumin in acid medium Pepsin-like and diastatic en- zymes "Amoebodiastase": trypsin- like, active in neutral or slightly alkaline medium, liquefies gelatin, coagulates albumin, inactive at 60°C. Diastatic enzyme Proteolytic enzyme in cultures Proteolytic enzyme, capable of hydrolyzing casein Proteolytic enzyme, capable of hydrolyzing casein Cellulase; Cellobiase Krukenberg (1886) Hartog and Dixon (1893) Mouton (1902) Glaessner (1908) Jahn (1931) Lwoff (1932) Elliott (1933) Cleveland et al. (1934) otic life in the intestine of the termite and of the wood roach, as dem- onstrated by Cleveland and his co-workers, digest by enzymes the cellulose which the host insect ingests. The assimilation products produced by an enormous number of these flagellates are seemingly sufficient to support the protozoans as well as the host. The cili- ate commensals inhabiting the stomach of ruminants also appar- ently digest the cellulose, since the faecal matter as a rule does not contain this substance. The digestion of fat by Protozoa had not been known, although oils and fat have been observed in numerous Protozoa, until Dawson and Belkin (1928) injected different oils into Amoeha duhia and found that from 1.4 to 8.3 per cent of the injected oil was digested. Mast (1938) noticed that the neutral fat 92 PROTOZOOLOGY globules of Colpidium are digested by Amoeba proteus and trans- formed into fatty acid and glycerine which unite and form neutral fat. The indigestible residue of the food is extruded from the body. The extrusion may take place at any point on the surface in many Sarcodina by a reverse process of the ingestion of food. But in pelli- cle-bearing forms, the defecation takes place either through the cytopyge located in the posterior region of the body or through an aperture to the vestibule (in Carchesium). Permanent cytopyge is lacking in some forms. In Fdbrea salina, Kirby (1934) noticed that a large opening is formed at the posterior end, the contents of food vacuoles are discharged, and the opening closes over. At first the margin of the body is left uneven, but soon the evenly rounded out- line is restored. The same seems to be the case with Spirostomum (Fig. 37), Blepharisma, etc. Fig. 37. Outline sketches showing the defecation process in Spirostomum ambiguum (Blattner). Holophytic (autotrophic, phytotrophic) nutrition. This is the type of nutrition in which the Protozoa are able to decompose carbon dioxide by means of chlorophyll contained in chromatophores (p. 78) in the presence of the sunlight, liberating the oxygen and combining the carbon with other elements derived from water and inorganic salts. The pyrenoids (p. 79) are inseparably connected with the reserve carbohydrate formation in this nutrition. Aside from the Phytomastigina, chromatophores were definitely observed in a cili- ate Cyclotrichium meunieri (Fig. 268, o) by Powers. In a number of other cases, the organism itself is without chromatophores but is apparently not holozoic, because of the presence of chlorophyll- bearing organisms within it. For example, in the testacean Paulinella (Fig. 182, c) in which occur no food vacuoles, chromatophores of peculiar shape are always present. The latter appear to be a species of alga which holds a symbiotic relationship with the testacean, and perhaps acts for the sarcodinan as the chromatophores of the Phyto- PHYSIOLOGY 93 mastigina. A similar relationship seems to exist between Paramecium hursaria and a zoochlorella, Paraeuplotes tortugensis and a zooxanthella and others (p. 25). Pringsheim showed that organic matters from zoochlorellae are passed on to their host, Paramecium hursaria, to be used as food. Through studies of relationships between zooxanthellae and invertebrates Yonge observed that the zooxan- thellae utilize carbon dioxide, nitrogen and phosphorus which are the cataboHc products of the host and supply in return oxygen, fats and carbohydrates to the host. Saprozoic (saprophytic) nutrition. In this nutrition, the Protozoa obtain nourishment by diffusion through the body surface. This is accomplished without any special organellae. Perhaps the only in- stance in which the saprozoic nutrition is accomplished through a special organella is the pusules (Figs. 107, 108) in marine dinoflagel- lates which, according to Kofoid and Swezy, appear to contain de- composed organic matter and aid the organisms in carrying on this process. The dissolved food matters are simpler compounds which originate in animal or vegetable matter due to the decomposing activities of bacterial organisms. Numerous free-living Zoomas- tigina nourish themselves with this method. Recently a number of investigators found that saprozoic Protozoa could be cultivated in bacteria-free media of known compositions. For example, Prings- heim observed in Polytoma uvella (Fig. 97, h) that sodium acetate is needed from which the starch among others is produced and carbo- hydrates have no direct bearing upon the nutrition, but fatty acids derived from them participate in the metabolism. Hall, Jahn, Loefer and others are following the same line of work which may lead to a better understanding of saprozoic nutrition as found in Proto- zoa. The Protozoa which live within the body of another organism are able to nourish themselves by absorbing the digested or decomposed substances of the host and could be considered as saprozoic, though the term parasitic has sometimes been used. Coelozoic Protozoa be- long to this group, as for example, Protociliata, astomatous ciliates, Trypanosomatidae, etc. In the case of cytozoic or certain histozoic forms, such as Cnidosporidia, the host cytoplasm is apparently liquefied or hydrolyzed by enzymes before being absorbed by them. The parasitic Protozoa, which actually feed on host tissue cells, such as Entamoeba histolytica, Balantidium coli, etc., or endocommensals, employ, of course, the holozoic nutrition. Many Protozoa nourish themselves by more than one method at the same or different times, subject to a change in external condi- 94 PROTOZOOLOGY tions. This is sometimes referred to as mixotrophic nutrition (Pfeif- fer). For example, Euglena gracilis, according to Zumstein (1900) and Lwoff (1932) may lose its green coloration and becomes Astasia- like in the dark, or even in the light when the culture medium is very abundant in decomposed organic substances, which may indicate that this organism is capable of carrying on both holophytic and saprozoic nutrition. With the introduction of bacteria-free culture technique in recent years, it has now become well established that a protozoan species exhibits conspicuous differences in form, size and structure, which are exclusively due to differences in the kind and amount of food material. For example, Kidder, Lilly and Claff (1940) noted in Tetrahymena vorax (Fig. 38), bacteria-feeders are tailed (50-75ju long), saprozoic forms are fusiform to ovoid (30-70iu long), forms feeding on sterile dead ciliates are fusiform (60-80ju long), and carni- vores and cannibals are irregularly ovoid (100-250^ long), in the latter form of which a large preparatory vacuole becomes developed. In Chilomonas Paramecium, Mast (1939) observed the individuals grown in sterile glucose-peptone solution were much smaller than those cultured in acetate-ammonium solution and moreover the former contained many small starch grains, but no fat, while the latter showed many larger starch grains and a little fat. Amoeba proteus when fed exclusively on Colpidium, became very large and extremely "fat" and sluggish, growing and multiplying slowly, but indefinitely; when fed on Chilomonas only, they grew and multi- plied for several days, then decreased in number and soon died, but lived longer on Chilomonas cultured in the glucose-peptone. Since the fact that endocrines influence greatly the metabolic ac- tivity and growth in higher animals became known, many workers undertook to determine the effects of various endocrines of verte- brates upon Protozoa. Nowikoff (1908) first noticed the apparent increase in number of Paramecium caudatum when cultured in a solu- tion of desiccated sheep thyroid as compared with the culture in a hay infusion. Shumway (1914, 1917) found that the emulsions of fresh thyroid, boiled thyroid and commercial powder, produced an increase of about 65 per cent in the division rate in Paramecium over common hay infusions. These animals were smaller and more ac- tively motile, and showed more vacuolated cytoplasm. Abderhalden and Schiffmann (1922) made a similar observation by using thyroid "optone." Shumway found further that suspensions of thymus, spleen, ovary, suprarenal, and pituitary body, did not have any ef- fect on Paramecium. By using freshly prepared thyroid extracts of PHYSIOLOGY 95 cat, bird, turtle, frog, and fish, on Paramecium and Stylonychia, Budington and Harve}^ (1915) noticed also that all extracts in- creased the division rate. Flather (1919) saw the contraction of the contractile vacuoles of Paramecium accelerated by adrenaline and pineal extract. Cori (1923) noted the acceleration (about 12 per cent) of the division Fig. 38. Form and size variation in Tetrahymena vorax, due to differ- ences in kind and amount of food material, as seen in life. X400 (Kidder, Lilly and Claff). a, bacteria-feeder; b, c, saprozoic forms; d, individual which has fed on killed Colpidium camjnjlum ; e, starved individual from a killed-Oolpidium culture; f-i, progressive form and size changes of saprozoic form in the presence of living Colpidium; j, a young carnivore which has been removed to a culture with living yeast. 96 PROTOZOOLOGY rate in P. putrinum occurred only in faintly alkaline thyroid ex- tract in hay infusions. Riddle and Torrey (1923) on the other hand found that the thja-oxine brought about a slight fall in the rate of division, an increased rate of pulsation of the contractile vacuoles, and a decrease in number of excretory crystals in Para- mecium. The two investigators suggested that the thyroxine acceler- ates catabolic, and not anabolic, processes. Woodruff and Swingle (1923, 1924), using a pedigree culture of P. aurelia found that (1) neither thyroid, pineal, nor pituitary material possesses intrinsic properties which accelerate division in Paramecium and (2) thyrox- ine does not accelerate the division and above certain concentrations, it depresses the division in this ciliate. Ball (1925) found different clones of P. caudatum and P. aurelia, respond differently to similar conditions and obtained following re- sults: (1) with an uncontrolled bacterial food supply, individuals of the same clone may divide at a higher rate in solutions of the desic- cated thyroid, liver, and hypophysis, than in hay infusions; (2) if ap- proximately equal number of bacteria are provided, thyroid does not bring about any significant increase in the division rate; and (3) the evidence indicates that thyroid accelerates the division rate of Paramecium by providing a favorable bacterial food supply, and not by any specific action of the thyroid hormone. Solutions of both the anterior and the posterior lobes of the pituitary gland produce no significantly higher rate of division than does a solution of liver. Unlike the observations made by some previous workers. Ball finds no demonstrable increase in the metabolic rate in thyroid-fed ani- mals as compared with those cultured in hay infusions. The importance of vitamins has abundantly been demonstrated in recent years for many higher animals. It is, therefore, natural to find attempts made to discover the influence of certain vitamins on Protozoa. Lwoff and Dusi (1937, 1938) found the growth of Chilo- monas Paramecium in asparagin media was favorably supported by thiamine, or by thiazole, and in ammonium acetate media, thiamine could be replaced by thiazole and pyrimidine. Thiamine has no ac- celerating effect on the growth of Euglena gracilis in light (Elliott, 1937), but either this substance or pyrimidine is necessary for the growth of the organism if cultured in asparagin and acetate media in darkness (Lwoff and Dusi). It is probable that chromatophore-bear- ing forms are able to synthesize thiamine from the constituents of inorganic media in sunlight. E. pisciformis on the other hand is said to require thiamine for growth even in light (Dusi, 1939). Many colorless flagellates require thiamine for growth as in Chilo- PHYSIOLOGY 97 monas 'Paramecium mentioned above, others appear to be capable of synthesizing it as in Polytoma uvella (Lwoff and Dusi). In a number of ciliates such as Colpidium campylum, C. striatum, Tetrahymena geleii, etc., thiamine seems to support growth according to several workers. For example, Elliott (1939) observed crystalline thiamine chloride to be a limiting factor for the growth of Colpidium striatum in media free from this substance. Vitamin Bi was found most effec- tive in concentration of 1 : 10,000 to 1 : 10,000,000. This worker further found that crystalline riboflavin and vitamin Be (concentrate) cannot supplant thiamine in the nutrition of this ciliate. According to Hall and Shottenfeld (1941), the density of population in bacteria-free cultures of Glaucoma pyriformis is related quantitatively to the con- centration of thiamine and the phases of death also are influenced markedly by the available concentration of this vitamin. For Col- pidium campylum, Hall (1942) observed thiamine and riboflavin to be essential for growth in a de-ashed gelatin medium, and thiamine to be necessary for growth in a silk-peptone culture which has been subjected to prolonged heating in strongly alkaline .solution. Lilly (1942) succeeded in growing bacteria-free several strains of Stylonychia pustulata and Pleurotricha lanceolata on ciliates (5 spe- cies), flagellates (3 species) and a strain of Saccharomyces cerevisiae, all cultured in sterile condition. He found that for continued growth of the two ciliates, it was necessary to supply in addition to the food organisms, a supplementary growth factor which was found in the largest quantity in yeast cells and less so in infusions of a wide vari- ety of plant materials, but which was not found in effective concen- trations in media made from animal organs or tissues. This substance was soluble in water and in 70 per cent alcohol, was stable to heat and alkali, and was adsorbed on charcoal and on Fuller's earth. The in- vestigator considers it as not identical with any of the known vita- mins of the B complex. Johnson and Baker (1943) have examined certain B vitamins in relation to populations of Tetrahymena geleii and found: (1) the addition of thiamine to fresh proteose-peptone medium produced a higher maximum population; (2) all of the long- time cultures containing added thiamine, had secondary increase in population, almost to the original peaks, after 64 days; (3) cultures with a mixture of thiamine, riboflavin, and pyridoxine, maintained the highest level of living population; and (4) cultures with para- aminobenzoic acid had higher maximum populations than those ob- tained under other conditions, but died out sooner than the other types of cultures (addition of thiamine prevented this early dying- out). 98 PROTOZOOLOGY Ascorbic acid has been found to be an essential factor for the growth in Leishmania donovani, L. tropica, Trypanosoma cruzi (Lwoff), Tritrichomonas foetus (Cailleau), etc. Haematin is said to be a necessary growth factor for the two species of Leishmania and Try- panosoma cruzi (Lwoff). Observations on the effect of vitamins as ap- plied to host infected by Protozoa are meager. Becker and his associ- ates (1941) have shown that when a ration somewhat restricted in vitamins Bi and Be was used as basal, the addition of moderate amounts of thiamine chloride resulted in a reduction in the number of oocysts of Eimeria nieschulzi eliminated by host rats. The same was true when vitamins Bi and Be were administered to infected rats intraperitoneally in normal salt solution. On the other hand, vitamin Be supplement alone produced an increase in the oocyst production. Certain substances which are sometimes called growth stimulants have also been applied to Protozoa in recent years. Elliott (1935) found that pantothenic acid brings about a doubling of the growth of Colpidium campylum in sterile culture at pH 5.5-6.6, but not at pH 7.0 or above, and it does not accelerate the growth of Haemato- coccus pluvialis in bacteria-free cultures. Thus it appears that panto- thenic acid has no effect on chlorophyll-bearers. As to the effect of auxins (plant growth substances), the same investigator demon- strated that Euglena gracilis grew faster in a medium at pH 5.6 in light and in the presence of auxins, while these substances did not bring about any noticeable effect on such colorless forms as Khawk- inea halli and Colpidium striatum. Hall (1939) examined the effect of pimelic acid on Colpidium campylum in sterile cultures and found that it exerts some sort of catalytic effect on the metabolism of this ciliate in peptone and gelatin media, as shown by an increase in the growth rate and in the density of population, but the effect may be masked, or perhaps, eliminated by the addition of dextrose to the medium. Fuller information on the relationships between endocrines, vita- mins and growth-promoting substances and Protozoa is dependent upon a greater application of sterile culture method and standard- ization of basal culture media to many Protozoa in future. The reserve food matter The anabolic activities of Protozoa result in the growth and in- crease in the volume of the organism, and also in the formation and storage of reserve food-substances which are deposited in the cy- toplasm to be utilized later for growth or reproduction. The re- serve food stuff is ordinarily glycogen or glycogen ous substances, PHYSIOLOGY 99 which seem to be present widely. Thus, in saprozoic Gregarinida, there occur in the cytoplasm numerous refractile bodies which stain brown to brownish-violet in Lugol's solution; are insoluble in cold water, alcohol, and ether; become swollen and later dissolved in boil- ing water; and are reduced to a sugar by boiling in dilute sulphuric acid. This substance which composes the refractile bodies is called paraglycogen (Biitschli) or zooamylon. The abundant glycogen bod- ies of Pelomyxa have already been mentioned (p. 91). Rumjantzew and Wermel demonstrated glycogen in Actinosphaerium. In loda- moeba, glycogen body is conspicuously present and is looked upon as a characteristic feature of the organism. The iodinophile vacuole of the spores of Myxobolidae is a conspicuously well-defined vacuole containing glycogenous substance and is also considered as possess- ing a taxonomic value. In many ciliates, both free-living (Parame- cium, Glaucoma, Vorticella, etc.) and parasitic (Ophryoscolecidae, Nyctotherus, Balantidium, etc.), glycogenous bodies are always present. According to MacLennan (1936), the development of the paraglycogen in Ichthyophthirius is associated with the chondrio- somes. In Eitneria tenella, glycogenous substance does apparently not occur in the schizonts, merozoites, or microgametocytes; but becomes apparent first in the macrogametoc3^te, and increases in amount with its development, a small amount being demonstrable in the sporozoites (Edgar et al., 1944). Fig. 39. a-d, two types of paramylon present in Euglena gracilis (Biitschli); e-h, paramylon of E. sanguinea, XllOO (Heidt). e, natural appearance; f, g, dried forms; h, strongly pressed bodies. The anabolic products of the holophytic nutrition are starch, paramylon, oil and fats. The paramylon bodies are of various forms among different species, but appear to maintain a certain character- istic form within a species and can be used to a certain extent in taxonomic consideration. According to Heidt (1937), the paramylon of Euglena sanguinea (Fig. 39) is spirally coiled which confirms Biitschli's observation. The paramylon appears to be a polysac- 100 PROTOZOOLOGY charide which is insoluble in boiling water, but dissolves in concen- trated sulphuric acid, potassium hydroxide, and slowly in formalde- hyde. It does not stain with either iodine or chlor-zinc-iodide and when treated with a dilute potassium hydroxide, the paramylon bodies become enlarged and frequently exhibit a concentric stratifi- cation. In the Chrysomonadina, the reserve food material is in the form of refractile bodies which are collectively called leucosin, probably a carbohydrate. Oils occur in various Protozoa and when there is a sufficient number of oil-producing forms in a body of water, the water may develop various odors. Table 4 shows kinds of odor pro- duced by certain Protozoa when they are present in the water in large numbers: Table 4. — Protozoa and odors of water Protozoa Odor produced by them Cryptomonas candied violets Mallomonas aromatic, violets, fishy Synura ripe cucumber, muskmelon, bitter and spicy taste Uroglenopsis fishy, cod-liver oil-like Dinobryon fishy, like rockweed Chlamydomonas fishy, unpleasant or aromatic Eudorina faintly fishy Pandorina faintly fishy Volvox fishy Ceratium vile stench Glenodinium fishy Peridinium fishy, like clam-shells Bursaria Irish moss, salt marsh, fishj^ (Whipple) Pelomyxa ripe cucumber (Schaeffer) Fats have also been detected in many Protozoa, such as Myxo- sporidia, Protociliata, certain Eucihata, Trypanosoma, etc. Accord- ing to Panzer, the fat content of Eimeria gadi was 3.55 per cent and Pratje reports that 12 per cent of the dry matter of Noctiluca scintil- lans appeared to be the fatty substance present in the form of granules and is said to give luminescence upon mechanical or chemi- cal stimulation. A number of other dinoflagellates, such as Peridi- nium, Ceratium, Gonyaulax, Gymnodinium, etc., also emit lumi- nescence. In other forms the fat may be hydrostatic in function, as is the case with a number of pelagic Radiolaria, many of which are also luminous. Another reserve food-stuff which occurs widely in Protozoa, ex- PHYSIOLOGY 101 cepting Ciliophora, is the so-called volutin or metachromatic gran- ule. It is apparently equally widely present in Protophyta. In fact it was first discovered in the protophytan Spirillum volutans. Meyer coined the name and held it to be made up of a nucleic acid. It stains deeply with nuclear dyes. Reichenow (1909) demonstrated that if Haematococcus pluvialis (Fig. 40) is cultivated in a phosphorus-free medium the volutin is quickly used up and does not reappear. If however, the organisms are cultivated in a medium rich in phos- phorus, the volutin increases greatly in volume and, as the culture becomes old, it gradually breaks down. In Polytomella agilis (Fig. 98, c, d), Doflein showed that an addition of sodium phosphate re- sulted in an increase of volutin. Reichenow, Schumacher, and others, hold that the volutin appears to be a free nucleic acid, and is a spe- cial reserve food material for the nuclear substance. Recently Sas- FiG. 40. Haematococcus pluvialis, showing the development of volutin in the medium rich in phosphorus and its disintegration in an exhausted medium, X570 (Reichenow). a, second day; b, third day; c, fourth day; d, e, sixth day; f, eighth day. suchin (1935) studied the volutin in Spirillum volutans and Sarcina flava and found that the volutin appears during the period of strong growth, nourishment and multiplication, disappears in unfavorable condition of nourishment and gives a series of characteristic carbo- hydrate reactions. Sassuchin considers that the volutin is not related to the nucleus, but is a reserve food material of the cell, and is composed of glycoprotein. Respiration In order to carry on various vital activities, the Protozoa, like all other organisms, must transform the potential energy stored in highly complex chemical compounds present in the cytoplasm, into various forms of active energy by oxidation. The oxygen involved in this process appears to be brought into contact with the sub- stances in two ways in Protozoa. The great majority of free-living, and certain parasitic forms absorb free molecular oxygen from the surrounding media. The absorption of oxygen appears to be carried on by the permeable body surface, since there is no special organella for this purpose. The polysaprobic Protozoa are known 102 PROTOZOOLOGY to live in water containing no free oxygen. For example, Noland (1927) observed Metopus es in a pool, 6 feet in diameter and 18 inches deep, filled with dead leaves which gave a strong odor of hj^drogen sulphide. The water in it showed pH 7.2 at 14°C., and contained no dissolved oxygen, 14.9 c.c. per liter of free carbon dioxide, and 78.7 c.c. per liter of fixed carbon dioxide. The parasitic Protozoa of metazoan digestive systems live also in a medium containing no molecular oxygen. All these forms appear to possess capacity of splitting complex oxygen-bearing substances present in the body to produce necessary oxygen. Several investigators studied the influence of abundance or lack of oxygen upon different Protozoa. For example, Putter demon- strated that several ciliates reacted differently when subjected to anaerobic condition, some perishing rapidly, others living for a con- siderable length of time. Death is said by Lohner to be brought about by a volume-increase due to accumulation of the waste prod- ucts. When first starved for a few days and then placed in anaerobic environment, Paramecium and Colpidium died much more rapidly than unstarved individuals. Putter, therefore, supposed that the dif- ference in longevity of aerobic Protozoa in anaerobic conditions was correlated with that of the amount of reserve food material such as protein, glycogen and paraglj^cogen present in the body. Putter fur- ther noticed that Paramecium is less affected by anaerobic condition than Spirostomum in a small amount of water, and maintained that the smaller the size of body and the more elaborate the contractile vacuole system, the organisms suffer the less the lack of oxygen in the water, since the removal of catabolic products depends upon these factors. The variety of habitats and results of artificial cultivations of various Protozoa indicate clearly that the oxygen requirements vary a great deal among different forms. Attempts were made in recent years to determine the oxygen requirement of Protozoa. The results of the observations are not always convincing. The oxygen consump- tion of Paramecium is said, according to Lund (1918) and Amberson (1928), to be fairly constant over a wide range of oxygen concentra- tion. Specht (1934) found the measurements of the oxygen con- sumption and carbon dioxide production in Spirostomum ambiguum vary because of the presence of a base produced by the organism. Soule (1925) observed in the cultural tubes of Trypanosoma lewisi and Leishmania tropica, the oxygen contained in about 100 c.c. of air of the test tube is used up in about 12 and 6 days respectively. A single Paramecium caudatum is said to consume in one hour at PHYSIOLOGY 103 21°C. from 0.0052 c.c. (Kalmus) to 0.00049 c.c. (Howland and Bern- stein) of oxygen. Amoeba proteus, according to Hiilpieu (1930), suc- cumbs slowly when the amount of oxygen in water is less than 0.005 per cent and also in excess, which latter confirms Putter's observation on Spirostomum. According to Clark (1942), a normal Amoeba pro- teus consumes 1.4X10~^ mm^ of oxj^gen per hour, while an enucle- ated amoeba only 0.2X10~^ mm.' He suggests that "the oxygen- carriers concerned wdth 70 per cent of the normal respiration of an amoeba are related in some way to the presence of the nucleus." The Hypermastigina of termites are killed, according to Cleveland, when the host animals are kept in an excess of oxygen. Jahn (1935) found that Chilomonas Paramecium in bacteria-free cultures in heavily buf- fered peptone-phosphate media at pH 6.0 required for rapid growth carbon dioxide which apparently brings about a favorable intracel- lular hydrogen-ion concentration. Excretion and secretion The catabolic waste material composed of water, carbon dioxide, urea and other nitrogenous compounds, all of which are soluble, pass out of the bod}^ by diffusion through the surface or by means of the contractile vacuole (p. 73). The protoplasm of the Protozoa is gen- erally considered to possess a molecular make-up which appears to be similar among those living in various habitats. In the freshwater Protozoa, the water diffuses through the body surface and so in- creases the water content of the body protoplasm as to interfere with its normal function. The contractile vacuole, which is invari- ably present in all freshwater forms, is the means of getting rid of this excess water from the body. On the other hand, marine or para- sitic Protozoa live in nearly isotonic media and there is no excess of water entering the body, hence the contractile vacuoles are not found in them. Just exactly w^hy all euciliates and suctorians possess the contractile vacuole regardless of habitat, has not fully been ex- plained. It is assumed that the pellicle of the ciliate is impermeable to salts and slowly permeable to water (Kitching). If this is true in all ciliates, it is not difficult to understand the universal occurrence of the contractile vacuole in the ciliates and suctorians. That the elimination of excess amount of water from the body is one of the functions of the contractile vacuole appears to be be- yond doubt judging from the observations of Zuelzer (1907), Finley (1930) and others, on Amoeba verrucosa which lost gradually its con- tractile vacuole as sodium chloride was added to the water, losing the organella completely in the seawater concentration. Herf (1922) 104 PROTOZOOLOGY studied the pulsation of the contractile vacuoles of Paramecium caudatum in fresh water as well as various salt concentrations, and obtained the following measurements: Per cent NaCl in water 0.25 0.5 0.75 1.00 Contraction period in second 6.2 9.3 18.4 24.8 163.0 Excretion per hour in body volumes 4.8 2.82 1.38 1.08 0.16 The contractile vacuole also serves to remove from the body part of soluble catabolic wastes, judged by numerous observations. Weatherby (1929) showed that the excretory vacuole of Spiros- tomum contains urea, and that of Didinium contains ammonia and occasionally trace of uric acid. The number of the contractile vacu- oles present in a given species as in various species of Paramecium, is not always constant. Nor is its size constant. According to Taylor (1920) the average size of the contractile vacuole of Euplotes patella is 29/x at maximum diastole, but may become 45-50m in diameter upon disturbance or after incision. The rate of pulsation is subject to change with temperature, physiological state of the organism, amount of food substances present in the water, etc. For example, Rossbach observed in the three ciliates mentioned below the pulsation of the contractile vacuole increased first rapidly and then more slowly with the rise of the temperature of the water: Time in seconds between two systoles at different temperature (C.) 5° 10° 15° 20° 25° 30° Euplotes char on 61 48 31 28 22 23 Stylonychia pustulata 18 14 10-11 6-8 5-6 4 Chilodonella cuculluhis 9 7 5 4 4 — In Amoeba mira, Hopkins (1938) found that small vacuoles (acid reaction) appear and coalesce with one another and also with bacteria taken in as food, thus giving rise to larger ones (alkaline reaction). These larger vacuoles after giving off substances to the protoplasm by diffusion, are discharged. Thus the vacuole system in this amoeba appears to perform not only digestive func- tion, but also excretory function as excess water, food residues and a substance stainable by janus green B, are extruded by way of this system. Aside from the soluble forms, there often occur in the protozoan body insoluble catabolic products in the forms of crystals and gran- ules of various kinds. Schewiakoff (1893) first noticed that Para- mecium often contained crystals (Fig. 41) composed of calcium phos- phate, which disappeared completely in 1-2 days when the organ- PHYSIOLOGY 105 isms were starved, and reappeared when food was given. Schewiakoff did not see the extrusion of these crystals, but considered that these crystals were first dissolved and excreted by the contractile vacuoles, as they were seen collected around the vacuoles. In Amoeba proteus, Schubotz (1905) noted that the crystals were of similar chemical composition and of usually bipyramidal or rhombic form, and that they measured about 2-5^ in length and doubly refractile. Schaeffer (1920) observed calcium phosphate cr3^stals in three species of B □ Fig. 41. Examples of crystals present in Protozoa, a-e, in Paramecium caudatum (Schewiakoff), (a-d, XlOOO, e, X2600); f, in Amoeba proteus; g, in A. discoides; h-1, in A. duhia (Schaeffer). Amoeba and was inclined to think that the forms and dimensions of these crystals were characteristic of each species. Thus in Amoeba proteus, they are truncate bipyramids, rarely fiat plates, up to 4.5jLt long; in A. discoides, abundant, truncate bipyramids, up to 2.5^ long; and in A. dubia, variously shaped (4 kinds), few, but large, up to 10m, 12/x, 30m long (Fig. 41). Rowland detected uric acid in Paramecium caudatum and Amoeba verrucosa. Luce and Pohl (1935) noticed that at certain times amoe- bae in culture are clear and contain relatively a few crystals but, as the culture grows older and the water becomes more neutral, the crystals become abundant and the organisms become opaque in transmitted light. These crystals are tubular and six-sided, and vary in length from 0.5 to 3.5m. They considered the crystals were com- posed of calcium chlorophosphate. Mast and Doyle (1935), on the other hand, noted in Amoeba proteus two kinds of crystals, plate- like and bipyramidal, which vary in size up to 7m in length and which are suspended in alkaline fluid to viscous vacuoles. These two authors believed that the plate-like crystals are probably leucine, while the bipyramidal crystals consist of a magnesium salt of a sub- stituted glycine. Other crystals are said to be composed of urate, carbonate, oxalate, etc. Another catabolic product is the haemozoin (melanin) grains 106 PROTOZOOLOGY which occur in many haemosporidians and which appear to be com- posed of a derivative of the haemoglobin of the infected erythrocyte. In certain Radiolaria, there occurs a brownish amorphous mass which is considered as cataboHc waste material and, in Foraminifera, the cytoplasm is frequently loaded with masses of brown granules which appear also to be catabolic waste and are extruded from the body periodically. While intracellular secretions are usually difficult to recognize, because the majority remain in fluid form except those which pro- duce endoskeletal structures occurring in Heliozoa, Radiolaria, cer- tain parasitic ciliates, etc., the extracellular secretions are easily recognizable as loricae, shells, envelopes, stalks, collars, mucous sub- stance, pigments which give the body a characteristic coloration (p. 38), etc. Furthermore, many Protozoa secrete, as was stated be- fore, certain substances through the pseudopodia, tentacles or tricho- cysts which possess paralyzing effect upon the preys. Movements Protozoa move about by means of the pseudopodia, flagella, or cilia, which may be combined with internal contractile organellae. Movement by pseudopodia. Amoeboid movements have long been studied by numerous observers. The first attempt to explain the movement was made by Berthold (1886), who held that the differ- ence in the surface tension was the cause of amoeboid movements, which view was supported by the observations and experiments of Biitschli (1894) and Rhumbler (1898). According to this view, when an amoeba forms a pseudopodium, there probably occurs a diminu- tion of the surface tension of the cytoplasm at that point, due to certain internal changes which are continuously going on within the body and possibly to external causes, and the internal pressure of the cytoplasm will then cause the streaming of the cytoplasm. This results in the formation of a pseudopodium which becomes attached to the substratum and an increase in tension of the plasma-mem- brane draws up the posterior end of the amoeba, thus bringing about the movement of the whole body. Jennings (1904) found that the movement of Amoeba verrucosa (Fig. 42, a) could not be explained by the surface tension theory, since he observed "in an advancing amoeba substance flows for- ward on the upper surface, rolls over at the anterior edge, coming in contact with the substratum, then remains quiet until the body of the amoeba has passed over it. It then moves upward at the posterior end, and forward again on the upper surface, continuing PHYSIOLOGY 107 in rotation as long as the amoeba continues to progress." Thus Amoeba verrucosa may be compared with an elastic sac filled with fluid. Bellinger (1906) studied the movement of Amoeba proteus, A. verrucosa and Difflugia spiralis. Studying in side view, he found Fig. 42. a, diagram showing the movement of Amoeba verrucosa in side view (Jennings) ; b, a marine limax-amoeba in locomotion (Pantin from Reichenow). ac, area of conversion; cet, contracting ectoplasmic tube; fe, fluid ectoplasm; ge, gelated ectoplasm. that the amoeba (Fig. 43) extends a pseudopod, ''swings it about, brings it into the line of advance, and attaches it" to the substratum and that there is then a concentration of the substance back of this point and a flow of the substance toward the anterior end. Bellinger Fig. 43. Outline sketches of photomicrographs of Amoeba proteus during locomotion, as viewed from side (Bellinger). held thus that ''the movements of amoebae are due to the presence of a contractile substance," which was said to be located in the endo- plasm as a coarse reticulum.' In the face of advancement of our knowledge on the nature of protoplasm, Rhumbler realized the difficulties of the surface tension 108 PROTOZOOLOGY Fig. 44. Diagram of Amoeba proteus, showing the solation and gelation of the cytoplasm during amoeboid movement (Mast), c, crystal; cy, con- tractile vacuole; f, food vacuole; he, hyaline cap; n, nucleus; pg, plasma- gel; pgs, plasmagel sheet; pi, plasmalemma; ps, plasmasol. PHYSIOLOGY 109 theory and later suggested that the conversion of the ectoplasm to endoplasm and vice versa were the cause of the cytoplasmic move- ments, which was much extended by Hyman (1917). Hyman con- sidered that: (1) a gradient in susceptibility to potassium cyanide exists in each pseudopodium, being the greatest at the distal end, and the most recent pseudopodium, the most susceptible; (2) the susceptibility gradient (or metabolic gradient) arises in the amoebae before the pseudopodium appears and hence the metabolic change which produces increased susceptibility, is the primary cause of pseudopodium formation; and (3) since the surface is in a state of gelation, amoeboid movement must be due to alterations of the col- loidal state. Solation, which is brought about by the metabolic change, is regarded as the cause of the extension of a pseudopodium, and gelation of the withdrawal of pseudopodia and of active con- traction. Schaeffer (1920) mentioned the importance of the surface layer which is a true surface tension film, the ectoplasm, and the streaming of endoplasm in the amoeboid movement. Pantin (1923) studied a marine limax-type amoeba (Fig. 42, h) and came to recognize acid secretion and absorption of water at the place where the pseudopodium was formed. This results in swelling of the cytoplasm and the pseudopodium is formed. Because of the acidity, the surface tension increases and to lower or reduce this, concentra- tion of substances in the "wall" of the pseudopodium follows. This leads to the formation of a gelatinous ectoplasmic tube which, as the pseudopodium extends, moves toward the posterior region where the acid condition is lost, gives up water and contracts finally becoming transformed into endoplasm near the posterior end. The contraction of the ectoplasmic tube forces the endoplasmic streaming to the front. This observation is in agreement with that of Mast (1923, 1926, 1931) who after a series of carefully conducted observations on Amoeba proteus came to hold that the amoeboid movement is brought about by "four primary processes; namely, attachment to the substratum, gelation of plasmasol at the anterior end, solation of plasmagel at the posterior end and the contraction of the plasmagel at the posterior end" (Fig. 44). As to how these processes work, Mast states: "The gelation of the plasmasol at the anterior end ex- tends ordinarily the plasmagel tube forward as rapidly as it is broken down at the posterior end by solation and the contraction of the plasmagel tube at the posterior end drives the plasmasol forward. The plasmagel tube is sometimes open at the anterior end and the plasmasol extends forward and comes in contact with the plasma- 110 PROTOZOOLOGY lemma at this end (Fig. 45, a), but at other times it is closed by a thin sheet of gel which prevents the plasmasol from reaching the anterior end (6). This gel sheet at times persists intact for consider- able periods, being built up by gelation as rapidly as it is broken down by stretching, owing to the pressure of the plasmagel against it. Usually it breaks periodically at various places. Sometimes the breaks are small and only a few granules of plasmasol pass through and these gelate immediately and close the openings (d). At other times the breaks are large and plasmasol streams through, filling the hyaline cap (c), after which the sol adjoining the plasmalemma gel- FiG. 45. Diagrams of varied cytoplasmic movements at the tip of a pseudopodium in Amoeba 'proteus (Mast), g, plasmagel; he, hyaline cap; hi, hyaline layer; pi, plasmalemma; s, plasmasol. ates forming a new gel sheet. An amoeba is a turgid system, and the plasmagel is under continuous tension. The plasmagel is elastic and, consequently, is pushed out at the region where its elasticity is weakest and this results in pseudopodial formation. When an amoeba is elongated and undergoing movement, the elastic strength of the plasmagel is the highest at its sides, lowest at the anterior end and intermediate at the posterior end, which results in continuity of the elongated form and in extension of the anterior end. If pressure is brought against the anterior end, the direction of streaming of plas- masol is immediately reversed, and a new hyaline cap is formed at the posterior end which is thus changed into a new anterior end. " Flagellar movement. The flagellar movement is in a few instances observable as in Peranema, but in most cases it is very difficult to observe in life. Since there is difference in the number, location, size, and probably structure (p. 45) of flagella occurring in Protozoa, it is supposed that there are varieties of flagellar movements. The first explanation was advanced by Biitschli, who observed that the flagel- lum undergoes a series of lateral movements and, in so doing, a pres- PHYSIOLOGY 111 sure is exerted on the water at right angles to its surface. This pres- sure can be resolved into two forces: one directed parallel, and the other at right angles, to the main body axis. The former will drive the organism forward, while the latter will tend to rotate the animal on its own axis. Gray (1928), who gave an excellent account of the movement of flagella, points out that "in order to produce propulsion there must be a force which is always applied to the water in the same direction and which is independent of the phase of lateral movement. There can be little doubt that this condition is satisfied in flagellated organ- isms not because each particle of the flagellum is moving laterally to and fro, but by the transmission of the waves from one end of the flagellum to the other, and because the direction of the transmission is always the same. A stationary wave, as apparently contemplated by Biitschli, could not effect propulsion since the forces acting on the water are equal and opposite during the two phases of the move- ment. If however the waves are being transmitted in one direction only, definite propulsive forces are present which alwaj^s act in a direction opposite to that of the waves." Because of the nature of the flagellar movement, the actual proc- ess has often not been observed. Verworn observed long ago that in Peranema trichophomm the undulation of the distal portion of flagel- lum is accompanied by a slow forward movement, while undulation along the entire length is followed by a rapid forward movement. Recently Krijgsman (1925) studied Monas sp. (Fig. 46) which he found in soil cultures, under the darkfield microscope and stated: (1) when the organism moves forward with the maximum speed, the flagellum starting from cl, with the wave beginning at the base, stretches back (c 1-6), and then waves back (d, e), which brings about the forward movement. Another type is one in which the flagellum bends back beginning at its base (/) until it coincides with the body axis, and in its effective stroke waves back as a more or less rigid structure (g); (2) when the organism moves forward with moderate speed, the tip of the flagellum passes through 45° or less (h-j); (3) when the animal moves backward, the flagellum under- goes undulation which begins at its base (k-o) ; (4) when the animal moves to one side, the flagellum becomes bent at right angles to the body and undulation passes along it from its base to tip (p); and (5) when the organism undergoes a slight lateral movement, only the distal end of the flagellum undulates (q). Ciliary movement. The cilia are the locomotor organella present permanently in the ciliates and vary in size and distribution among 112 PROTOZOOLOGY different species. Just as flagellates show various types of move- ments, so do the ciliates. Individual cilium on a progressing ciliate bends throughout its length and strikes the water so that the organ- ism tends to move in a direction opposite to that of the effective beat, while the water moves in the direction of the beat (Fig. 47, Fig. 46. Diagrams illustrating ftagellar movements of Monas sp. (Krijgsman). a-g, rapid forward movement (a, b, optical image of the movement in front and side view; c, preparatory and d, e, effective stroke; f, preparatory and g, effective stroke); h-j, moderate forward movement (h, optical image; i, preparatory and j, effective stroke); k-o, undulatory movement of the fiagellum in backward movement; p, lateral movement; q, turning movement. PHYSIOLOGY 113 a-d). In the Protociliata and the majority of holotrichous and heterotrichous cOiates, the ciHa are arranged in longitudinal, or oblique rows and it is clearly noticeable that the cilia are not beating in the same phase, although they are moving at the same rate. A cilium (Fig. 47, e) in a single row is slightly in advance of the cilium behind it and shghtly behind the one just in front of it, thus the cilia on the same longitudinal row beat metachronously. On the other hand, the cilia on the same transverse row beat synchronously, the condition clearly being recognizable on Opahna among others, which is much like the waves passing over a wheat field on a wind}^ ,,i /'"^^ 7 1 2 w/'r.i^im^^^ii Fig. 47. Diagrams illustrating ciliary movements (Verworn). a-d, movement of a marginal cilium of Urostyla grandis (a, preparatory and b, effective stroke, resulting in rapid movement; c, preparatory, and d, effective stroke, bringing about moderate speed) ; e, metachronous move- ments of cilia in a longitudinal row. day. The organized movements of cilia, cirri, membranellae and un- dulating membranes are probably controlled by the neuromotor system (p. 54) which appears to be conductile as judged by the results of micro-dissection experiments of Taylor (p. 57). The Protozoa which possess myonemes are able to move by con- traction of the body or of the stalk, and others combine this with the secretion of mucous substance as is found in Haemogregarina and Gregarinida. Irritability Under natural conditions, the Protozoa do not behave always in the same manner, because several stimuli act upon them usually in combination and predominating stimulus or stimuU vary under dif- 114 PROTOZOOLOGY ferent circumstances. Many investigators have, up to the present time, studied the reactions of various Protozoa to external stimula- tions, full discussion of which is beyond the scope of the present work. Here one or two examples in connection with the reactions to each of the various stimuli will only be mentioned. Of various responses expressed by a protozoan against a stimulus such as changes in body form, movement, structure, behavior, etc., the movement is the most clearly recognizable one and, therefore, free- swimming forms, particularly ciliates, have been the favorite ob- jects of study. We consider the reaction to a stimulus in pratozoans as the movement response, and this appears in one of the two direc- tions: namely, toward, or away from, the source of the stimulus. Here we speak of positive or negative reaction. In forms such as Amoeba, the external stimulation is first received by the body sur- face and then by the whole protoplasmic body. In flagellated or ciliated Protozoa, the flagella or cilia act in part sensory; in fact in a number of cihates are found non-vibratile cilia which appear to be sensory in function. In a comparatively small number of forms, there are sensory organellae such as stigma, ocellus, statocysts, concretion vacuoles, etc. In general, the reaction of a protozoan to any external stimulus depends upon its intensity so that a certain chemical substance may bring about entirely opposite reactions on the part of the protozoans in different concentrations and, even under identical conditions, different individuals of a given species may react diffferently. Reaction to mechanical stimuli. One of the most common stimuli a protozoan would encounter in the natural habitat is that which comes from contact with a solid object. When an amoeba which Jennings observed, came in contact with the end of a dead algal filament at the middle of its anterior surface (Fig. 48, a), the amoe- boid movements proceeded on both sides of the filament (5), but soon motion ceased on one side, while it continued on the other, and the organism avoided the obstacle by reversing a part of the current and flowing in another direction (c). When an amoeba is stimulated mechanically by the tip of a glass rod (d), it turns away from the side touched, by changing endoplasmic streaming and forming new pseudopodia (e). Positive reactions are also often noted, when a suspended amoeba (/) comes in contact with a solid surface with the tip of a pseudopodium, the latter adheres to it by spreading out (g). Streaming of the cytoplasm follows and it becomes a creeping form (h). Positive reactions toward solid bodies account of course for the ingestion of food particles. PHYSIOLOGY 115 In Paramecium, according to Jennings, the anterior end is more sensitive than any other parts, and while swimming, if it comes in contact with a soHd object, the response may be either negative or positive. In the former case, avoiding movement (Fig. 49, c) follows and in the latter case, the organism rests with its anterior end or the whole side in direct contact with the object, in which position it ingests food particles through the cytostome. Fig. 48. Reactions of amoebae to mechanical stimuli (Jennings), a-c, an amoeba avoiding an obstacle; d, e, negative reaction to mechanical stimulation; f-h, positive reaction of a floating amoeba. Reaction to gravity. The reaction to gravity varies among dif- ferent Protozoa, according to body organization, locomotor organ- ellae, etc. Amoebae, Testacea and others which are usually found attached to the bottom of the container, react as a rule positively toward gravity, while others manifest negative reaction as in the case of Paramecium (Jensen; Jennings), which explains in part why Paramecium in a culture jar are found just below the surface film in mass, although the vertical movement of P. caudatum is undoubt- edl.y influenced by various factors, as was pointed out by Dem- bowski (1929). Reaction to current. Free-swimming Protozoa appear to move or orientate themselves against the current of water. In the case of Paramecium, Jennings observed the majority place themselves in line with the current, with anterior end upstream. The mycetozoan is said to exhibit also a well-marked positive reaction. 116 PROTOZOOLOGY Reaction to chemical stimuli. When methylgreen, methylene blue, or sodium chloride is brought in contact with an advancing amoeba, the latter organism reacts negatively (Jennings). Jen- nings further observed various reactions of Paramecium against chemical stimulation. This ciliate shows positive reaction to weak solutions of many acids and negative reactions above certain con- centrations. For example, Paramecium enters and stays wdthin the >M:^^M«. Fig. 49. Reactions of Paramecium (Jennings), a, collecting in a drop of 0.02% acetic acid; b, ring-formation around a drop of a stronger solu- tion of the acid; c, avoiding reaction. area of a drop of 0.02 per cent acetic acid introduced to the prepara- tion (Fig. 49, a) ; and if stronger acid is used, the organisms collect about its periphery where the acid is diluted by the surrounding water (Fig. 49, h). The reaction to chemical stimuli is probably of the greatest importance for the existence of Protozoa, since it leads them to proper food substances, the ingestion of which is the found- ation of metabolic activities. In the case of parasitic Protozoa, possibly the reaction to chemical stimuli results in their finding specifi.c host animals and their distribution in different organs and tissues within the host body. Recent investigations tend to indicate that chemotaxis plays an important role in the sexual reproduction in Protozoa. PHYSIOLOGY 117 Reaction to light stimuli. Most Protozoa seem to be indifferent to the ordinary light, but when the light intensity is suddenly in- creased, there is usually a negative reaction. Verworn saw the di- rection of movements of an amoeba reversed when its anterior end was subjected to a sudden illumination; Rhumbler observed that an amoeba, which was in the act of feeding, stopped feeding when it was subjected to strong light. According to Mast, Amoeba pro- tens ceases to move when suddenly strongly illuminated, but con- tinues to move if the increase in intensity is gradual and if the il- lumination remains constant, the amoeba begins to move. According to Jennings, Stentor coeruleus reacts negatively against light. The positive reaction to light is most clearly shown in stigma- bearing Mastigophora, as is well observable in a jar containing Euglena, Phacus, etc., in which the organisms collect at the place where the light is strongest. If the light is excluded completely, the organisms become scattered throughout the container, inac- tive and sometimes encyst, although the mixotrophic forms would continue activities by saprozoic method. The positive reaction to light by chromatophore-bearing forms enables them to find places in the water where photosynthesis can be carried on to the maximum degree. All Protozoa seem to be more sensitive to ultraviolet rays. Inman found that amoeba shows a greater reaction to the rays than others and Hertel observed that Paramecium which was indifferent to an ordinary light, showed an immediate response (negative reaction) to the rays. MacDougall brought about mutations in Chilodonella by means of these rays (p. 181). When ciliates are vitally stained with eosin, erythrosin, etc., they react sometimes positively or negatively as in Paramecium (Metzner), or always negatively, as in Spiro- stomum(Blattner). According toEfimoff, this "induced phototaxis" is not limited to fluorescent dyes, but also is possessed by all vital- staining dyes. Zuelzer (1905) studied the effects of radium rays upon various Protozoa and found that the effect was not the same among different species. For example, limax amoeba was more resistant than others. In all cases, however, long exposure to the rays was fatal to Protozoa, the first ejffect of exposure being shown by accele- rated movement. Halberstaedter and Luntz (1929) studied injuries and death of Eudorina elegans by exposure to radium rays. Joseph and Prowazek (1902) found Paramecium and Volvox gave negative response to the rontgen-ray. Reaction to temperature stimuli. As was stated before, there seems to be an optimum temperature range for each protozoan, 118 PROTOZOOLOGY although it can withstand temperatures which are lower or higher than that range. As a general rule, the higher the temperature, the greater the metabolic activities, and the latter condition results in turn in a more rapid growth and more frequent reproduction. It has been suggested that change to different phases in the life-cycle of a protozoan in association with the seasonal change may be largely due to temperature changes of the environment. 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L. 1938 The vacuoles and vacuolar activity in the marine amoeba, Flabellida mira Schaeffer and the nature of the neutral red system in Protozoa. Biodynamica, No. 34. HowLAND, R. B. 1928 The pH of gastric vacuoles. Protoplasma, Vol. 5. and A. Bernstein 1931 A method for determining the oxy- gen consumption of a single cell. Jour. Gen. Physiol., Vol. 14. HuLPiEU, H. R. 1930 The effect of oxygen on Amoeba proteus. Jour. Exp. Zool., Vol. 56. Hyman, L. H. 1917 Metabolic gradients in amoeba and their rela- tion to the mechanism of amoeboid movement. Jour. Exp. Zool., Vol. 24. Jennings, H. S. 1904 Contributions to the study of the behavior of the lower organisms. Publ. Carnegie Inst. Washington, No. 16. 1906 Behavior of the lower organisms. New York. 120 PROTOZOOLOGY Johnson, W. H. 1941 Nutrition in the Protozoa. Quart. Rev. Biol., Vol. 16. and E. G. S. Baker 1943 Certain B vitamins on popula- tions of Tetrahymena geleii. Physiol. Zool., Vol. 16. Khainsky, a. 1910 Zur Morphologie unci Physiologic einiger In- fusorien (Paramecium caudaium) auf Grund einer neuen his- tologischen Methode. Arch. f. Protistenk., Vol. 21. Kidder, G. W., D. M. Lilly and C. L. Claff. 1940 Growth stud- ies on ciliates. IV. Biol. Bull., Vol. 78. KiRBY, H. Jr., 1934 Some ciliates from salt marshes in California. Arch. f. Protistenk., Vol. 82. KoFoiD, C. A. and O. Swezy 1921 The free-living unarmored Dino- fiagellata. Mem. Univ. Calif., Vol. 5. Krijgsman, B. J. 1925 Beitrage zum Problem der Geisselbe- wegung. Arch. f. Protistenk., Vol. 52. Kudo, R. R. 1921 On the nature of structures characteristic of cnidosporidian spores. Trans. Amer. Micr. Soc, Vol. 40. Lilly, D. M. 1942 Nutritional and supplementary factors in the growth of carnivorous ciliates. Physiol. Zool., Vol. 15. Mast, S. O. 1923 Mechanics of locomotion in amoeba. Proc. Nat. Acad. Sci., Vol. 9. 1926 Structure, movement, locomotion, and stimulation in amoeba. Jour. Morph. Physiol., Vol. 41. 1931 Locomotion in Amoeba proteus. Protoplasma, Vol. 14. 1938 Digestion of fat in Amoeba proteus. Biol. Bull., Vol. 75. 1939 The relation between kind of food, growth, and struc- ture in Amoeba. Ibid., Vol. 77. 1942 The hydrogen ion concentration of the content of the food vacuoles and the cytoplasm in Amoeba and other phe- nomena concerning the food vacuoles. Biol. Bull., Vol. 83. and W. L. Doyle 1934 Ingestion of fluid by amoeba. Ibid., Vol. 20. 1935 Structure, origin and function of cytoplasmic constituents in Amoeba proteus with special reference to mito- chondria and Golgi substance. Arch. f. Protistenk., Vol. 86. Meldrum, N. U. 1934 Cellular respiration. London. Metalnikoff, S. 1912 Contribution a I'etude de la digestion intra- cellulaire chez les protozoaires. Arch. zool. exp. (ser. 5), Vol. 9. Mouton, H. 1902 Recherches sur la digestion chez les amibes et sur leur diastase intracellulaire. Ann. Inst. Pasteur. Vol. 16. Nirenstein, E. 1925 Ueber die Natur und Starke der Saurebildung in den Nahrungsvakuolen von Paramecium caudatum. Zeitschr. wiss. Zool., Vol. 125. Noland, L. E. 1927 Conjugation in the ciliate Metopus sigmoides. Jour. Morph. Physiol., Vol. 44. Pantin, C. F. a. 1923 On the physiology of amoeboid movement I. Jour. Marine Biol. Assn. Plymouth, N. S., Vol. 13. Panzer, T. 1913 Beitrag zur Biochemie der Protozoen. Hoppe- Seylers Zeitschr. phys. Chemie, Vol. 86. Powers, P. B. A. 1932 Cyclotrichium meunieri sp. nov. ; cause of red water in the gulf of Maine. Biol. Bull., Vol. 63. PHYSIOLOGY 121 Pratjb, a. 1921 Makrochemische, quantitative Bestimmung des Fettes und Cholesterins, sowie ihrer Kennzahlen bei Noctiluca miliaris. Biol. Zentralbl., Vol. 21. Pringsheim, E. G. 1923 Zur Physiologie saprophytischer Flagella- ten. Beitr. allg. Bot., Vol. 2. Putter, A. 1905 Die Atmung der Protozoen. Zeitschr. allg. Physiol, Vol. 5. 1908 Methoden zur Erforschung des Lebens der Protistenk. Tigerstedt's Handb. physiol. Methodik, Vol. 1. RosKiN, G. and L. Levinsohn 1926 Die Oxydasen und Peroxy- dasen bei Protozoen. Arch. f. Protistenk., Vol. 56. Rhumbler, L. 1910 Die verschiedenartigen Nahrungsaufnahmen bei Amoeben als Folge verschiedener Colloidalzustande ihrer Oberflachen. Arch. Entw. Organism., Vol. 30. Sassuchin, D. N. 1935 Zum Studium der Protisten- und Bak- terien-kerne. Arch. f. Protistenk., Vol. 84. Schaeffer, a. a. 1920 Amoeboid movement. Princeton. ScHEWiAKOFF, W. 1894 Ueber die Natur der sogenannten Exkret- korner der Infusorien. Zeitschr. wiss. Zool., Vol. 57. ScHOPFER, W. H. (translated by N. L. Noecker). 1943 Plants and vitamins. Waltham, Mass. Shapiro, N. N. 1927 The cycle of hydrogen-ion concentration in the food vacuoles of Paramecium, Vorticella, and Stylonychia. Trans. Amer. Micro. Soc, Vol. 46. SouLE, M. H. 1925 Respiration of Trypanosoma Icwisi and Leish- mania tropica. Jour. Infec. Dis., Vol. 36. Stolc, a. 1900 Beobachtungen und Versuche ueber die Verdauung und Bildung der Kohlenhydrate bei einen amoebenartigen Or- ganismen, Pclomyxa palustris. Zeitschr. wiss. Zool. Vol. 68. Verworn, M. 1889 Psycho-physiologische Protisten-studien. Jena. 1903 Allgemeine Physiologie. 4te Aufl. Jena. Weatherby J. H. 1929 Excretion of nitrogenous substances in Pro- tozoa. Physiol. Zool., Vol. 2. Whipple, G. C. 1927 The microscopy of drinking water. 4 ed. New York. Woodruff, L. L. and W. W. Swingle 1924 The effects of thyroid and some other endocrine products on Paramecium. Amer. Jour. Physiol., Vol. 69. Zuelzer, M. 1907 Ueber den Einfluss des Meerwassers auf die pul- sierende Vacuole. Berhn. Sitz.-Ber. Ges. naturf. Freunde. Zumstein, H. 1900 Zur Morphologic und Physiologie der Euglena gracilis Klebs. Pringsheims Jahrb. wiss. Botanik, Vol. 34. Chapter 5 Reproduction THE mode of reproduction in Protozoa is highly variable among different groups, although it is primarily a cell division. The reproduction is initiated by the nuclear division in all cases, which will therefore be considered first. Nuclear division Between a simple direct division on the one hand and a com- plicated indirect division which is comparable with the typical metazoan mitosis on the other hand, all types of nuclear division occur. Direct nuclear division. Although not so widely found as it was thought to be in former years, amitosis occurs normally and regu- larly in many forms. While the micronuclear division of the Cilio- phora is mitotic (p. 137), the macronuclear division is invariably amitosis. The sole exception to this general statement appears to be the so-called promitosis reported by Ivanic (1938) in the macro- nucleus in the "Vermehrungsruhe" stage of Chilodenella uncinata in which chromosomes and spindle-fibers were observed. In Para- mecium caudatum (Fig. 50), the micronucleus initiates the division by mitosis and the macronucleus elongates itself without any visible changes in its internal structure. The elongated nucleus becomes constricted through the middle and two daughter nuclei are pro- duced. It is assumed that the nuclear components undergo solation during division, since the formed particles of nucleus which are stationary in the resting stage, manifest a very active Brownian movement as was observed in vivo in Endamoeba blattae (Fig. 51). Furthermore, in some cases the nuclear components may undergo phase reversal, that is to say, the chromatin granules which are dispersed phase in the non-staining fluid dispersion medium in the resting nucleus, be- come dispersion medium in which the latter is suspended as dis- persed phase. By using Feulgen's nucleal reaction, Reichenow (1928) demonstrated this reversal phenomenon in the division of the macronucleus of Chilodonella cucullulus (Fig. 52). The macronucleus becomes at the time of its division somewhat enlarged and its chromatin granules are more deeply stained than before. But chromosomes which characterize the mitotic division are entirely absent, although in a few forms in which mating types 122 REPRODUCTION 123 occur, the type difference and certain other characters, according to Sonneborn and Kimball, appear to be under control of genie consti- tuents of the macronucleus. Since the number of chromatin granules appear approximately the same in the macronuclei of different gen- erations of a given species, the reduced number of chromatin gran- FiG. 50. Nuclear and cytosomic division of Paramecium caudatum as seen in stained smears, X260 (Kudo). ules must be restored sometime before the next division takes place. Calkins (1926) is of the opinion that "each granule elongates and divides into two parts, thus doubling the number of chromomeres." Reichenow (1928) found that in Chilodonella cucullulus the lightly Feulgen positive endosome appeared to form chromatin granules and Kudo (1936) maintained that the large chromatin spherules of Fig. 51. Division of Endamoeba blattae as seen in life, X250 (Kudo). The entire process took one hour and seven minutes. Fig. 52. The solation of chromatin during the macronuclear division of Chilodonella cucullulus, as demonstrated by Feulgen's nucleal reaction, Xl800(Reichenow). REPRODUCTION 125 the macronucleus of Nyctotherus ovalis probablj^ produce smaller spherules in their alveoli. When the macronucleus is elongated as in Spirostomum, Stentor, Euplotes, etc., the nucleus becomes condensed into a rounded form prior to its division. When the macronuclear material is distrib- uted throughout the cytoplasm as numerous grains as in Dileptus anser (Fig. 239, c), ''each granule divides where it happens to be and with the majority of granules both halves remain in one daugh- ter cell after division" (Calkins). Hayes noticed a similar division, but at the time of simultaneous division prior to cell division, each macronucleus becomes elongated and breaks into several small nuclei. During the "shortening period" of the elongated macronuclei prior to division, there appear 1-3 characteristic zones which have Macronuclear reorganization before division in Euplotes X240 (Turner), a, reorganization band appearing at a tip of the macronucleus; b-d, later stages. been called by various names, such as nuclear clefts, reconstruction bands, reorganization bands, etc. In Euplotes patella (E. eurystomus) , Turner (1930) observed prior to division of the macronucleus a re- organization band consisting of a faintly staining zone ("recon- struction plane") and a deeply staining zone ("solution plane"), ap- pears at each end of the nucleus (Fig. 53, a) and as each moves to- ward the center, a more chromatinic area is left behind (b-d). The two bands finally meet in the center and the nucleus assumes an ovoid form. This is followed by a simple division into two. In the T-shaped macronucleus of E. woodruffi, according to Pierson (1943), a reorganization band appears first in the right arm and the posterior tip of the stem of the nucleus. When the anterior band reaches the junction of the arm and stem, it splits into two, one part moving along the left arm to its tip, and the other entering and passing down 126 PROTOZOOLOGY the stem to join the posterior band. According to Summers (1935) a process similar to that of E. eurystomus occurs in Diophrys ap- pendiculata and Stylonychia pustulata; but in Aspidisca lynceus (Fig. 54) a reorganization band appears first near the middle region of the macronucleus (6), divide into two and each moves toward an end, leaving between them a greater chromatinic content of the Fig. 54. Macronuclear reorganization prior to division in Aspidisca lynceus, X1400 (Summers), a, resting nucleus; b-i, successive stages in reorganization process; j, a daughter macronucleus shortly after division. reticulum (c-i). Summers suggested that "the reorganization bands are local regions of karyolysis and resynthesis of macronuclear materials with the possibility of an elimination of physically or possibly chemically modified nonstaining substances into the cyto- plasm." The extrusion of a certain portion of the macronuclear material during division has been observed in a number of species. In Urolep- tus halseyi, Calkins actually noticed each of the eight macronuclei REPRODUCTION 127 is "purified" by discarding a reorganization band and an "x-body" into the cytoplasm before fusing into a single macronucleus which then divides into two nuclei. In the more or less rounded macro- nucleus that is commonly found in many ciliates, no reorganization band has been recognized. A number of observers have however noted that during the nuclear division there appears and persists a small body within the nuclear figure, Ipcated at the division plane as in the case of Loxocephalus (Behrend), Eupoterion (MacLennan and 't^M^. Fig. 55. Macronuclear division in Coyichophlhirus mytili, X440 (Kidder). Connell) and even in the widely different protozoan, Endamoeha blattae (Kudo) (Fig. 51). We owe Kidder for a careful comparative study of this body. Kidder (1933) observed that during the division of the macronucleus of Conchophthirus mytili (Fig. 55), the nucleus "casts out a part of its chromatin at every vegetative division," which "is broken down and disappears in the cytoplasm of either daughter organism." A similar phenomenon has since been found further in C. anodontae, C. curtus, C. magna (Kidder), Urocentrum turbo, Colpidium colpoda, C. campylum, Glaucoma scintillans (Kidder and Diller), and Allosphaerium convexa (Kidder and Summers). Kidder and his associates believe that the process is probably 128 PROTOZOOLOGY elimination of waste substances of the prolonged cell-division, since chromatin extrusion does not take place during a few divisions sub- sequent to reorganization after conjugation in Conchophthirus mytili and since in Colpidium and Glaucoma, the chromatin elimination appears to be the cause of high division rate and infrequency of con- jugation. Woodruff and Erdmann (1914)^ observed that in Pararnecium aurelia (Fig. 56, a) at regular intervals of about 30 days, the old Fig. 56. Diagram showing the endomixis in Paramecium aurelia (Wood- ruff), a, normal individual; b, degeneration of macronucleus and first micronuclear division; c, second micronuclear division; d, degeneration of 6 micronuclei; e, cell division; f, g, first and second reconstruction micro- nuclear divisions; h, transformation of 2 micronuclei into 2 macronuclei; i, micronuclear and cell divisions; j, typical nuclear condition is restored. macronucleus breaks down and disappears, while each of the two micronuclei divides twice, forming eight nuclei (b, c). Of these, six disintegrate. At this point the organism divides into two, each daughter individual receiving one micro nucleus {d, e). This nucleus soon divides twice into four, two of which develop into macronuclei if-h), and the other two divide again. Here the organisms divide once more by binary fission (i), each bearing cne macronucleus and two micronuclei (j). This process which is "a complete periodic nuclear reorganization without cell fusion in a pedigreed race of Parame- REPRODUCTION 129 cium" was called by the two authors endomixis. In the case of P. caudatum, they found endomixis occurs at intervals of about 60 days. Endomixis has since been reported in Spathidium spathula, Euplotes longipes, Chilodonella uncinata, Didinium nasutum, Para- mecium multimicronucleatum, Urostyla grandis, Paraclevelandia sim- plex, etc. It appears to be another process of nuclear reorganization. As has already been stated, two types of nuclei: macronucleus and micronucleus, occur in Euciliata and Suctoria. The macro- nucleus is the center of the whole metabolic activity of the organism and in the absence of this nucleus, the animal perishes. The waste substances which become accumulated in the macronucleus through its manifold activities, are apparently eliminated at the time of division, as has been cited above in many species. On the other hand, it is also probable that under certain circumstances, the macro- nucleus becomes impregnated with waste materials which cannot be eliminated through this process. Prior to and during conjugation (p. 154) and autogamy (p. 161), the macronucleus becomes trans- formed, in many species, into irregularly coiled thread-like sti;ucture (Fig. 79) which undergoes segmentation into pieces and finally is absorbed by the cytoplasm. New macronuclei are formed from some of the division-products of micronuclei (synkarya) by probably in- corporating the old macronuclear material. In most cases this sup- position is not demonstrable. However, Kidder (1938) has shown in the encysted Paraclevelandia simplex, an endocommensal of the colon of certain wood-feeding roaches, this is actually the case; namely, one of the divided micronuclei fuses directly with a part of macronucleus (endomixis) to form a macronuclear anlage which then develops into a macronucleus after passing through "ball-of- yarn" stage similar to that which appears in an exconjugant of Nyctotherus (Fig. 79). Since the macronucleus originates in a micronucleus, it must con- tain all structures which characterize the micronucleus. Why then does it not divide mitotically as does the micronucleus? During conjugation or autogamy in a ciliate, the macronucleus degenerates, disintegrates and finally becomes absorbed in the cytoplasm. In Paramecium aurelia, Sonneborn (1940, 1942) observed that in amicronucleate animals or when the micronuclei fail to give rise to macronuclei, many (40 or more) pieces of the disintegrated old macronucleus do not degenerate, but instead regenerate into new macronuclei, which are segregated out to daughter individuals formed at successive divisions, until one such regenerated macro- nucleus is present in each individual. These macronuclei grow and 130 PROTOZOOLOGY behave in the same way as do those which arise from microniiclei. Thus the macronucleus in this ciHate appears to be a compound structure with its 40 or more component parts, each containing all that is needed for development into a complete macronucleus. From these observations, Sonneborn concludes that the macronucleus in P. aurelia appears to undergo amitosis, since it is a compound nu- cleus composed of 40 or more "sub-nuclei" and since at fission all that is necessary to bring about genetically equivalent functional macronuclei is to segregate these multiple subnuclei into two random groups. a b c d e Fig. 57. Amitosis of the vegetative nucleus in the trophozoite of Myxosoma catostonii, X2250 (Kudo). Other examples of amitosis are found in the vegetative nuclei in the trophozoite of Myxosporidia, as for example, Myxosoma catos- tomi (Fig. 57), Thelohanellus notatus (Debaisieux), etc., in which the endosome divides first, followed by the nuclear constriction. In Strehlomastix strix, the compact elongated nucleus was found to undergo a simple division by Kof oid and Swezy. Indirect nuclear division. The indirect division which occurs in the protozoan nuclei is of manifold types as compared with the mitosis in the metazoan cell, in which, aside from minor variations, the change is of a uniform pattern. Chatton, Alexeieff and others, have proposed several terms to designate the various types of indirect nuclear division, but no one of these types is sharply defined. For our purpose, mentioning of examples will suffice. A veritable mitosis was noted by Dobell in the heliozoan Oxnerella maritima (Fig. 58), which possesses an eccentrically situated nucleus containing a large endosome and a central centriole, from which radiate many axopodia (a). The first sign of the nuclear division is the slight enlargement, and migration toward the centriole, of the nucleus (6). The centriole first divides into two (c, d) and the nucleus becomes located between the two centrioles (e). Presently spindle fibers are formed and the nuclear membrane disappears (/, g) . After passing through an equatorial-plate stage, the two groups of 24 chromosomes move toward the opposite poles {g-4). As the spindle fibers become indistinct, radiation around the centrioles becomes REPRODUCTION 131 conspicuous and the two daughter nuclei are completely recon- structed to assume the resting phase (j-l). The mitosis of another heliozoan Acanthocystis aculeata is, according to Schaudinn and Stern, very similar to the above. Aside from these two species, the centriole has been reported in many others, such as Hartmanella Fig. 58. Nuclear and cytosomic division in Oxnerella maritima, X about 1000 (Dobell). a, a living individual; b, stained specimen; c-g, prophase; h, metaphase; i, anaphase; j, k, telophase; 1, division completed. (Arndt), Euglypha, Monocystis (Belaf), Aggregata (Dobell; Belaf; Naville), various Hypermastigina (Kofoid; Duboscq, Grasse; Kirby; Cleveland and his associates). In numerous species the division of the centriole (or blepharo- plast) and a connecting strand between them, which has been called desmose (centrodesmose or paradesmose), have been observed. Ac- 132 PROTOZOOLOGY Fig. 59. Mitosis in Trichonympha campanula, XSOO (Kofoid and Swezy). a, resting nucleus; b-g, prophase; h, metaphase; i, j, anaphase; k, telophase; 1, a daughter nucleus being reconstructed. cording to Kofoid and Swezy (1919), in Trichonympha campanula (Fig. 59), the prophase begins early, during which 52 chromosomes are formed and become split. The nucleus moves nearer the anterior end where the centriole divides into two, between which develops a I REPRODUCTION 133 desmose. From the posterior end of each centriole; astral rays extend out and the spHt chromosomes form loops, pass through "tangled skein" stage, and emerge as 26 chromosomes. In the metaphase, the equatorial plate is made up of V-shaped chromosomes as each of the Fig. 60. Development of spindle and astral rays during the mitosis in Barbulanympha, X930 (Cleveland), a, interphase centrioles and centro- somes; b, prophase centrioles with astral rays developing from their distal ends through the centrosomes; c, meeting of astral rays from two cen- trioles; d, astral rays developing into the early central spindle; e, a later stage showing the entire mitotic figure. split chromosomes are still connected at one end, which finally be- comes separate in anaphase, followed by reformation of two daugh- ter nuclei. 134 PROTOZOOLOGY As to the origin and development of the achromatic figure, vari- ous observations and interpretations have been advanced. Certain Hypermastigina po.ssess very large filiform centrioles and a large rounded nucleus. In Barbulanympha (Fig. 60), Cleveland (1938a) found that the centrioles vary from 15 to 30/x in length in the four species of the genus which he studied. They can be seen, according to Cleveland, in life as made up of a dense hyaline protoplasm. When stained, it becomes apparent that the two centrioles are joined at their anterior ends by a desmose and their distal ends 20 to 30m apart, each of which is surrounded by a special centrosome (a). In the resting stage no fibers extend from either centriole, but in the prophase, astral rays begin to grow out from the distal end of each centriole (6). As the rays grow longer (c), the two sets soon meet and the individual rays or fibers join, grow along one another and over- lap to form the central spindle (d). In the resting nucleus, there are large irregular chromatin granules which are connected by fibrils with one another and also with the nuclear membrane. As the achro- matic figure is formed and approaches the nucleus, the chromatin be- comes arranged in a single spireme imbedded in matrix. The spireme soon divides longitudinally and the double spireme presently breaks up transversely into paired chromosomes. The central spindle begins to compress the nuclear membrane and the chromosomes become shorter and move apart. The intra- and extra-nuclear fibrils unite as the process goes on (e), the central spindle now assumes an axial position, and two groups of V-shaped chromosomes are drawn to opposite poles. In the telophase, the chromosomes elongate and be- come branched, thus assuming conditions seen in the resting nucleus. In the unique resting nucleus of Spirotrichomjmpha polygyra (Fig. 61), Cleveland (1938) found four chromosomes, each of which con- tains a distinct coil within a sheath and its one end connected with the anterior margin of the nuclear membrane by an intranuclear chromosomal fiber, and the other end with a deeply staining endo- some (a). The spindle fibers appear between the separating flagellar bands which come in contact with the nuclear membrane. Soon some of the astral rays become connected with the intranuclear chromosomal fibers and one long and one short chromosomes which become thicker and shorter move toward each pole. During the telo- phase, each chromosome splits lengthwise and forms the resting nucleus (g). In Lophomonas hlattarum, the nuclear division (Fig. 62) is initiated by the migration of the nucleus out of the calyx. On the nuclear membrane is attached the centriole which probably originates in the REPRODUCTION 135 blepharoplast ring; the centriole divides and the desmose which grows, now stains very deeply, the centrioles becoming more con- spicuous in the anaphase when new flagella develop from them. Chromatin granules become larger and form a spireme, from which 6-8 chromosomes are produced. Two groups of chromosomes move toward the opposite poles, and when the division is completed, each centriole becomes the center of formation of all motor organellae. Fig. 61. Mitosis in Spirotrichonympha polygyra (Cleveland), a, resting nucleus with 4 chromosomes; b, c, prophase; d, chromosomes moving apart; e, elongation of nucleus; f, telophase; g, a daughter nucleus in which the chromosomes are splitting, a-e, X3800; f, g, X2400. In some forms, such as Noctiluca (Calkins), Actinophrys(Bglaf), etc., there may appear at each pole, a structureless mass of cyto- plasm (centrosphere), but in a very large number of species there appear no special structures at poles and the spindle fibers become stretched seemingly between the two extremities of the elongating 136 PROTOZOOLOGY nuclear membrane. Such is the condition found in Cryptomonas (Belaf), Rhizochrysis (Doflein), Aulacantha (Borgert), and in micro- nuclear division of the majority of Euciliata and Suctoria. The behavior of the endosome during the mitosis differs among different species as are probably their functions. In Eimeria schuhergi (Schaudinn), Euglena viridis (Tschenzoff), Oxyrrhis marina (Hall), Colacium vesiculosum (Johnson), Haplosporidium limnodrili (Gran- FiG. 62. Nuclear division in Lophomonas hlattarum, X1530 (Kudo), a, resting nucleus; b, c, prophase; d, metaphase; e-h, anaphase; i-k, telo- phase. ata), etc., the conspicuously staining endosome divides by elongation and constriction along with other chromatic elements, but in many other cases, it disappears during the early part of division and reap- pears when the daughter nuclei are reconstructed as observed in Monocystis, Dimorpha, Euglypha, Pamphagus (Belaf), Acantho- cystis (Stern), Chilomonas (Doflein), Dinenympha (Kirby), etc. In the vegetative division of the micronucleus of Conchophthirus anodontae (Fig. 63), Kidder (1934) found that prior to division the micronucleus moves out of the pocket in the macronucleus and the REPRODUCTION 137 chromatin becomes irregularly disposed in a reticulum; swelling continues and the chromatin condenses into a twisted band, a spireme, which breaks into many small segments, each composed of large chromatin granules. With the rapid development of the spindle fibers, the twelve bands become arranged in the equatorial plane and condense. Each chromosome now splits longitudinally and two groups of 12 daughter chromosomes move to opposite poles and m m feSsS^iaSt Fig. 63. Mitosis of the micronucleus of Conchophthirus anodontae, X2640 (Kidder), a-c, prophase; d, e, metaphase; f, g, anaphase; h, i, telophase. transform themselves into two compact daughter nuclei. In Zeller- iella intermedia (Fig. 64), Chen (1936) saw the formation of 24 chromosomes, each of which is connected with a fiber of the intra- nuclear spindle and splits lengthwise in the metaphase. While in the majority of protozoan mitosis, the chromosomes split longitudinally, there are observations which suggest a transverse division. As exam- 138 PROTOZOOLOGY pies may be mentioned the chromosomal divisions in Astasia laevis (Belaf), Entosiphon sulcatum (Lackey), and a number of ciliates. In a small number of species observations vary within a species, as, for example, in Peranema trichophorum in which the chromosomes were observed to divide transversely (Hartmann and Chagas) as well as longitudinally (Hall and Powell; Brown). It is inconceivable that the division of the chromosome in a single species of organism is haphazard. The apparent transverse division might be explained Fig. 64. Stages in mitosis in Zelleriella inter iriedia, X1840 (Chen), a, early prophase; b, metaphase; c, anaphase; d, telophase. by assuming, as Hall (1937) showed in Euglena gracilis, that the splitting is not completed at once and the pulling force acting upon them soon after division brings forth the long chromosomes still connected at one end. Thus the chromosomes remain together before the anaphase begins. In the instances considered on the preceding pages, the so-called chromosomes found in them, appear to be essentially similar in structure and behavior to typical metazoan chromosomes. In many other cases, the so-called chromosomes or "pseudochromosomes" are slightly enlarged chromatin granules which differ from the ordin- ary chromatin granules in their time of appearance and movement only. In these cases it is of course not possible at present to deter- mine how and when their division occurs before separating to the respective division pole. In Table 5 are listed the number of the "chromosomes" which have been reported by various investigators in the Protozoa that are mentioned in the present work: REPRODUCTION Table 5. — Chromosomes in Protozoa 139 Protozoa Number of chromosomes Observers Rhizochrysis scherffeli 22 Doflein H aematococcus pluvialis 20-30 Elliott Polytomella agilis 5 Doflein Chlamydomonas spp. 10 (haploid) Pascher Polytoma uvella 16 (diploid); 8 (haploid) Moewus Euglena pisciformis 12-15(?) Dangeard E. viridis 30 or more Dangeard Phacus pyrurn 30-40 Dangeard Menoidium incurimm About 12 Hall Vacuolaria virescens About 30 Fott Syndinium turbo 5 Chatton Anthophysis vegetans 8-10 Dangeard Gercomonas longicauda 4-5 Dangeard Collodictyon triciliatum About 20 Belaf Chilomastix gallinarum About 12 Boeck and Tanabe Eidrichomastix serpentis 5 Kofoid and Swezy Dinenympha fimbricata 25-30 Kirby Metadevescovina debilis About 4 Light Trichomonas elongatum 3 Hinshaw Tritrichomonas batrachorum 4 or 8 Kuczynski 6 Bishop T. augusta 5 Kofoid and Swezy 4 or 8 Kuczynski Hexamita salmonis 5 or 6 Davis Giardia intestinalis 4 Kofoid and Swezy G. muris 4 Kofoid and Christiansen Colony mpha grassii 4 or 5 Janicki Spirotrichonympha polygyra 2 doubles Cup 2 Cleveland Lophomonas blattarum 16 or 8 doubles Janicki 8 or 6 Kudo 12 or 6 doubles Belaf L. striata 12 or 6 doubles Belaf Barbtdanynipha laurabuda 40 Cleveland B. uf alula 50 Cleveland Rhynchonympha tarda 19 Cleveland Urinympha talea 14 Cleveland Staurojoenia assimilis 24 Kirby Trichonympha campanula 52 or 26 doubles Kofoid and Swezy T. grandis 22 Cleveland Plasmodiophora brassicae 8 (diploid); 4 (haploid) Terby Dimastig amoeba bistadialis 16-18 Kuhn 140 PROTOZOOLOGY Table 5. — Continued Protozoa Number of chromosomes Observers Endamoeba disparata About 12 Kirby Entamoeba histolytica 6 Kofoid and Swezy; Uribe E. coli 6 Swezy; Stabler E. gingivalis 5 Stabler Dientamoeba fragilis 4 Wenrich 6 Dobell Hydramoeba hydroxena 8 Reynolds and Threlkeld Spirillina vivipara 12 (diploid); 6 (haploid) Myers Patellina corrugata 24 (diploid) ; 12 (haploid) Myers Pontigulasia vas 8-12 Stump Actinophrys sol 44 (diploid); 22 (haploid) Belaf Oxnerella maritima About 24 Dobell Thalassicolla nucleata 4 Belaf Aulacantha scolymantha More than 1600 Borgert 4 in gamogony Belaf Zygosoma globosum 12 (diploid); 6 (haploid) Noble Diplocystis schneideri 6 (diploid) ; 3 (haploid) Jameson Gregarina blattarum 6 (diploid) ; 3 (haploid) Sprague Nina gracilis 5 (haploid) Leger and Duboscq Actinocephalus parvus 8 (diploid); 4 (haploid) Weschenfelder Aggregata eberthi 12 (diploid); Dobell and Jameson; 6 (haploid) Belaf; Naville Merocystis kathae 6 (haploid) Patten Adelea ovata 8-10 (diploid); 4-5 (haploid) Greiner Adelina deronis 20 (diploid) ; 10 (haploid) Hauschka Orcheobius herpobdellae 10-12 Kunze Chloromyxum leydigi 4 (diploid) ; 2 haploid) Naville Sphaerospora polymorphc I 4 (diploid); 2 (haploid) Kudo Myxidium lieberkuhni 4 Bremer M. serotinum 4 (diploid) ; 2 (haploid) Kudo Sphaeromyxa sabrazesi 6 Debaisieux; Belaf 4 Naville S. balbianii 4 Naville REPRODUCTION 141 Table 5. — Continued Protozoa Number of chromosomes Observers Myxobolus pfeifferi 4 Keysselitz; Mercier; Georgevitch Protoopalina intestinalis 8 (diploid); 4 (haploid) Metcalf Zelleriella antilliensis 2(?) Metcalf Z. intermedia 24 Chen Didinium nasutum 16 (diploid); 8 (haploid) Prandtl Chilodonella uncinata 4 (dilpoid); 2 (haploid) Enrique; MacDougall C. uncinata (tetraploid) 8; 4 MacDougall Conchophthirus anodontae 12 (diploid) Kidder C. mytili 16 (diploid); 8 (haploid) Kidder Ancistruma isseli About 5 (haploid) Kidder Paramecium aurelia 30-40 Diller Stentor coeruleus 28 (diploid) ; 14 (haploid) Mulsow Oxytricha fallax 24 (diploid) ; 12 (haploid) Gregory Uroleptus halseyi 24 (diploid); 12 (haploid) Calkins Pleurotricha lanceolata About 40 (dipl.); 20 (haploid) Manwell Stylonychia pustulata 6 Prowazek Euplotes patella 6 (diploid) Yocom; Ivanic 8 (diploid) ; Turner 4 (haploid) Carchesium polypinum 16 (diploid); 8 (haploid) Popoff Trichodina sp. 4-6 Diller In man}^ other Protozoa, the division figure, especially the achromatic figure, suggests strongly a mitosis, but the chromatin substance which makes up the equatorial plate can hardly be called chromosomes. A typical example of this type is found in the nuclear division of Amoeba proteus (Fig. 65). According to Chalkley and Daniel (1933), the conspicuous granules present, in the resting nu- cleus, under the membrane contain very little chromatin, while abundant chromatin is lodged in the central area. The peripheral granules appear to give rise to achromatic figure. At the beginning of division, the chromatin granules become aggregated in a zone (6); they then assume a ring-form along the periphery of the central mass 142 PROTOZOOLOGY of network (c); at this stage, the cytoplasm around the nucleus is much vacuolated. A little later appears a discoid equatorial plate or ring which is connected with the nuclear membrane by numerous fibrils, and the nucleus becomes markedly flattened with its mem- brane still intact (d), which is considered as the end of the prophase. In the metaphase, the nuclear membrane becomes extremely faint and the portion over one side of the plate is without it (e). At the Fig. 65. Nuclear division in Amoeba proteus, Xl80 (Chalkley and Daniel), a, resting stage; b-d, prophase; e, metaphase; f, g, anaphase; h, a daughter nucleus. anaphase the membrane completely disappears, the equatorial plate splits and each half contracts in the plane of the plate, producing two daughter-plates. In some specimens a faint spindle formation is noted. At about this time, vacuolated condition of the perinuclear cytoplasm disappears (/). In later phases of anaphase the plates are more widely separated and are slightly less in diameter as compared with earlier stages. There are distinct polar caps of fibrillar material at the poles of the spindle (g) , fi.nally each plate transforms itself into a resting nucleus (h). The two investigators added that if the chro- matin granules located in the equatorial plate are chromosomes, "they must be extremely numerous." Liesche (1938) estimates the number of these granules which he called chromosomes as between 500 and 600. REPRODUCTION 143 Cytosomic division Binary fission. As in metazoan cells, the binary fission occurs very widely among the Protozoa. It is a division of the body through middle of the extended long axis into two nearly equal daughter individuals (Fig. 51). In Amocha proteus, Chalkley and Daniel found that there is a definite correlation between the stages of nuclear divi- sion and external morphological changes (Fig. 66). During the pro- \j^':. b o\SS^ j(..-.^-. '^-'i5/^:f '^i^- r^d ^\, ■ Fig. 66. External morphological changes during division of Amoeba proteus, as viewed in life in reflected light, X about 20 (Chalkley and Daniel), a, shortly before the formation of the division sphere; b, a later stage; c, prior to elongation; d, further elongation; e, division almost completed. phase, the organism is rounded, studded with fine pseudopodia and exhibits under reflected light a clearly defined hyaline area near its center (a), which disappears in the metaphase (6, c). During the anaphase the pseudopodia rapidly become coarser; in the telophase the elongation of body, cleft formation, and return to normal pseudopodia, take place. In Testacea, one of the daughter individuals remains, as a rule, within the old test, while the other moves into a newly formed one, as in Arcella, Pj^xidicula, Euglypha, etc. According to Doflein, the division plane coincides with the axis of body in Cochliopodium, Pseudodifflugia, etc., and the delicate homogeneous test also divides 144 PROTOZOOLOGY into two parts. In the majority of the Mastigophora, the division is longitudinal, as is shown by that of Menoidium incurvum (Fig. 67). In certain dinoflagellates, such as Ceratium, Cochliodinium, etc., the division plane is oblique, while in forms such as Oxyrrhis (Dunk- erly; Hall), the fission is transverse. In Strehlomastix strix (Kofoid and Swezy), Lophomonas striata (Kudo), Spirotrichomjmpha hispira Fig. 67. Nuclear and cytosomic division in Menoidium incurvum, X about 1400 (Hall), a, resting stage; b, c, prophase; d, equatorial plate; 8, f, anaphase; g, telophase. (Cleveland), etc., the division takes place transversely but the polar- ity of the posterior individual is reversed so that the posterior end of the parent organism becomes the anterior end of the posterior daughter individual. In the ciliate Bursaria, Lund (1917), observed reversal of polarity in one of the daughter organisms at the time of division of normal individuals and also in those which regenerated after being cut into one-half the normal size. In the Ciliophora the division is as a rule transverse (Fig. 50), in which the cytosome without any enlargement or elongation divides by constriction through the middle so that the two daughter indivi- duals are about half as large at the end of division. Both individuals usually retain their polarity. Multiple division. In multiple division the body divides into a number of daughter individuals, with or without residual cyto- REPRODUCTION 145 plasmic masses of the parent body. In this process the nucleus may undergo either simultaneous multiple division, as in Aggregata, or more commonly, repeated binary fission, as in Plasmodium (Fig. 225) to produce large numbers of nuclei, each of which becomes the center of a new individual. The number of daughter individuals often varies, not only among the different species, but also within one and the same species. Multiple division occurs commonly in the Fora- minifera (Fig. 184); the Radiolaria (Fig. 194), and various groups of Sporozoa in which the trophozoite multiplies abundantly by this method. Budding. Multiplication by budding which occurs in the Proto- zoa is the formation of one or more smaller individuals from the parent organism. It is either exogenous or endogenous, depending upon the location of the developing buds or gemmules. Exogenous budding has been reported in Acanthocystis, Noctiluca (Fig. 107), Myxosporidia (Fig. 68, h), astomatous ciliates (Fig. 266), Chono- tricha, Suctoria (Fig. 331, k), etc. Endogenous budding has been found in Testacea, Gregarinida, Myxosporidia (Figs. 247, e; 249, j), and other Sporozoa as well as Suctoria (Fig. 331, A). Collin observed a unique budding in Tokophrya cyclopum in which the entire body, excepting the stalk and pellicle, transforms itself into a yoUng ciliated bud which leaves sooner or later the parent pellicle as a swarmer. Plasmotomy. Occasionally the multinucleate body of a protozoan divides into two or more small, mutinucleate individuals, the cyto- somic division taking place independently of nuclear division. This has been called plasmotomy by Doflein. It has been observed in the trophoxoites of several coelozoic myxosporidians, such as Chloro- myxum leydigi (Fig. 68), Sphaeromyxa halbianii (Fig. 68), etc. It occurs further in Mycetozoa (Fig. 156), Protociliata and certain Sarcodina (Pelomyxa). Colony formation When the division is repeated without a complete separation of the daughter individuals, a colonial form is produced. The compon- ent individuals of a colony may either have protoplasmic connections among them or be grouped within a gelatinous envelope if completely separated. Or, in the case of loricate or stalked forms, these exo- skeletal structures may become attached to one another. Although varied in appearance, the arrangement and relationship of the com- ponent individuals are constant, and this makes the basis for dis- tinguishing the types of protozoan colonies, as follows: 146 PROTOZOOLOGY Catenoid or linear colony. The daughter individuals are attached endwise, forming a chain of several individuals. It is of compara- tively rare occurrence. Examples: Astomatous ciliates such as Radiophrya (Fig. 266), Protoradiophrya (Fig. 266) and dinoflagel- lates such as Ceratium, Haplozoon (Fig. 109) and Polykrikos (Fig. 110). Arboroid or dendritic colony. The individuals remain connected with one another in a tree-form. The attachment may be by means '^^"^^^^^ ml <^mv--j Fig. 68. a, b, budding in Myxidium lieberkuhni; c, d, plasmotomy in Chloromijxum leydigi; e, plasmotomy in Syhaeromyxa balbianii. of the lorica, stalk, or gelatinous secretions. It is a very common colony found in different groups. Examples: Dinobryon (Fig. 92), Hyalobryon (Fig. 92), etc. (connection by lorica); Colacium (Fig. 102), many Peritricha (Figs. 322; 324), etc. (by stalk); Poterioden- dron (Fig. 116), Stylobryon (Fig. 130), etc. (by lorica and stalk); Hydrurus (Fig. 93), Spongomonas (Fig. 129), Cladomonas (Fig. 129) and Anthophysis (Fig. 130) (by gelatinous secretions). Discoid colony. A small number of individuals are arranged in a single plane and grouped together by a gelatinous substance. Exam- REPRODUCTION 147 pies: Cyclonexis (Fig. 92), Gonium (Fig. 99), Platydorina (Fig. 100), Protospongia (Fig. 114), Bicosoeca (Fig. 116), etc. Spheroid colony. The individuals are grouped in a spherical form. Usually enveloped by -a distinct gelatinous mass, the component individuals may possess protoplasmic connections among them. Examples: Uroglena (Fig. 92, c), Uroglenopsis (Fig. 92, d), Volvox (Fig. 99), Pandorina (Fig. 100,/), Eudorina (Fig. 100, h), etc. Such forms as Stephanoon (Fig. 100, a) appear to be intermediate between this and the discoid type. The component cells of some spheroid colonies show a distinct differentiation into somatic and reproductive individuals, the latter developing from certain somatic cells during the course of development. The gregaloid colony, which is sometimes spoken of, is a loose group of individuals of one species, usually of Sarcodina, which become attached to one another by means of pseudopodia in an ir- regular form. Asexual reproduction The Protozoa nourish themselves by certain methods, grow and multiply, by the methods described in the preceding pages. This phase of the life-cycle of a protozoan is the vegetative stage or the trophozoite. The trophozoite repeats its asexual reproduction process under favorable circumstances. Generally speaking, the Sporozoa increase to a much greater number by schizogony and the tropho- zoites are called schizonts. Under certain conditions, the trophozoite undergoes encystment (Fig. 69). Prior to encystment, the trophozoites cease to ingest, and extrude remains of, food particles, resulting in somewhat smaller forms which are usually rounded and inactive. This phase is some- times called the precystic stage. The whole organism becomes de- differentiated; namely, various cell organs such as cilia, cirri, flagella, axostyle, peristome, etc., become absorbed. Finally the organism secretes substances which become solidified into a resistant wall, and thus the cyst is formed. In this condition, the protozoan is apparently able to maintain its vitality for a certain length of time under unfavorable conditions. The causes of encystment are still the matter which many investigators are attempting to comprehend. It appears certain at least in some cases that the encystment is brought about by changes in temperature, chemical composition, amount of water, food material, and catabolic waste substances, etc., in the medium in which the organisms live. In some cases, the organisms encyst temporarily in order to undergo nuclear reorgani- 148 PROTOZOOLOGY zation and multiplication as in Colpoda cucullus (Kidder and Claff, 1938). Because of the latter condition and also of the failure in at- tempting to cause certain Protozoa to encyst under experimental conditions, some suppose that certain internal factors play as great Encystment of Lopho)nonas blattarum, X1150 (Kudo). a part as do the external conditions in the phenomenon of encyst- ment. Ordinarily a single cyst wall seems to be sufficient to protect the protoplasm against unfavorable external conditions. In some cases there may be a double cyst wall, the inner one usually being more delicate. The cyst wall is generally composed of homogeneous substances, but it may contain calcareous scales as in Euglypha (Fig. 70). While chitin is the common material of which the cyst wall Fig. 70. Encystment of Euglypha acanthophora, X320 (Kiihn). is composed, cellulose makes up the cyst membrane of numerous Phytomastigina. The capacity of Protozoa to produce cyst is probably one of the reasons why they are so widely distributed over the surface of the globe. The minute protozoan cysts are easily carried from place to REPRODUCTION 149 place by wind, attached to soil particles, debris, etc., by the flowing water of rivers or the current in oceans or by insects, birds, other animals to which they become readily attached. When a cyst en- counters a proper environment, a redifferentiation process takes place within the cyst. Various organellae which characterize the organism, are regenerated and reformed, and the trophozoite excysts. The emerged organism once more returns to its trophic phase of cm ® ® ® Fig. 71. Diagram illustrating the life-cycle of Thelohania legeri (Kudo). a, extrusion of the polar filament in gut of anopheline larva; b, emerged amoebula; c-f, schizogony in fat body; g-m, sporont-formation; m-x, stages in spore-formation. existence. Although encystment is a general occurrence among Protozoa, there are some species in which this phenomeonon has never been observed. Paramecium belongs to this group (p. 600). In Sporozoa, no encystment occurs. Here at the end of active schizogony, sexual reproduction usually initiates the production of large numbers of the spores (Fig. 71). Sexual reproduction and life-cycles Besides reproducing bj' the asexual method, numerous Protozoa reproduce themselves in a manner comparable with the sexual re- 150 PROTOZOOLOGY production which occurs universally in the Metazoa. Various types of sexual reproduction have been reported in literature, of which a few will be considered here. The sexual fusion or syngamy which is a complete union of two gametes, has been reported from various groups, while the conjugation which is a temporary union of two individuals for the purpose of exchanging the nuclear material, is found almost exclusively in the Ciliophora. Sexual fusion. The gametes which develop from trophozoites, may be morphologically alike (isogametes) or unlike (anisogametes), both of which are, in well-studied forms, physiologically different as judged by their behavior toward each other. If a gamete does not meet with another one, it perishes. Anisogametes are called micro- gametes and macrogametes. Difference between them is comparable in many instances (Figs. 74; 76; 225) with that which exists between Fig. 72. a, macrogamete, and b, microgamete of Volvox aureus, XlOOO (Klein). the spermatozoa and ova of Metazoa. The microgametes are motile, relatively small and usually numerous, while the macrogametes are usually not motile, much more voluminous and fewer in number. Therefore, they have sometimes been referred to as male and female gametes (Fig. 72). While morphological differences between the gametes have long been known and studied by many workers, whatever information we possess on physiological differences between them is of recent origin. Since 1933, Moewus and his co-workers have published a series of papers based upon their extended studies of bacteria-free cultures of many species (and strains) of Chlamydomonas (p. 217) which throw some light on the gamete differentiation among these phytomonadinans. The gametes in Chlamydomonas are mostly isogamous, except in a few forms. Sexual fusion takes place in the majority of species and strains between the gametes produced in different clones, and there is no gametic fusion within a single clone. Moewus obtained "sex substances" from some of the cultures and showed that these are chemotactic substances. Each gamete secretes substances that attract the other and each reacts to the substances REPRODUCTION 151 secreted by the other. Kuhn, Moewus and Wendt (1939) recognized "hormones," and named them, termones (sex-determining hor- mones), anderotermone (male-determining hormone) and gynoter- mone (female-determining hormone). In a few strains or species of Chlamydomonas, sexual fusion is found to take place among the gametes that develop within a single clone. Moewus considers in these cases there exist two types of gametes in a clone. However, Pascher, Pringsheim, and others ob- tained results which seem to indicate that there is no physiological or sex differentiation between the fusing gametes. In the much- studied Sporozoa, for example, Plasmodium, the two gametes are both morphologically and physiologically differentiated, and sexual fusion always takes place between two anisogametes. Fig. 73. Sexual fusion in Coprompnas subtilis, X1300 (Dobell). The isogamy is typically represented by the flagellate Copro- monas suhtilis (Fig. 73), in which there occurs, according to Dobell, a complete nuclear and cytoplasmic fusion between two isogametes. Each nucleus, after casting off a portion of its nuclear material, fuses with the other, thus forming a zygote containing a synkaryon. In Stephanosphaera pluvialis (Fig. 74), both asexual and sexual re- productions occur, according to Hieronymus. Each individual multiplies and develops into numerous biflagellate gametes, all of which are alike. Isogamy between two gametes results in formation of numerous zygotes which later develop into trophozoites. Anisogamy has been observed in certain Foraminifera. It perhaps occurs in the Radiolaria also, although positive evidence has yet to be presented. Anisogamy seems to be more widely distributed. In Pandorina niorum (Fig. 75), Pringsheim observed that each cell de- velops asexually into a young colony (a, b) or into anisogametes (c) which undergo sexual fusion {d-g) and encyst (/i).-The organism emerged from the cyst, develops into a young trophozoite (i-m). A similar life-cycle was found by Goebel in Eudorina elegans (Fig. 76). 152 PROTOZOOLOGY Among the Sporozoa, anisogamy is of common occurrence. In Coccidia, the process was well studied in Eimeria schuhergi (Fig. 215), Aggregata eberthi (Fig. 217), Adelea ovata (Fig. 222), etc., and the resulting products are the oocysts (zygotes) in which the spores Fig. 74. The life-cycle of Stephanosphaera pluvialis (Hieronymus). a-e, asexual reproduction; f-m, sexual reproduction. or sporozoites develop. Similarly in Haemosporidia such as Plasmo- dium vivax (Fig. 225), anisogamy results in the formation of the ookinetes or motile zygotes which give rise to a large number of sporozoites. Among Myxosporidia, a complete information as to how the initiation of sporogony is associated with sexual reproduc- REPRODUCTION 153 Fig. 75. The life-cycle of Pandorina morum, X400 (Pringsheim). a, b, asexual reproduction; c-m, sexual reproduction. ■^')^i- Fig. 76. The life-cycle of Eudorina elegans (Goebel). a, asexual repro- duction; b, sexual reproduction, a female colony with clustered and iso- lated microgametes. 154 PROTOZOOLOGY tion, is still lacking. Naville, however, states that in the trophozoite of Sphaeromyxa sahrazesi (Fig. 245), micro- and macro-gametes develop, each with a haploid nucleus. Anisogamy, however, is pe- culiar in that the two nuclei remain independent. The microgametic nucleus divides once and the two nuclei remain as the vegetative nuclei of the pansporoblast, while the macrogamete nucleus multi- plies repeatedly and develop into two spores. Anisogamy has been suggested to occur in some members of Amoebina, particularly in Endamoeha hlattae. Mercier (1909) believed that in this amoeba there occurs anisogamy soon after excystment in the host's intestine, but this still awaits confirmation. Cultural studies of various para- sitic amoebae in recent years show no evidence of sexual reproduc- tion. Among the Ciliophora, the sexual fusion occurs only in Protociliata (Fig. 263). Conjugation. The conjugation is a temporary union of two indivi- duals of one and the same species for the purpose of exchanging part of the nuclear material and occurs almost exclusively in the Euci- liata and Suctoria. The two individuals which participate in this process may be either isogamous or anisogamous. In Paramecium caudatum (Fig. 77), two similar individuals come in contact on their oral surface (a). The micronucleus in each conjugant divides twice (h-e), forming four micro nuclei, three of which degenerate and do not take active part during further changes (f-h). The remaining micronucleus divides once more, producing a wandering pronucleus and a stationary pronucleus (/, g). The wandering pronucleus in each of the conjugants enters the other individual and fuses with its sta- tionary pronucleus (h, r). The two conjugants now separate from each other and become exconjugants. In each exconjugant, the synkaryon divides three times in succession (i-m) and produces eight nuclei (n), four of which remain as micronuclei, while the other four develop into new macronuclei (o). Cytosomic fision follows then, producing first, two individuals with four nuclei (p) and then, four small indivi- duals, each containing a micronucleus and a macronucleus (a). Ac- cording to Jennings, however, of the four smaller nuclei formed in the exconjugant indicated in Fig. 77, o, only one remains active, and the other three degenerate. This active nucleus divides prior to the cytosomic division so that in the next stage {p), there are two de- veloping macronuclei and one micronucleus which divides once more before the second and last cytosomic division (q). During these changes the original macronucleus disintegrates, degenerates, and finally becomes absorbed in the cytoplasm. In 1937, Sonneborn discovered that in certain races of P. aurelia, I REPRODUCTION 155 Fig. 77. Diagram illustrating the conjugation of Paramecium caudatum. a-q, X about 130 (Calkins); r, a synkaryon, X1200 (Dehorne). there are two classes of individuals with respect to "sexual" differ- entiation and that the members of different classes conjugate with each other, while the members of each class do not. These classes 156 PROTOZOOLOGY were called the mating types. Soon a similar phenomenon has been reported by several workers in five other species of the genus ; namely, P. hursaria, P. caudatum, P. trichium, P. calkinsi, and P. multimi- cronucleatum. When organisms which belong to different mating types are brought together, they adhere to one another in large clumps ("agglutination") of numerous individuals (Fig. 78, h). --7; J'' l- 1 - ■> ' J - ' ' -%». *^-. V V *- « o ' r • 4 '> ;% ♦ v« • Fig. 78. Mating behavior of Paramecium hursaria (Jennings), a, indi- viduals of a single mating type; b, 6 minutes after individuals of two mat- ing types have been mixed; c, after about 5 hours, the large masses have been broken down into small masses; d, after 24 hours, paired conjugants. After a few to several hours, the large masses break down into small masses (Fig. 78, c) and still later, conjugants appear in pairs (Fig. 78, d). The only other ciliate in which mating types are definitely known to occur is Euplotes patella in which, according to Kimball (1939), there occurs no agglutination mating reaction. How widely mating types occur is not known at present. But as REPRODUCTION 157 was pointed out by Jennings, the mating types may be of general oc- currence among ciliates; for example, Maupas (1889) observed that in Lionotus {Loxophyllum) fasciola, Leucophrys patula, Siylonychia pustulata, and Onychodromus graridis, conjugation took place be- tween the members of two clones of different origin, and not among the members of a single clone. Precise information on the occurrence among different ciliates depends on future research. In Paramecium aurelia, Sonneborn distinguishes seven varieties which possess the same morphological characteristics. There occurs no conjugation between the clones of different varieties. Within each of the six varieties, there are two mating types, while there is only one type in the seventh variety. Animals belonging to the same variety, but to different mating types only conjugate when put together (Table 6). In P. hursaria, Jennings (1938, 1939) finds three varieties, but each of two varieties contains four mating types and in the third variety eight mating types occur (Table 6). In Euplotes patella, Kimball (1939) observed six mating types (Table 6). These mating types cannot be considered as the true sex types, since the conjugants mutually fertilize each other. Recent studies of mating types have revealed much information regarding conjugation. Conjugation usually does not occur in well- fed or extremely starved animals, and appears to take place shortly after the depletion of food. Temperature also plays a role in con- jugation, as it takes place within a certain range of temperature which varies even in a single species among different varieties (Sonneborn). Light seems to have different effects on conjugation in different varieties of P. aurelia. The time between two conju- gations also varies in different species and varieties. In P. hursaria, Jennings found that in some races the second conjugation would not take place for many months after the first, while in others such an "immature" period may be only a few weeks. In P. aurelia, in some varieties there is no "immature" period, while in others there is 6 to 10 days' "immaturity." Very little is known about the physiological state of conjugants as compared with vegetative individuals. Several investigators ob- served that animals which participate in conjugation show much viscous body surface. Boell and Woodruff (1941) found that the mating individuals of Paramecium calkinsi show a lower respiratory rate than not-mating individuals. Neither is the mechanism of con- jugation understood at present. Kimball (1942) discovered in Euplotes patella, the fluid taken from cultures of animals of one type induces conjugation among the animals of other types. Pre- 158 PROTOZOOLOGY sumably certain substances are secreted by the organisms and be- come diffused in the culture fluid. In the case of P. aurelia, Sonne- born considers that there are also some substances which however do not diffuse into the surrounding medium, and possibly transported from one individual to another by contact and subsequent migration. Fuller understanding of the phenomenon of mating types depends upon future investigations. When the ciliate possesses more than one micronucleus, the first division ordinarily occurs in all and the second may or may not take place in all, varying apparently even among individuals of the same species. According to Woodruff, in Paramecium aurelia, of the eight micronuclei formed by two fissions of the two original micjonuclei, only one undergoes the third division to produce two pronuclei. This is the case with the majority, although more Table 6. — Varieties and mating types in Paramecium aurelia, P. bursaria and Ewplotes patella. + indicates that conjugation occurs; — indicates that it does not. Paramecium aurelia (Sonneborn) Va- riety 1 2 3 4 5 6 7 mat- ing type I II III IV V VI VII VIII IX X XI XII XIII 1 I II + + - - - - - - - - - - - 2 III IV - - - + + - - - - - - - - - - 3 V VI - - - - - + + - - - - - - - - 4 VII VIII - - - - - - - + + - - - - - - 5 IX X - - - - - - - + + - - - - 6 XI XII - - - - - - - - - + + - - 7 XIII - - _ - - - - - - - - - REPRODUCTION Table 6. — Continued Paramecium hursaria (Jennings) 159 Variety 1 2 3 Type A B c D E F G H J K L M N P Q A _ + + + _ — - - _ - - - - _ _ _ 1 B C D + + + + + + + + + — _ - _ _ - - - - _ _ _ E _ — _ _ - + + + + + + + _ _ _ _ F - - - - + - + + + + + + - - - - G — — — — + + — + + + + + — _ _ _ 2 H - - - - + + + - + + + + - _ _ _ J — — — — + + + + - + + + — _ _ _ K - - - - + + + + + - + + - _ _ _ L - - - - + + + + + + - + - - - - M - - - - + + + + + + + - - - - - N _ _ _ _ _ _ _ _ _ _ _ _ _ + + + 3 P Q - - _ - - - - - + + + - + + + - + + + - Euplotes patella (Kimball) Type I II III IV V VI I _ + + + + + II + - + + + + III + + - + + + IV + + + - + + V + + + + + VI + + + + + - than one micronucleus may divide for the third time to produce several pronuclei, for example, two in Euplotes patella, Stylonychia pustulata ; two to three in Oxytricha fallax and two to four in Uro- leptus mobilis. This third division is always characterized by long extended nuclear membrane stretched between the division prod- ucts. Ordinarily the individuals which undergo conjugation appear to be morphologically similar to those that are engaged in the trophic activity, but in some species, the organism divides just prior to conjugation. According to Wichterman (1936), conjugation in 160 PROTOZOOLOGY Nyctotherus cordiformis (Fig. 79) takes place only among those which live in the tadpoles undergoing metamorphosis (f-j). The conjugants are said to be much smaller than the ordinary tropho- FiG. 79. The life-cycle of Nyctotherus cordiformis in Hyla versicolor (Wichterman). a, a cyst; b, excystment in tadpole; c, d, division is repeated until host metamorphoses; e, smaller preconjugant; f-j, con- jugation; k, exconjugant; 1, amphinucleus divides into 2 nuclei, one micro- nucleus and the other passes through the "spireme ball" stage before developing into a macro nucleus; k-n, exconjugants found nearly exclu- sively in recently transformed host; o, mature trophozoite; p-s, binary fission stages; t, precystic stage. REPRODUCTION 161 zoites, because of the preconj ligation fission {d~e). The micronuclear divisions are similar to those that have been described for Para- mecium caudatum and finally two pronuclei are formed in each con- jugant. Exchange and fusion of pronuclei follow. In each excon jug- ant, the synkaryon divides once to form the micronucleus and the macronuclear anlage {k-l) w^hich develops into the "spireme ball" and finally into the macronucleus (m-o). A sexual process which is somewhat intermediate between the sexual fusion and conjugation, is noted in several instances. Ac- cording to Maupas' classical work on Vorticella nehuUfera, the or- dinary vegetative form divides twice, forming four small individuals, which become detached from one another and swim about inde- pendently. Presently each becomes attached to one side of a stalked individual. In it, the micronucleus divides three times and produces eight nuclei, of which seven degenerate; and the remaining nucleus divides once more. In the stalked form the micronucleus divides twice, forming four nuclei, of which three degenerate, and the other dividing into two. During these changes the cytoplasm of the two conjugants fuse completely. The wandering nucleus of the smaller conjugant unites with the stationary nucleus of the larger conjugant, the other two pronuclei degenerating. The synkaryon divides several times to form a number of nuclei, from some of which macronuclei are differentiated and exconjugant undergoes multiplication. Another example of this type has been observed in Metopus es (Fig. 80). According to No land (1927), the conjugants fuse along the anterior end (a), and the micronucleus in each individual divides in the same way as was observed in Paramecium caudatum (h-e). But the cytoplasm and both pronuclei of one conjugant pass into the other (/), leaving the degenerating macronucleus and a small amount of cytoplasm behind in the shrunken pellicle of the smaller conjugant which then separates from the other (j). In the larger exconjugant, two pronuclei fuse, and the other two degenerate and disappear (g, h). The synkaryon divides into two nuclei, one of which condenses into the micronucleus and the other grows into the macro- nucleus (i, k-m). This is followed by the loss of cilia and encystment. Automixis. In certain Protozoa, the fusion occurs between two nuclei which originate in a single nucleus of an individual. This process has been called automixis by Hartmann, in contrast to the amphimixis (Weismann) which is the complete fusion of two nuclei originating in two individuals, as was discussed in the preceding pages. If the two nuclei w^hich undergo a complete fusion are present in a single cell, the process is called autogamy, but, if they are in two 162 PROTOZOOLOGY different cells, then paedogamy. The autogamy is of common occur- rence in the myxosporidian spores. The young sporoplasm contains two nuclei which fuse together prior to or during the process of ger- mination in the alimentary canal of a specific host fish, as for exam- ple in Sphaeromyxa sabrazesi (Figs. 244; 245) and Myxosoma cato- stomi (Fig. 243). In the Microsporidia, autogamy appears to initiate the spore-formation at the end of schizogonic activity of individuals as in Thelohania legeri (Fig. 71). Fig. 80. Conjugation of Metopus es (Noland). a, early stage; b, first micronuclear division; c, d, second micronuclear division; e, third micro- nuclear division; f, migration of pronuclei from one conjugant into the other; g, large conjugant with two pronuclei ready to fuse; h, large con- jugant with the synkaryon, degenerating pronuclei and macronucleus; i, large exconjugant with newly formed micronucleus and macronucleus j, small exconjugant with degenerating macronucleus; k-m, development of two nuclei, a, X290; b-j, X250, k-m, X590. REPRODUCTION 163 Diller (1936) observed in solitary Paramecium aurelia (Fig. 81), certain micronuclear changes similar to those which occur in conjugating individuals. The two micronuclei divide twice, form- ing eight nuclei (a-d), some of which divide for the third time (e), producing two functional and several degenerating nuclei (/). The two functional nuclei then fuse in the "paroral cone" and form the Fig. 81. Diagram illustrating autogamy in Paramecium aurelia (Diller). a, normal animal; b, first micronuclear division; c, second micronuclear division; d, individual with 8 micronuclei and macronucleus preparing for skein formation; e, two micronuclei dividing for the third time; f, two gamete-nuclei formed by the third division in the paroral cone; g, fusion of the nuclei, producing synkaryon; h, i, first and second division of synkaryon; j, with 4 nuclei, 2 becoming macronuclei and the other 2 re- maining as micronuclei; k, macronuclei developing, micronuclei dividing; 1, one of the daughter individuals produced by fission. synkaryon {g, h) which divides twice into four {i, j). The original macronucleus undergoes fragmentation and becomes absorbed in the cytoplasm. Of the four micronuclei, two transform into the new macronuclei and two remain as micronuclei (k) each dividing into two after the body divided into two (l). Another sexual process appears to have been observed by Diller (1934) in conjugating Paramecium trichium in which there was no nuclear exchange between the two conjugants. Wichterman (1939, 1940) observed a similar process in P. caudatum and named it cytogamy. Two small (about 200m long) individuals of P. caudatum 164 PROTOZOOLOGY fuse on their oral surfaces. There occur three micronuclear divisions as in the case of conjugation, but there is no nuclear exchange be- tween the members of the pair. The two gametic nuclei in each indi- vidual are said to fuse and form a synkaryon as in autogamy. The paedogamy occurs in at least two species of Myxosporidia, namely, Leptotheca ohlmacheri (Fig. 247) and Unicapsula muscularis (Fig. 248). The spores of these myxosporidians contain two uninu- cleate sporoplasms which are independent at first, but prior to emergence from the spore, they undergo a complete fusion to meta- :^I ^sS^?-^ ^U Fig. 82. Paedogamy in Adinophrys sol, X460 (Belaf). a, withdrawal of axopodia; b, c, division into two uninucleate bodies, surrounded by a common gelatinous envelope; d-f, the first reduction division; g-i, the second reduction division; j-1, synkaryon formation. morphose into a uninucleate amoebula. Perhaps the classical exam- ple of the paedogamy is that which was found by Hertwig (1898) in Actinosphaerhim eichhorni. The organism encysts and the body di- vides into numerous uninucleate secondary cysts. Each secondary cyst divides into two and remains together within a common cyst- wall. In each the nucleus divides twice, and forms four nuclei, one of which remains functional, the remaining three degenerating. The paedogamy results in formation of a zygote in place of a secondary cyst. Belaf (1922) observed a similar process in Adinophrys sol (Fig. 82). This heliozoan withdraws its axopodia and divides into two uninucleate bodies which become surrounded by a common REPRODUCTION 165 gelatinous envelope. Both nuclei divide twice and produce four nu- clei, three of which degenerate. The two daughter cells, each with one haploid nucleus, undergo paedogamy and the resulting individual now contains a diploid nucleus. In Paramecium aurelia, Diller (1936) found simple fragmentation of the macronucleus which was not correlated with any special micronuclear activity and which could not be stages in conjugation or autogamy. Diller suggests that if conjugation or autogamy is to create a new nuclear complex, as is generally held, it is conceivable that somewhat the same result might be achieved by 'purification act' (through fragmentation) on the part of the macronucleus itself, without involving micronuclei. He coined the term hemixis to in- clude these reorganizations. Fig. 83. Mitotic and meiotic micronuclear divisions in conjugating Didinium nasutum. (Prandtl, modified), a, normal micronucleus;b, equa- torial plate in the first (mitotic) division; c, anaphase in the first division; d, equatorial plate in the second division; e, anaphase in the second (meiotic) division. Meiosis. In the foregoing sections, references have been made to the divisions which the nuclei undergo prior to sexual fusion or con- jugation. In all Metazoa, during the development of the gametes, the gametocytes undergo reduction division or meiosis, by which the number of chromosomes is halved; that is to say, each fully mature gamete possesses half (haploid) number of chromosomes typical to the species (diploid). In the zygote, the diploid number is reestab- lished. In the Protozoa in which sexual reproduction occurs during their life-cycle, meiosis presumably takes place to maintain the con- stancy of chromosome-number, but the process is understood only in a small number of species. In conjugation, the meiosis seems to take place in the second micronuclear division, although in some, for example, Oxytricha fallax, according to Gregory, the actual reduction occurs during the first division. Prandtl (1906) was the first to note a reduction in number of chromosomes in the Protozoa. In conjugating Didinium nasutum (Fig. 83), he observed 16 chromosomes in each of the 166 PROTOZOOLOGY daughter micronuclei during the first division, but only 8 in the second division. Since that time, the fact that meiosis occurs during the second micronuclear division has been observed in Chilodonella uncinata (Enrique; MacDougall), Carchesium polypinum (Popoff), Uroleptus halseyi (Calkins), etc. (note the ciliates in Table 5 on p. 141). In various species of Paramecium and many other forms, the number of chromosomes appears to be too great to allow a precise counting, but Sonneborn's work on the mating types in Parame- cium as quoted elsewhere (p. 186), indicates clearly the occurrence of meiosis during conjugation. Information on the meicsis involved in the complete fusion of gam- etes is even more scanty and fragmentary. In Monocystis rostrata, a parasite of the earthworm, Mulsow (1911) noticed that the nuclei of two gametocytes which encyst together, multiply by mitosis in which eight chromosomes are constantly present, but in the last division in gamete formation, each daughter nucleus receives only 4 chromosomes. In another species of Monocystis, Calkins and Bowl- ing (1926) observed that the diploid number of chromosomes was 10 and that haploid condition is established in the last gametic division thus confirming Mulsow's finding. In the paedogamy of Actinophrys sol, Belaf finds 44 chromosomes in the first nuclear division, but after two meiotic divisions, the remaining functional nucleus contains only 22 chromosomes so that when paedogamy is completed the diploid number is restored. In Polytoma uvella, Moewus finds each of the two gametes is haploid (8 chromosomes) and the zygotes are diploid. The synkaryon divides twice, and during the first division reduction division takes place. In the coccidian Aggregata eherthi (Fig, 217), according to Dobell and Jameson, Belar, and Naville, and in the gregarine Diplocystis schneideri, according to Jameson, there is no reduction in the number of chromosomes during the gamete-formation, but the first zygotic division is meiotic, 12 to 6 and 6 to 3, respectively. A similar reduc- tion takes place also in Gregarina hlattarum (6 to 3, after Sprague, 1941) and in Adelina deronis (20 to 10, after Hauschka, 1943). Thus in these forms, the zygote is the only stage in which diploid nucleus occurs, while the nuclei in stages in the remainder of the life-cycle are haploid. Some sixty years ago Weismann pointed out that a protozoan grows and muliplies by binary fission or budding into two equal or unequal individuals without loss of any protoplasmic part and these in turn grow and divide, and that thus in Protozoa there is neither senescence nor natural death which occur invariably in Metazoa in REPRODUCTION 167 which germ and soma cells are differentiated. Since that time, the problem of potential immortality of Protozoa has been a matter which attracted the attention of numerous investigators. Because of large dimensions, rapid growth and reproduction, and ease with which they can be cultivated in the laboratory, the majority of Protozoa used in the study of the problem have been free-living freshwater ciliates that feed on bacteria and other microorganisms. The very first extended study was made by Maupas (1888) who isolated Stylonychia pustulata on Februar}^ 27, 1886, and observed 316 binary fissions until July 10. During this period, there was noted a gradual decrease in size and increasing abnormality in form and Fig. 84. Degeneration or aging in Stylonychia pustulata. X340 (Maupas, modified), a, Beginning stage with reduction in size and completely atrophied micronucleus; b, c, advanced stages in which disappearance of the frontal zone, reduction in size, and fragmentation of the macronucleus occurred; d, final stage before disintegration. structure, until the animals could no longer divide and died (Fig. 84). A large number of isolation culture experiments have since been carried on numerous species of ciliates by many investigators. The results obtained are not in agreement. However, the bulk of ob- tained data indicates that the vitality of animals decreases with the passing of generations until finally the organisms suffer inevitable death, and that in the species in which conjugation or other sexual reproduction occurs, the declining vitality becomes restored. Perhaps the most thorough experiment was carried on by Calkins (1919, 1933) with Uroleptus mohilis. Starting with an exconjugant on 168 PROTOZOOLOGY November 17, 1917, a series of pure-line cultures was established by the daily isolation method. It was found that no series lived longer than a year, but when two of the progeny of a series were allowed to conjugate after the first 75 generations, the exconjugants repeated the history of the parent series, and did not die when the parent series died. In this way, lines of the same organism have lived for more than 12 years, passing through numerous series. In a series, the average division for the first 60 days was 15.4 divisions per 10 days, but the rate gradually declined until death. Woodruff and Spencer (1924) also found the isolation cultures of Spathidium spatula (fed on Colpidium colpoda) died after a gradual decline in the division rate, but were inclined to think that improper environ- mental conditions rather than internal factors were responsible for the decline. On the other hand. Woodruff (1932) found that 5071 generations produced by binary fission from a single individual of Paramecium aurelia between May 1, 1907 and May 1, 1915, did not manifest any decrease in vitality after eight years of uninterrupted asexual reproduction without conjugation. With a race of P. caudatum, Metalnikov (1924) observed a similar continued asexual reproduc- tion. Dawson (1919) subjected an amicronucleate race of Oxytricha hymenostoma to isolation culture and found that it declined in divi- sion-rate and finally died out; but in mass cultures, the organisms lived indefinitely. He attributed the decline in isolation culture to improper environmental conditions. With Actinophrys sol Belaf (1924) carried on isolation cultures (thus preventing paedogamy (p. 164) for 1244 generations for a period of 32 months and noticed no decline in the division rate. Hartmann (1921) made a similar obser- vation on Eudorina elegans. It would appear that in these forms the life continues indefinitely without apparent decrease in vital activity. As has been noted in the beginning part of the chapter, the macronucleus in the ciliates undergoes, at the time of binary fission a reorganization process before dividing into two parts and undoubt- edly, there occurs at the same time extensive cytoplasmic reorgani- zation as judged by the degeneration and absorption of the old, and formation of the new, organellae. It is reasonable to suppose that this reorganization of the whole body structure at the time of divi- sion is an elimination process of waste material accumulated by the organism during the various phases of vital activities as was con- sidered by Kidder and others (p. 127) and that this elimination, though not complete, enables the protoplasm of the products of divi- sion to carry on their metabolic functions more actively. REPRODUCTION 169 As the generations are multiplied, the general decline in vitality is manifest not only in the decreased division-rate, slow growth, abnormal form and function of certain organellae, etc., but also in inability to complete the process involved in conjugation. Jennings found that when individuals of aging stock of Paramecium hursaria conjugate with those of a young vigorous stock, certain numbers of exconjugants die without multiplication, or give rise to weak and abnormal descendants. As the stock grows older, the number of ex- conjugants that die or are weak increases until a period arrives at which time all the exconjugants die without multiplication. How- ever, if conjugation takes place before the decline in vitality becomes too great, such stocks in some cases are able to restore to high vital ity. Endomixis and autogamy have also been considered to have a similar effect on the vitality of the progeny of the individuals in which they occur. Experimental data indicate that conjugation of still vigorous young stocks does not bring about any greater vitality to their progeny, but that of older stocks results in many strains with restored vitality. Sonneborn (1942) mentions the appearance of varied characters after macronuclear regeneration (p. 129) in P. aurdia. The animals become smaller and of different form; they reproduce more slowly ; they are less viable and die out after a period of few weeks or months. Thus regenerated macronuclei are unable to produce high vitality. It is probable that the process of replacing old macronuclei by micronuclear material which are derived from the products of fusion of two micronuclei of either the same (autogamy) or two different animals (conjugation), would perhaps result in a complete elimina- tion of waste substances from the newly formed macronuclei, and divisions which follow this fusion may result in shifting the waste substances unequally among different daughter individuals. Thus in some individuals there may be a complete elimination of waste material and consequently a restored high vitality, while in others the influence of waste substances present in the cytoplasm may offset or handicap the activity of new macronuclei, giving rise to stocks of low vitality which will perish sooner or later. In addition in conjuga- tion, the union of two haploid micronuclei produces diverse genetic constitutions which would be manifest in progeny in manifold ways. Experimental evidences indicate clearly such is actually the case. In many ciliates, the elimination of waste substances at the time of binary fission and sexual reproduction (conjugation, and autog- amy), seemingly allow the organisms continued existence through 170 PROTOZOOLOGY a long chain of generations indefinitely. Jennings (1929, 1942) who has recently reviewed the whole problem states: "Some Protozoa are so constituted that they are predestined to decline and death after a number of generations. Some are so constituted that decline occurs, but this is checked or reversed by substitution of reserve parts for those that are exhausted; they can live indefinitely, but are dependent on this substitution. In some the constitution is such that life and multiplication can continue indefinitely without visible substitution of a reserve nucleus for an exhausted one ; but whether this is due to the continued substitution, on a minute scale, of re- serve parts for those that are outworn cannot now be positively stated. This perfected condition, in which living itself includes con- tinuously the necessary processes of repair and elimination, is found in some free cells, but not in all." Regeneration The capacity of regenerating the lost parts, though variable among different species, is characteristic of all Protozoa from simple forms to those with highly complex organizations, as shown by ob- servations of numerous investigators. Brandt (1877) studied regen- eration in Actinosphaerium eichhorni and found that only nucleate portions containing at least one nucleus regenerated, and enucleate portions or isolated nuclei degenerated. Similarly Gruber (1886) found in Amoeba proteus the nucleate portion regenerated completely, while enucleate part became rounded and perished in a few days. The parts which do not contain nuclear material, may continue to show certain metabolic activities such as locomotion, contraction of contractile vacuoles, etc., for some time; for example, Grosse-Aller- mann (1909) saw enucleate portions of Amoeba verrucosa alive for 20 to 25 days, while Stole (1910) found enucleate Amoeba proteus living for 30 days. Clark (1942, 1943) showed that Amoeba proteus lives for about seven days after it has been deprived of its nucleus. Enucleated individuals show a 70 per cent depression of respiration and are unable to digest food due to the failure of zymogens to be activated in the dedifferentiating cytoplasm. It is now a well estab- lished fact that when a protozoan is cut into two parts and the parts are kept under proper environmental conditions, the enucleated portion is able to carry on catabolic activities, but unable to under- take anabolic activities, and consequently degenerates sooner or later. In Arcella (Martini; Hegner) and Difflugia (Verworn; Penard), when the tests are partially destroyed, the broken tests remain un- REPRODUCTION 171 changed. Verworn considered that in these testaceans test-forming activity of the nucleus is limited to the time of asexual reproduction of the organisms. On the other hand several observers report in Foraminifera the broken shell is completely regenerated at all times. Verworn pointed out that this indicates that here the nucleus con- trols the formation of shell at any time. In a radiolarian, Thalassi- colla nucleata, the central capsule, if dissected out from the rest of body, will regenerate into a complete organism (Schneider). A few regeneration studies on Sporozoa have not given any results to be considered here, because of the difficulties in finding suitable media for cultivation in vitro. An enormous number of regeneration experiments have been con- ducted on more than 50 ciliates by numerous investigators. Here also the general conclusion is that the nucleus is necessary for re- generation. In many cases, the macronucleus seems to be the only essential nucleus for regeneration, as judged by the continued divi- sion on record of several amicronucleate ciliates and by experiments such as Schwartz's in which there was no regeneration in Stentor coeruleus from which the whole macronucleus had been removed. A remarkably small part of a protozoan is known to be able to re- generate completely if nuclear material is included. For example, Sokoloff found 1/53-1/69 of Spirostomum amhiguum and 1/70-1/75 of Dileptus anser regenerated and Philips showed portions down to 1/80 of an amoeba were able to regenerate. In Stentor coeruleus, Lillie and Morgan found pieces as small as 1/27 and 1/64 respect- ively of the original organisms regenerated. Burnside cut 27 speci- mens of this ciliate belonging to a single clone, into two or more parts in such a way that some of the pieces contained a large portion of the nucleus while others a small portion. These fragments re- generated and multiplied, giving rise to 268 individuals. No dimen- sional differences resulted from the different amounts of nuclear material present in the cut specimens. Apparently regulatory pro- cesses took place and in all cases normal size was restored, re- gardless of the amount of the nuclear material in ancestral pieces. Thus biotypes of diverse sizes are not produced by causing inequali- ties in the proportions of nuclear material in different individuals. Information on regeneration in individuals in division or encyst- ment is incomplete. While Calkins observed that the regenerative reaction in Uronychia was slowed down in late division stages, in Stentor and Paramecium other investigators found no difference in regeneration between vegetative individuals and those under- going fission. 172 PROTOZOOLOGY In addition to these restorative regenerations, there are physio- logical regenerations in which as in the case of asexual and sexual re- production, various organellae such as cilia, flagella, cytostome, contractile vacuoles, etc., are completely regenerated due to certain internal conditions. References Balamuth, W. 1940 Regeneration in Protozoa: a problem of mor- phogenesis. Quart. Rev. Biology, Vol. 15. Belar, K. 1924 Untersuchungen an Actinophrijs sol Ehrenberg. II. Beitrage zur Physiologic des Formwechsels. Arch. f. Protistenk., Vol. 48. 1926 Der Formwechsel der Protistenkerne. Ergebn. u. Fortsch. Zool., Vol. 6. BoELL, E. J. and L. L. Woodruff 1941 Respiratory metabolism of mating types in Paramecium calkinsi. Jour. Exp. Zool., Vol. 87. BuRNSiDE, L. H. 1929 Relation of body size to nuclear size in Stentor coerulens. Ibid., Vol. 54. Calkins, G. N. 1919 Uroleptus mohilis Engelm. II. Renewal of vi- tality through conjugation. Ibid., Vol. 29. 1933 The biology of the Protozoa. Second edition. Philadel- phia. and R. C. Bowling 1926 Gametic meiosis in Monocystis. Biol. Bull, Vol. 51. and F. M. Summers, editors. 1941 Protozoa in biological re- search. New York. Chalkley, H. W. and G. E. Daniel. 1933 The relation between the form of the living cell and the nuclear phases of division in Amoeba proteus. Physiol. Zool., Vol. 6. Chen, T. T. 1936 Observations on mitosis in opalinids. I. Proc. Nat. Acad. Sci., Vol. 22. Clark, A. M. 1942 Some effects of removing the nucleus from Amoeba. Austral. Jour. Exp. Biol. & Med. Sci., Vol. 20. 1943 Some physiological functions of the nucleus in Amoeba, investigated by micrurgical methods. Ibid., Vol. 21. Cleveland, L. R. 1938 Longitudinal and transverse division in two closely related flagellates. Biol. Bull., Vol. 74. 1938a Origin and development of the achromatic figure. Ibid. , S. R. Hall, E. P. Sanders and J. Collier 1934 The wood- feeding roach Cryptocercus, its Protozoa, and the symbiosis be- tween Protozoa and roach. Mem. Amer. Acad. Arts and Sci., Vol. 17. Dawson, J. A. 1919 An experimental study of an amicronucleate Oxytricha. Jour. Exp. Zool., Vol. 29. DiLLER, W. F. 1936 Nuclear reorganization processes in Parame- cium aurelia, with descriptions of autogamy and 'hemixis.' Jour. Morph., Vol. 59. DoBELL, C. 1908 The structure and life history of Copromonas suh- tilis. Quart. Jour. Micr. Sci., Vol. 52. REPRODUCTION 173 1917 On Oxnerclla maritima, nov. gen., nov. spec, a new heliozoan, and its method of division, with some remarks on the centroplast of the Heljozoa. Ibid., Vol. 62. 1925 The Hfe-history and chromosome cycle of Aggregata eberthi. Parasitology, Vol. 17. GiESE, A. C. 1939 Studies on conjugation in Paramecium multi- micronucleatum. Amer. Nat., Vol. 73. Oilman, L. C. 1941 Mating types in diverse races of Paramecium caudatum. Biol. Bull., Vol. 80. Hall, R. P. 1923 Morphology and binary fission of Menoidium incurvum. Univ. California Publ. Zool., Vol. 20. Hartmann, M. 1917 Ueber die dauernde rein agame Zuchtung von Eudorina elegans and ihre Bedeutung fiir das Befruchtungs- und Todproblem. Ber. preuss. Akad. Wiss., Phys.-Math. Klasse, pp. 760-776. Hauschka, T. S. 1943 Life history and chromosome cycle of the coccidian, Adelina deronis. Jour. Morph., Vol. 73. IvANic, M. 1938 Ueber die mit der Chromosomenbildung verbun- dene promitotische Orosskernteilung bei den Vermehrungsruhe Stadien von Chilodon uncinatus. Arch. f. Protistenk., Vol. 91. Jameson, A. P. 1920 The chromosome cycle of gregarines with spe- cial reference to Diplocystis schneideri. Quart. Jour. Micr. Sci., Vol. 64. Jennings, H. S. 1929 Oenetics of the Protozoa. Bibliogr. Oen., Vol. 5. 1938 Sex reaction types and their inheritance in Parame- cium hursaria. I. II. Clones collected from natural habitats. Proc. Nat. Acad. Sci., Vol. 24. 1939 Oenetics of Paramecium hursaria. I. Oenetics, Vol. 24. 1941 Oenetics of Paramecium hursaria. II. Proc. Amer. Phil. Soc, Vol. 85. ■ — 1942 Genetics of Parameciumhursaria. III. Oenetics, Vol. 27. 1942 Senescence and death in Protozoa and invertebrates. In E. V. Cowdry: Problems of ageing. Second edition. Balti- more. , D. Raffel, R. S. Lynch and T. M. Sonneborn 1932 The diverse biotypes produced by conjugation within a clone of Paramecium aurelia. Jour. Exp. Zool., Vol. 62. Kidder, O. W. 1933 Studies on Conchophthirus mytili de Morgan. I. Arch. f. Protistenk., Vol. 79. 1938 Nuclear reorganization without cell division in Para- clevelandia simplex, an endocommensal ciliate of the wood-feed- ing roach, Panesthia. Ibid., Vol. 91. and C. L. Claff 1938 Cytological investigations of Col- poda cucullus. Biol. Bull., Vol. 74. and W. F. Diller 1934 Observations on the binaiy fission of four species of common free-living ciliates, with special refer- ence to the macronuclear chromatin. Ibid., Vol. 67. and F. M. Summers 1935 Taxonomic and cytological stud- ies on the ciliates associated with the amphipod family Orchesti- idae from the Woods Hole district. Ibid., Vol. 68. 174 PROTOZOOLOGY Kimball, R. F. 1939 Change of mating type during vegetative re- production in Paramecium aurelia. Jour. Exp. Zool., Vol. 81. ■ 1939 Mating types in Euplotes. Amer. Nat., Vol. 73. 1942 The nature and inheritance of mating tj^pes in Eu- plotes patella. Genetics, Vol. 27. 1943 Mating types in the ciliate Protozoa. Quart. Rev. Biology, Vol. 18. KoFOiD, C. A. and O. Swezy 1919 On Strehlomastix strix, a poly- mastigote flagellate with a linear plasmodial phase. Univ. Calif. Publ. Zool., Vol. 20. — 1919 On Trichonympha campanula sp. nov. Ibid. KoRSCHELT, E. 1927 Regeneration und Transpla7itation. Vol. 1. Ber- lin. Kudo, R. R. 1925 Observations on Endmnoeha hlattae. Amer. Jour. Hyg., Vol. 6. 1926 Observations on Lophomonas hlattarum, a flagellate inhabiting the colon of the cockroach, Blatta orientalis. Arch. f. Protistenk., Vol. 53. 1926 A cytological stud}^ of Lophomonas striata. Ibid., Vol. 55. ■ — 1936 Studies on Nyctotherus ovalis Leidy, with special refer- ence to its nuclear structure. Ibid., Vol. 87. LiESCHE, W. 1938 Die Kern- und Fortpflanzungsverhaltnisse von Amoeba proteus. Ibid., Vol. 91. Lund, E. J. 1917 Reversibility of morphogenetic processes in Bur- saria. Jour. Exp. Zool., Vol. 24. Maupas, E. 1888 Recherches experimentales sur la multiplication des infusoires cilies. Arch. zool. exp. et gen. (2), Vol. 6. — 1889 Le rejeunissement karyogamique ches les cilies. Ibid., Vol. 7. Mulsow, K. 1911 Ueber Fortpflanzungserschneinungen bei Mono- cystis rostrata n. sp. Arch. f. Protistenk., Vol. 22. Noland, L. E. 1927 Conjugation in the ciliate Metopus sigrnoides. Jour. Morph. Physiol., Vol. 44. Prandtl, H. 1906 Die Konjugation von Didinium nasidum. Arch, f. Protistenk., Vol. 7. Reichenow, E. 1928 Ergebnisse mit der Nuclealfarbung bei Pro- tozoen. Ibid., Vol. 61. SoKOLOFF, B. 1924 Das Regenerationsproblem bei Protozoen. Ibid., Vol. 47. SoNNEBORN, T. M. 1937 Sex, sex inheritance and sex determina- tion in Paramecium aurelia. Proc. Nat. Acad. Sci., Vol. 23. 1938 Mating types in Paramecium aurelia: diverse condi- tions for mating in different stocks; occurrence, number and in- terrelations of the types. Proc. Amer. Phil. Soc, Vol. 79. 1940 The relation of macronuclear regeneration in Parame- cium aurelia to macronuclear structure, amitosis and genetic determination. Anat. Record, Vol. 78. 1942 Sex hormones in unicellular organisms. Cold Spring Harbor Symp. Quant. Biol., Vol. 10. REPRODUCTION 175 1942a Inheritance in ciliate Protozoa. Amer. Nat., Vol. 76. Sprague, V. 1941 Studies on Gregarina hlattarum with particular reference to the chromosome cycle. 111. Biol. Monogr., Vol. 18. Summers, F. M. 1935 The division and reorganization of the macronuclei of Aspidisca lynccus, Diophrys appendiculata and Stylonychia pustulata. Arch. f. Protistenk., Vol. 85. Turner, J. P. 1930 Division and conjugation in Euplotes patella with special reference to the nuclear phenomena. Univ. Calif. Publ. ZooL, Vol. 33. WiCHTERMAN, R. 1936 Divisiou and conjugation in Nydotherus cordiformis with special reference to the nuclear phenomena. Jour. Morph., Vol. 60. 1940 Cytogamy: a sexual process occurring in living joined pairs of Paramecium caudatum and its relation to other sexual phenomena. Ibid., Vol. 66. Woodruff, L. L. 1932 Paramecium aurelia in pedigree culture for twenty-five years. Trans. Amer. Micr. Soc, Vol. 51. and R. Erdmann 1914 A normal periodic reorganization process without cell fusion in Paramecium. Jour. Exp. ZooL, Vol. 17. and H. Spencer 1921 The survival value of conjugation in the life history of Spathidium spathida. Proc. Soc. Exp. Biol, and Med., Vol. 18. Chapter 6 Variation and heredity IT IS generally recognized that individuals of all species of organ- ism show a greater or less variation in morphological and physio- logical characteristics. Protozoa are no exceptions, and manifest a wide variation in size, form, structure, and physiological characters among the members of a single species. The different groups in a species are spoken of as the races, varieties, strains, etc. It is well known that dinoflagellates show a great morphological variation in different localities. Schroder (1914) found at least nine varieties of Ceraiium hirundinella (Fig. 85) occurring in various waters of Europe, and List found that the organisms living in shallow ponds possess a marked morphological difference from those living in deep ponds. Cyphoderia ampulla is said to vary in size among those in- habiting the same deep lakes; namely, individuals from the deep water may reach 200m in length, while those from the surface layer measure only about 100m long. In many species of Foraminifera, the shell varies in thickness ac- cording to the part of ocean in which the organisms live. Thus the strains which live floating in surface water have a much thinner shell than those that dwell on the bottom. For example, according to Rhumbler, Orhulina universa inhabiting surface water has a com- paratively thin shell, 1.28-18m thick, while individuals living on the bottom have a thick shell, up to 24m in thickness. According to Uyemura, a species of Amoeba living in thermal waters, showed a distinct dimensional difference in different springs. It measured 10-40m in diameter in sulphurous water and 45-80m in ferrous water; in both types of water the amoebae were larger at 36-40°C. than at 51°C. ' Such differences or varieties appear to be due to the influence of diverse environmental conditions, and will continue to exist under these conditions; but when the organisms of different varieties are subjected to a similar environment, the strain differences disappear sooner or later. That the differences in kind and amount of foods bring about extremely diverse individuals in Tetrahymena vorax and Chilomonas Paramecium in bacteria-free cultures has already been mentioned(p. 94). Chlamydomonas dcbaryana are represented by many races differing in form, size, and structure, in various localities as well as under different laboratory conditions. Moewus (1934) dis- 176 VARIATION AND HEREDITY 177 tinguished 12 such varieties and showed that any variety could be changed into another by using different culture media. This trans- formation, however, did not occur at the same rate among different Fig. 85. Varieties of Ceratiuvi hirundinella from various European waters (Schroder), a, furcoides-type (130-300/x by 30-45ju); b, brachy- ceroides-type (130-145ju by 30-45^t); c, silesiaciun-type (148-280/x by 28-34/x); d, carinthiacum-type (120-145/x by 45-60/i); e, gracile-type (140-200M by 60-75m); f, austriacum-type (120-160^ by 45-60)u); g, robustum-type (270-310^ by 45-55/i); h, scotticum-type (160-210/x by 50-60/i); i, piburgense-type (180-260^ by 50-60)u). races. It was found that the longer a strain has remained under con- ditions producing a given type, the greater the time and the number of generations needed to change it to a new type under a new condi- tion, as is shown in Table 7. While in many species, the races or varieties have apparently been brought about into being under the influence of environmental con- ditions, in others the inherited characters persist for a long period, and still in others the biotype may show different inherited char- 178 PROTOZOOLOGY acters. To the last-mentioned category belongs perhaps a strain of Tetrahymena geleii in which, according to Fiirgason (1940), a pure- line bacteria-free culture derived from a single individual was found to be composed of individuals differing in shape and size which be- came more marked in older cultures. The first comprehensive study dealing with the variation in size and its inheritance in uniparental or vegetative reproduction of Table 7. — Relation between the number of days cultivated in peptone medium and the number of days \cultivated in salt-sugar medium needed to change from type\l tojype 5\in Chlamydomonas]debaryana (^Moewus). Days in peptone medium Days in salt-sugar medium needed as type 1 to change to type 5 28 28 140 49 273 133 441 175 567 231 609 370 644 459 672 531 690 534 Protozoa was conducted by Jennings (1909). From a "wild" lot of Paramecium caudaium, eight races or biotypes with the relative mean lengths of 206, 200, 194, 176, 142, 125, 100, and 45/i were isolated. It was found that within each clone derived from a single parent, the size of individuals varies greatly (which is attributable to growth, amount of food, and other environmental conditions), any one of which may give rise to progenj^ of the same mean size. Thus selection within the pure race has no effect on the size, and the differ- ences brought about merely by environment are not inherited. Jen- nings (1916) examined the inheritance of the size and number of spines, size of shell, diameter of mouth, and size and number of teeth of the testacean Difflugia coro/m, and showed that "a popula- tion consists of many hereditarily diverse stocks, and a single stock, derived from a single progenitor, gradually differentiates into such hereditarily diverse stocks, so that by selection marked results are produced." Root (1918) with Centropyxis aculeata, Hegner (1919) with Arcella dentata, and Reynolds (1924) with A. polypora, ob- tained similar results. Jennings (1937) studied the inheritance of teeth in Difflugia corona in normal fission and by altering through VARIATION AND HEREDITY 179 operation, and found that operated mouth or teeth were restored to normal form in 3 or 4 generations and that three factors appeared to determine the character and number of teeth: namely, the size of the mouth, the number and arrangement of teeth in the parent, and "something in the constitution of the clone (its genotype) which tends toward the production of a mouth of a certain size, with teeth of a certain form, arrangement, and number." Numerous strains have been recognized in all intensively studied parasitic Protozoa such as Entamoeba, Trypanosoma, Plasmodium, etc. For example, Dobell and Jepps (1918) noticed five races in Entamoeba histolytica on the basis of differences in the size of cysts. Spector (1936) distinguished two races in the trophozoite of this amoeba. The large strain was found to be pathogenic to kittens, but the small strain was not. Meleney and Frye (1933, 1935) and Frye and Meleney (1939) also hold that there is a small race in Entamoeba histolytica which has a weak capacity for invading the intestinal wall and not pathogenic to man. Sapero, Hakansson and Louttit (1942) similarly notice two races which can be distinguished by the diameters of cysts, the division line being 10m and 9/i in living and balsam-mounted specimens respectively. The race with large cysts gives rise to trophozoites which are more actively motile, ingest erythrocytes, culture easily and is pathogenic to man and kitten, while the race with small cysts develops into less actively motile amoebae which do not ingest erythrocytes, difficult to culture, and is not pathogenic to hosts, thus not being histozoic. Recent investigations by Boyd and his co-workers show beyond doubt that the species of Plasmodium are composed of many strains which vary in diverse physiological characters. In an extended study on Try-pavosoma lewisi, Taliaferro (1921-1926) found that this flagel- late multiplies only during the first ten days in the blood of a rat after inoculation, after which the organisms do not reproduce. In the adult trypanosomes, the variability for total length in a population is about 3 per cent. Inoculation of the same pure line into different rats some- times brings about small but significant differences in the mean size and passage through a rat-flea generally results in a significant vari- ability of the pure line. It is considered that some differences in dimensions among strains are apparently due to environment (host), but others cannot be considered as due to this cause, since they per- sist when several strains showing such differences are inoculated into the same host. Hoare (1943) reviewed recently the "biological" races of certain parasitic Protozoa. Jollos (1921) subjected Paramecium caudatum to various environ- 180 PROTOZOOLOGY mental influences such as temperature and chemicals, and found that the animals develop tolerance which is inherited through many gen- erations even after removal to the original environment. For exam- ple, one of the clones which tolerated only 1.1% of standard solution of arsenic acid, was cultivated in gradually increasing concentrations for four months, at the end of which the tolerance for this chemical was raised to 5%. After being removed to water without arsenic acid, the tolerance changed as follows: 22 days, 5%; 46 days, 4.5%; 151 days, 4%; 166 days, 3%; 183 days, 2.5%; 198 days, 1.25% and 255 days, 1%. As the organisms reproduced about once a day, the acquired increased tolerance to arsenic was inherited for about 250 generations. There are also known inherited changes in form and structure which are produced under the influence of certain environmental conditions. Jollos designated these changes long-lasting modifica- tions (Dauermodifikationen) and maintained that a change in en- vironmental conditions, if applied gradually, brings about a change, not in the nucleus, but in the cytoplasm, of the organism which when transferred to the original environment, is inherited for a number of generations. These modifications are lost usually during sexual processes at which time the whole organism is reorganized. The long-lasting morphological and physiological modifications induced by chemical substances have long been known in parasitic Protozoa. Werbitzki (1910) discovered that Trypanosoma hrucei loses its blepharoplast when inoculated into mice which have been treated with pyronin, acridin, oxazin and allied dyes. Laveran and Roudsky (1911) found that these dyes have a special affinity for, and bring about the destruction by auto-oxidation of, the blepharo- plast. Such trypanosomes lacking blepharoplast behave normally and remain in that condition during many passages through mice. When subjected to small doses of certain drugs repeatedly, species of Trypanosoma often develop into drug-fast or drug-resistant strains which resist doses of the drug greater than those used for the treatment of the disease for which they are responsible. These modi- fications may also persist for several hundred passages through host animals and invertebrate vectors, but are eventually lost. Long-lasting modifications have also been produced by several investigators by subjecting Protozoa to various environmental in- fluences during the nuclear reorganization at the time of fission, conjugation, or autogamy. In Stentor (Popoff) and Glaucoma (Chatton), long-lasting modifications appeared during asexual divi- sions. Calkins (1924) observed a double-type Uroleptus mohilis VARIATION AND HEREDITY 181 which was formed by a complete fusion of two conjugants. This ab- normal animal underwent fission 367 times for 405 days, but finally reverted back to normal forms, without reversion to double form. Jennings (1941) outlined five types of long-lasting inherited changes during vegetative reproduction, as follows: (1) changes that occur in the course of normal life history, immaturity to sexual ma- turity which involves many generations; (2) degenerative changes resulting from existence under unfavorable conditions; (3) adaptive changes or inherited acclimitization or immunity ; (4) changes which are neither adaptive nor degenerative, occurring under specific en- vironmental conditions; and (5) changes in form, size, and other characters, which are apparently not due to environment. Whatever exact mechanism by which the long-lasting modifica- tions are brought about may be, they are difficult to distinguish from permanent modification or mutation, since they persist for hundreds of generations, and cases of mutation have in most instan- ces not been followed by sufficiently long enough pure-line cultures to definitely establish them as such. Jollos observed that if Paramecium were subjected to environ- mental change during late stages of conjugation, certain individuals, if not all, become permanently changed. Possibly the recombining and reorganizing nuclear materials are affected in such a way that the hereditary constitution or genotype becomes altered. MacDougall subjected Chilodonella uncinata to ultraviolet rays and produced many changes which were placed in three groups: (1) abnormalities which caused the death of the organism; (2) temporary variations which disappeared by the third generation ; and (3) variations which were inherited through successive generations and hence considered as mutations. The mutants were triploid, tetraploid, and tailed diploid forms (Fig. 86), which bred true for a variable length of time in pure-line cultures, either being lost or dying off finally. The tailed form differed from the normal form in the body shape, in the number of ciliary rows and contractile vacuoles, and in the mode of move- ment, but during conjugation it showed the diploid number of chro- mosomes as in the typical form. The tailed mutant remained true and underwent 20 conjugations during ten months. In biparental inheritance, the nuclei of two individuals partici- pate in producing new combinations which would naturally bring about diverse genetic constitutions. The new combination is ac- complished either by sexual fusion in Sarcodina, Mastigophora, and Sporozoa, or by conjugation in Euciliata and Suctoria. The genetics of sexual fusion is only known in a few forms. Perhaps 182 PROTOZOOLOGY the most complete information was obtained by Moewus through his extended studies of certain Phytomonadina. In Polytoma (p. 222), Chlamydomonas (p. 217), and allied forms, the motile indi- viduals are usually haploid. Two such individuals (gametes) fuse with each other and produce a diploid zygote which becomes en- cysted. The zygote later undergoes at least two divisions within the cyst wall, in the first division of which chromosome reduction takes place. These swarmers when set free become trophozoites and Fig. 86. Chilodonella uncinata (MacDougall). a, b, ventral and side view of normal individual; c, d, ventral and side view of the tailed mutant. multiply asexually by division for many generations, the descend- ants of each s warmer giving rise to a clone. Moewus (1935) demonstrated the segregation and independent as- sortment of factors by hybridization of Polytoma. He used two va- rieties each of two species: P. uvella and P. pascheri, both of which possess 8 haploid chromosomes. Their constitutions were as follows: P. uvella Form A: Oval (F), without papilla (p), with stigma (S), large (D) (Fig. 87, a). Form B: Oval (F), without papilla (p), without stigma (s), large (D) (Fig. 87, 6). P. pascheri Form C: Pyriform (f), with papilla (P), without stigma (s), large (D)(Fig.87,c). Form D: Pyriform (f), with papilla (P), without stigma (s), small (d)(Fig.87,6;). VARIATION AND HEREDITY 183 Thus six different crosses were possible from the four pairs of characters. When A (FpSD) and B (FpsD) fuse, the zygote divides into four swarmers, two swarmers have stigma (S), and the other two lack this cell organ, which indicates the occurrence of segrega- tion of the two characters (S, s) during the reduction division. When B (FpsD) is crossed with C (fPsD), thus differing in two pairs of characters, two swarmers possess one combination or type and the other two another combination. Different pairs of combinations are E Fig. 87. a, b. Polytoma uvella. a, Form A; b, Form B. c, d. P. pascheri. c, Form C; d, Form D. e, f. Crosses between Forms B and C. (Moewus) of course found. It was found that about half the zygotes gives rise to the two parental combinations (Fig. 87, h, c), while the other half gives rise to FPsD (Fig. 87, e) and fpsD (Fig. 87,/). When B (FpsD) is crossed with D (fPsd) or A (FpSD) is crossed with D (fPsd), only two types of swarmers are also formed from each zygote, and in the case of BXD, eight different combinations are produced, while in the case of AXD, sixteen different combina- tions, which appear in about equal numbers, are formed. Thus these four factors or characters show independent assortment during divi- sions of the zygote. Furthermore, Moewus noticed that certain other characters ap- peared to be linked with some of the four characters mentioned above. For example, the length of flagella, if it is under control of a factor, is linked on the same chromosome with the size-controlling factors (D, d), for large individuals have invariably long flagella and small individuals short flagella. During the experiments to de- termine this linkage, it was found that crossing over occurs between two entire chromosomes that are undergoing synapsis. 184 PROTOZOOLOGY In certain races of Polytoma pascheri and Chlamydomonas euga- metos, the sexual fusion takes place between members of different clones only. The zygote gives rise as was stated before to four swarm- ers by two divisions, which are evenly divided between the two sexes, which shows that the sex-determining factors are lodged in a single chromosome pair. In a cross between Chlamydomonas para- doxa and C. pseudoparadoxa, both of which produce only one type of gamete in a clone, the majority of the zygotes yield four clones, two producing male gametes and the other two female gametes; but a small number of zygotes gives rise to four clones which contain both gametes. It is considered that this is due to crossing-over that brought the two sex factors (P and M) together into one chromo- some, and hence the "mixed" condition, while the other chromosome which is devoid of the sex factors gives rise to individuals that soon perish. In crosses between Chlamydomonas eugametos which possesses a stigma and 10 haploid chromosomes and C. paupera which lacks a stigma and 10 haploid chromosomes, 12 pairs of factors including sex factor are distinguishable. Consequently at least two chromo- somes must have two factors in them. Thus adaptation to acid or alkaline culture media was found to be linked with differences in the number of divisions in zygote. That there occurs a sex-linked in- heritance in Chlamydomonas was demonstrated by crossing stigma- bearing C. eugametos of one sex with stigma-lacking C. paupera of the opposite sex. The progeny that were of the same sex as C. euga- metos parent possessed stigma, while those that were of the same sex as C. paupera parent lacked stigma. Thus it is seen that the sex factor and stigma factor are located in the same chromosome. The genetics of conjugation which takes place between two diploid conjugants has been studied by various investigators. Pure-line cultures of exconjugants show that conjugation brings about diverse inherited constitutions in the clones characterized by difference in size, form, division-rate, mortality-rate, vigor, resistance, degene- ration, etc. The diversities brought about by autogamy are not as varied as those produced by conjugation. In Paramecium much in- formation has in recent years been brought to light through the studies of Sonneborn, Jennings, Kimball, and others on the mating type (p. 156). The mating type is as a rule inherited without change to descendants through vegetative reproduction. Sonneborn (1939) has made extended studies of variety 1 of Paramecium aurelia (p. 158) and found that genetically diverse ma- terials show different types of inheritance, as follows: VARIATION AND HEREDITY . 185 (1) Stocks containing two mating types. When types I and II conjugate, among a set of exconjugants some produce all of one mating type, others all of the other mating type and still others both types (one of one type and the other of the other type) . In the last mentioned exconjugants, the types segregate usually at the first division, since of the two individuals produced by the first divi- sion, one and all its progeny, are of one mating type, and the other and all its progeny are of the other mating type. A similar change was also found to take place at autogamy. Sonneborn considers that the mating types are determined by macronuclei, as judged by segregation at first or sometimes second division in exconjugants and by the influence of temperature during conjugation and the first division. (2) Stocks containing only one mating type. No conjugation oc- curs in such stocks. Autogamy does not produce any change in type which is always type I. Stocks that contain type II only have not yet been found. (3) Hybrids between stocks containing one and two mating types. When the members of the stock containing both types I and II (two-type condition) conjugate with those of the stock containing one type (one-type condition), all the descendants of the hybrid exconjugants show two-type condition, which shows the dominancy of two-type condition over one-type condition. The factor for the two-type condition may be designated A and that for the one-type condition a. The parent stocks are AA and aa, and all Fi hybrids Aa. When the hybrids (Aa) are backcrossed to recessive parent (aa) (158 conjugating pairs in one experiment), approximately one-half (81) of the pairs give rise to two-type condition (Aa) and the remain- ing one-half (77) of the pairs to one-type condition (aa), thus showing a typical Mendelian result. When Fi hybrids (Aa) were interbred by 120 conjugating pairs, each exconjugant in 88 of the pairs gave rise to two-type condition and each exconjugant in 32 pairs pro- duced one-t3^pe condition, thus approximating an expected Men- delian ratio of 3 dominants to 1 recessive. That the F2 dominants are composed of two-thirds heterozygotes (Aa) and one-third homo- zygotes (AA) was confirmed by the results obtained by allowing F2 dominants to conjugate with the recessive parent stock (aa). Of 19 pairs of conjugants, 6 pairs gave rise to only dominant progeny, which shows that they were homoz^^gous (AA) and their progeny heterozygous (Aa), while 13 pairs produced one-half dominants and one-half recessives, which indicates that they were heterozygous (Aa) and their progeny half homozygous (aa) and half heterozygous 186 . PROTOZOOLOGY (Aa). Thus the genie agreement between two conjugants of a pair and the relative frequency of various gene combinations as shown in these experiments confirm definitely the occurrence of meiosis and chromosomal exchange during conjugation which have hitherto been known only on cytological ground. In Euplotes patella, Kimball (1942) found that the six mating types (p. 159) are determined by six possible combinations of a series of three allelic genes. There is no dominance among these alleles, the three heterozygous combinations determining three mating types being different from one another and from the three determined by homozygous combination. Kimball (1939, 1941) had shown that the fluid obtained free of Euplotes from a culture of one mating type will induce conjugation among animals of certain other mating types. When all possible combinations of fluids and animals are made it was found that the fluid from any of the heterozygous types in- duces conjugation among animals of any types other than its own and the fluid from any of the homozygous types induces conjugation only among animals of the types which do not have the same allele as the type from which the fluid came. These reactions are explained by an assumption that each of the mating type alleles is responsible for the production by the animal of a specific conjugation-inducing substance. Thus the two alleles in a heterozygote act independently of each other; each brings about the production by the animal of a substance of its own. Thus heterozygous animals are induced to con- jugate only by the fluids from individuals which possess an allele not present in the heterozygotes. The relation between the cytoplasm and nucleus in respect to in- heritance has become better known in recent years in some ciliates. De Garis (1935) succeeded in bringing about conjugation in Para- mecium caudatum, between the members of a large clone (198m long) and of a small clone (73/i long). The exconjugants of a pair are dif- ferent only in the cytoplasm as the nuclei are alike through exchange of a haploid set of chromosomes. The two exconjugants divide and give rise to progeny which grow to size characteristic of each parent clone, division continuing at the rate of once or twice a day. How- ever, as division is repeated, the descendants of the large clone be- come gradually smaller after successive fissions, while the descend- ants of the small clone become gradually larger, until at the end of 22 days (in one experiment) both clones produced individuals of inter- mediate size (about 135^ long) which remained in generations that followed. Since the exconjugants differed in the cytoplasm only, it must be considered probable that at first the cytoplasmic character VARIATION AND HEREDITY 187 was inherited through several vegetative divisions, but ultimately the influence of the new nucleus gradually changed the cytoplasmic character. The ultimate size between the two clones is not always midway between the mean sizes of the two parent clones, and is ap- parently dependent upon the nuclear combinations brought about by conjugation. It has also become known that different pairs of con- jugants between the same two clones give rise to diverse progeny, similar to those of sexual reproduction in Metazoa, which indicates that clones of Paramecium caudatum are in many cases heterozygous for size factors and recombination of factors occur at the time of conjugation. In P. aurelia, Kimball (1939) observed that there occasionally occurs a change of one mating type into another following autogamy. When the change is from type II to type I, not all animals change type immediately. Following the first few divisions of the product of the first division after autogamy there are present still some type II animals, although ultimately all become transformed into type I. Here also the cytoplasmic influence persists and is inherited through vegetative divisions. Jennings (1941) in his recent review writes as follows: "The primary source of diversities in inherited characters lies in the nucleus. But the nucleus by known material interchanges impresses its constitution on the cytoplasm. The cytoplasm retains the constitution so impressed for a considerable length of time, dur- ing which it assimilates and reproduces true to its impressed char- acter. It may do this after removal from contact with the nucleus to which its present constitution is due, and even for a time in the presence of another nucleus of different constitution. During this period, cytoplasmic inheritance may occur in vegetative reproduc- tion. The new cells produced show the characteristics due to this cytoplasmic constitution impressed earlier by a nucleus that is no longer present. But in time the new nucleus asserts itself, impressing its own constitution on the cytoplasm. Such cycles are repeated as often as the nucleus is changed by conjugation." Sonneborn (1943) has recently found in the four races of variety 4 of P. aurelia a pair of characters which he designated as "killer" and "sensitive." Fluid in which the killer race has lived kills individuals of the sensitive races. Race 51 is a killer, while races 29, 32, and 47 are sensitive. It appears that the killer and sensitive characters never occur together in the same individual. All progeny in race 51 are killers, and all progeny of the sensitive races are sensitive. When the pure killer race 51 is crossed with the pure sensitive race 32, the two exconjugants of each pair produce phenotypically different 188 PROTOZOOLOGY clones: one is a killer and the other is sensitive. He was able to dem- onstrate that the Fi killer clones are those that derive their cyto- plasm from the killer parent and the Fi sensitive clones are those which contain the cytoplasm of the sensitive parent — a result con- tradictory to the hitherto prevailing notion that the two exconju- gants possess the same genotype and should produce clones alike in their hereditary characters. Through a series of experiments, he has come to realize that there exist certain "relations between a gene and a cytoplasmic substance, both of which are required for the development of a hereditary character. When some of the cyto- plasmic substance is present, the gene controls its continued produc- tion; but when the cytoplasmic substance is absent, the gene cannot initiate its production. Addition of the cytoplasmic substance to an organism, lacking the character dependent on it, but containing the required gene, results in the continued production of the substance, in the development of the character determined by the combined presence of gene and cytoplasmic substance, and in the hereditary maintenance of the character in successive generations." References Calkins, G. N. 1924 Uroleptus mohilis. V. Jour. Exp. Zool., Vol. 41. De Garis, C. F. 1935 Heritable effects of conjugation between free individuals and double monsters in diverse races of Paramecium. Ibid., Vol. 71. FuRGASON, W. H. 1940 The significant cytostomal pattern of the "Glaucoma-Colpidium group," and a proposed new genus and species, Tetrahymena geleii. Arch. f. Protistenk., Vol. 94. Hegner, R. W. 1919 Heredity, variation, and the appearance of diversities during the vegetative reproduction of Arcella dentata Genetics, Vol. 4. HoARE, C, A. 1943 Biological races in parasitic Protozoa. Biol. Re- views, Vol. 18. Jennings, H. S. 1909 Heredity and variation in the simplest or- ganisms. Amer. Nat., Vol. 43. 1916 Heredity, variation and the results of selection in the uniparental reproduction of Difflugia corona. Genetics, Vol. 1. 1929 Genetics of the Protozoa. BibUographia Genetica, Vol. 5. 1937 Formation, inheritance and variation of the teeth in Difflugia corona. Jour. Exp. Zool., Vol. 77. 1939 Genetics of Paramecium hursaria. I. Mating types and groups, their interrelations and distribution; mating behavior and self-sterility. Genetics, Vol. 24. 1941 Inheritance in Protozoa. In G. N. Calkins and F. M. Summers (editors) : Protozoa in biological research. New York. VARIATION AND HEREDITY 189 , D. Raffel, R. S. Lynch and T. M. Sonneborn 1932 The diverse biotypes produced by conjugation within a clone of Paramecium. Jour. Exp. Zool., Vol. 63. JoLLOS, V. 1913 Experimentelle Untersuchungen an Infusorien Biol. Zentralbl, Vol. 33. — \ 1921 Experimentelle Protistenstudien. I. Untersuchungen iiber Variabilitat und Vererbung bei Infusorien. Arch. f. Pro- tistenk., Vol. 43. 1934 Dauermodifikationen und Mutationen bei Protozoen. Ibid., Vol. 83. Kimball, R. F. 1939 A delayed change of phenotype following a change of genotype in Paramecium aurelia. Genetics, Vol. 24. — 1939 Mating types in Euplotes. Amer. Nat., Vol. 73. 1942 The nature and inheritance of mating types in Eu- plotes 'patella. Genetics, Vol. 27. MacDougall, M. S. 1929 Modifications in Chilodon uncinatus produced by ultraviolet radiations. Jour. Exp. Zool., Vol. 54. 1931 Another mutation of Chilodon uncinatus produced by ultraviolet radiation, with a description of its maturation pro- cess. Ibid., Vol. 58. MoEWUS, F. 1934 Ueber Dauermodifikation bei Chlamydomona- den. Arch. f. Protistenk., Vol. 83. 1935 Ueber die Vererbung des Geschlechts bei Polytoma pascheri und bei Polytoma uvella. Zeitschr. Induk. Abstamm.- u. Vererb., Vol. 69. 1936 Faktorenaustausch, insbesondere der Realisatoren bei Chlamydomonas-Kreuzungen. Berichte deutsch. Bot. Ges., Vol. 54. 1938 Vererbung des Geschlechts bei Chlamydomonas eu- gametos und verwandten Arten. Biol. Zentralbl., Vol. 58 Reynolds, B. D. 1924 Interactions of protoplasmic masses in rela- tion to the study of heredity and environment in Arcella poly- pora. Biol. Bull,., Vol. 46. Root, F. M. 1918 Inheritance in the asexual reproduction in Cen- tropyxis aculeata. Genetics, Vol. 3. Sapero, J. J., E. G. Hakansson and C. M. Louttit 1942 The occurrence of two significantly distinct races of Endanioeha his- tolytica. Amer. Jour. Trop. Med., Vol. 22. Sonneborn, T. M. 1937 SeX; sex inheritance and sex determina- tion in Paramecium aurelia. Proc. Nat. Acad. Sci., Vol. 23. 1939 Paramecium aurelia: mating types and groups; lethal interactions; determination and inheritance. Amer. Nat., Vol. 73. — 1942 Inheritance in cihate Protozoa. Ibid., Vol. 76. 1943 Gene and Cytoplasm. I and II. Proc. Nat. Acad. Sci. Vol. 29. Taliaferro, W. H. 1926 Variability and inheritance of size in Try- panosoma lewisi. Jour. Exp. Zool., Vol. 43. 1929 The immunology of parasitic infections. New York. and C. G. Huff 1940 The genetics of the parasitic Pro- tozoa. Amer. Assoc. Adv. Sci., Publication No. 12. PART II: TAXONOMY AND SPECIAL BIOLOGY Chapter 7 Major groups and phylogeny of Protozoa THE Protozoa are grouped into two subphyla: Plasmodroma (p. 198) and Ciliophora (p. 545). The Plasmodroma are more primi- tive Protozoa and subdivided into three classes: Mastigophora (p. 198), Sarcodina (p. 328), and Sporozoa (p. 427). The Ciliophora possess more complex body organizations, and are divided into two classes : Ciliata (p. 545) and Suctoria (p. 695). In classifying Protozoa, the natural system would be one which is based upon the phylogenetic relationships among them in conform- ity with the doctrine that the present day organisms have descended from primitive ancestral forms through organic evolution. Unlike Metazoa, the great majority of Protozoa now existing do not possess skeletal structures, which condition also seemingly prevailed among their ancestors, and when they die, they disintegrate and leave nothing behind. The exceptions are Foraminifera (p. 394) and Radiolaria (p. 417) which produce multiform varieties of skeletal structures composed of inorganic substances and which are found abundantly preserved as fossils in the earliest fossiliferous strata. These fossils show clearly that the two classes of Sarcodina were already w^ell-difTerentiated groups at the time of fossilization. The sole information the palaeontological record reveals for our reference is that the differentiation of the major groups of Protozoa must have occurred in an extremely remote period of the earth history. There- fore, consideration of phylogeny of Protozoa had to depend ex- clusively upon the data obtained through morphological, physio- logical, and developmental observations of the present-day forms. The older concept which found its advocates until the beginning of the present century, holds that the Sarcodina are the most primi- tive of Protozoa. It was supposed that at the very beginning of the living world, there came into being undifferentiated mass of pro- toplasm which later became differentiated into the nucleus and the cytosome. The Sarcodina represented by amoebae and allied forms do not have any further differentiation and lack a definite body wall, they are, therefore, able to change body form by forming pseudopodia. These pseudopodia are temporary cytoplasmic proc- esses and formed or withdrawn freely, even in the more or less permanent axopodia. On the other hand, flagella and cilia are per- manent cell-organs possessing definite structural plans. Thus from 193 194 PROTOZOOLOGY the morphological viewpoint, the advocates of this concept main- tained that the Sarcodina are the Protozoa which were most closely related to ancestral forms and which gave rise to Mastigophora, Cihata, and Sporozoa. This concept is however difficult to follow, since it does not agree with the general belief that the plant came into existence before the animal ; namely, holophytic organisms living on inorganic substances anteceded holozoic organisms living on organic substances. There- fore, from the physiological standpoint the Mastigophora which include a vast number of chlorophyll-bearing forms, must be con- sidered as more primitive than the holozoic Sarcodina. The class Mastigophora is composed of Phytomastigina (chromatophore-bear- ing flagellates and closely related colorless forms) and Zoomastigina (colorless flagellates). Of the former, Chrysomonadina (p. 200) are mostly naked, and are characterized by possession of 1-2 flagella, 1-2 yellow chromatophores and leucosin. Though holophytic nutri- tion is general, many are also able to carry on holozoic nutrition. Numerous chrysomonads produce pseudopodia of different types; some possess both flagellum and pseudopodia; others such as Chrys- amoeba (p. 203) may show flagellate and ameoboid forms (Klebs; Scherffel); still others, for example, members of Rhizochrysidina (p. 209), may lack flagella completely, though retaining the char- acteristics of Chrysomonadina. When individuals of Rhizochrysis (p. 210) divide, Scherffel (1901) noticed unequal distribution of the chromatophore resulted in the formation of colorless and colored individuals (Fig. 94, a, h). Pascher (1917) also observed that in the colonial chrysomonad, Chrysarachnion (p. 210), the division of component individuals produces many in which the chromatophore is entirely lacking (Fig. 94, c, d). Thus these chrysomonads which lack chromatophores, resemble Sarcodina rather than the parent Chryosomonadina. Throughout all groups of Phytomastigina, there occur forms which are morphologically alike except the presence or absence of chromatophores. For example, Cryptomonas (p. 214) and Chilo- monas (p. 214), the two genera of Cryptomonadina, are so mor- phologically alike that had it not been for the chromatophore, the former can hardly be distinguished from the latter. Other examples are Euglena, Astasia, and Khawkinea; Chlorogonium and Hyalo- gonium; Chlamydomonas and Polytoma; etc. The chromatophores of various Phytomastigina degenerate read- ily under experimental conditions. For instance, Zumstein (1900) showed that Euglena gracilis loses its green coloration even in light MAJOR GROUPS AND PHYLOGENY 195 if cultured in fluids rich in organic substances ; in a culture fluid with a small amount of organic substances, the organisms retain green color in light, lose it in darkness; and when cultured in a pure inor- ganic culture fluid, the flagellates remain green even in darkness. Therefore, it would appear reasonable to consider that the mor- phologically similar forms with or without chromatophores such as are cited above, are closely related to each other phylogenetically, that they should be grouped together in any scheme of classification, and that the apparent heterogeneity among Ph3^tomastigina is due to the natural course of events. The newer concept which is at pres- ent followed widely is that the Mastigophora are the most primitive unicellular animal organisms. Of Mastigophora, Phytomastigina are to be considered on the same ground more primitive than Zoomastigina. According to the studies of Pascher, Scherffel and others, Chrysomonadina appear to be the nearest to ancestral forms from which other groups of Phyto- mastigina arose. Among Zoomastigina, Rhizomastigina possibly gave rise to Protomonadina, from which Polymastigina and Hyper- mastigina later arose. The last-mentioned group is the most highly advanced one of Mastigophora in which an increased number of flagella is an outstanding characteristic. As to the origin of Sarcodina, many arose undoubtedly from vari- ous Zoomastigina, but there are indications that they may have evolved directly from Phytomastigina. As was stated already, Rhizochrysidina possess no flagella and the chromatophore often de- generates or is lost through unequal distribution during division, apparently being able to nourish themselves by methods other than holophytic nutrition. Such forms may have given rise to Amoebina. Some chrysomonads such as Cyrtophora (p. 203) and Palatinella, have axopodia, and it may be considered that they are closer to the ancestral forms from which Heliozoa arose through stages such as shown by Actinomonas (p. 265), Dimorpha (p. 265), and Pteri- domonas (p. 265) than any other forms. Another chrysomonad, Porochrysis (p. 204), possesses a striking resemblance to Testacea. The interesting marine |chrysomonad, Chrysothylakion (p. 210) that produces a brownish calcareous test from which extrudes an- astomosing rhizopodial network, resembling a monothalamous foraminiferan, and forms such as Distephanus (p. 209) with siliceous skeletons, may depict the ancestral forms of Foraminifera and Radiolaria respectively. The flagellate origin of these two groups of Sarcodina is also seen in the appearance of flagellated swarmers dur- ing their development. The Mycetozoa show also flagellated phase 196 PROTOZOOLOGY during their life cycle, which perhaps suggests their origin in flagel- lated organisms. In fact, in the chrysomonad Myxochrysis (p. 205), Pascher (1917) finds a multinucleate and chromatophore-bearing organism (Fig. 90, e-j) that stands intermediate between Chr3^so- monadina and Mycetozoa. Thus there are a number of morpho- logical, developmental, and physiological observations which sug- gest the flagellate origin of various Sarcodina. The Sporozoa appear to be equally polyphyletic. The Telosporidia contain three groups in which flagellated microgametes occur, which suggests their derivation from flagellated organisms. Leger and Duboscq even considered them to have arisen from Bodonidae (p. 289) on the basis of flagellar arrangement. Obviously Gregarinida are the most primitive of the three groups. The occurrence of such a form as Selenococcidium (p. 466), would indicate the gregarine- origin of the Coccidia and the members of Haemogregarinidae (p. 480) suggest the probable origin of the Haemosporidia in the Coc- cidia. The Cnidosporidia are characterized by multinucleate tro- phozoites and by the spore in which at least one polar capsule with a coiled filament occurs. Some consider them as having evolved from Mycetozoa-like organisms, because of the similarity in multi- nucleate trophozoites, while others compare the polar filament with the flagellum. It is interesting to note here that the nematocyst, similar to the polar capsule, occurs in certain Dinoflagellata (p. 245) independent of flagella. The life cycle of Acnidosporidia is still in- completely known, but the group may have differentiated from such Sarcodina as Mycetozoa. The Ciliata and Suctoria are distinctly separated from the other groups. They possess the most complex body organization seen among Protozoa. All ciliates possess cilia or cirri which differ from flagella essentially only in size. Apparently Protociliata and Eucili- ata have different origins, as judged by their morphological and physiological differences. It is probable that Protociliata arose from forms which gave rise to Hypermastigina. Among Euciliata, one finds such forms as Coleps, Urotricha, Plagiocampa, Microregma, Trimyema, Anophrys, etc., which have, in addition to numerous cilia, a long flagellum-like process at the posterior end, and Ileonema that possesses an anterior vibratile flagellum and numerous cilia, which also indicates flagellated organisms as their ancestors. It is reasonable to assume that Holotricha are the most primitive ciliates from which Spirotricha, Chonotricha, and Peritricha evolved. The Suctoria are obviously very closely related to Ciliata and most prob- ably arose from ciliated ancestors by loss of cilia during adult stage MAJOR GROUPS AND PHYLOGENY 197 and by developing tentacles in some forms from cytostomes as was suggested by Collin (Fig. 13). References BuTSCHLi, O. 1883-1887 Bronn's Klassen und Ordnungen des Thierreichs. Vol 1. DoFLEiN, F. and E. Reichenow 1929 Lehrhuch der Protozoenkunde. Jena. MiNCHiN, E. A. 1912 Introduction to the study of the Protozoa. Lon- don. Pascher, a. 1912 Ueber Rhizopoden- und Palmellastadien bei Flagellaten. Arch. f. Protistenk.,, Vol. 25. 1917 Rhizopodialnetz als Fangvorrichtung bei einer Plas- modialen Chrysomonade. Ibid., Vol. 37. 1917 Fusionsplasmodien bei Flagellaten und ihre Bedeut- ung fiir die Ableitung der Rhizopoden von den Flagellaten. Ibid. 1917 Flagellaten und Rhizopoden in ihren gegenseitigen Beziehungen. Ibid., Vol. 38. ScHERFFEL, A. 1901 Kleiner Beitrag zur Phylogenie einiger Grup- pen niederer Organismen. Bot. Zeit., Vol. 59. ZuMSTEiN, H. 1900 Zur Morphologic von Euglena gracilis Klebs. Pringsheims Jahrb., Vol. 34. T Chapter 8 Phylum Protozoa Goldfuss Subphylum 1 Plasmodroma Doflein HE Plasmodroma possess pseudopodia which are used for loco- motion and food-getting or flagella that serve for cell-organs of locomotion. In Sporozoa, the adult stage does not possess any cell- organs of locomotion. The body structure is less complicated than that of Ciliophora. In some groups, are found various endo- and exo-skeletons. The nucleus is of one kind, but may vary in number. Nutrition is holozoic, holophytic, or saprozoic. Sexual reproduction is exclusively by sexual fusion ; asexual reproduction is by binary or multiple fission or budding. The majority are free-living, but numer- ous parasitic forms occur, Sporozoa being all parasitic. The Plasmodroma are subdivided into three classes as follows: Trophozoite with flagellum Class 1 Mastigophora Trophozoite with pseudopodium Class 2 Sarcodina (p. 328) Without cell-organs of locomotion; producing spores; all parasitic Class 3 Sporozoa (p. 427) Class 1 Mastigophora Diesing The Mastigophora includes those Protozoa which possess one to several flagella. Aside from this common characteristic, this class makes a very heterogeneous assemblage and seems to prevent a sharp distinction between the Protozoa and the Protophyta, as it includes Phytomastigina which are often dealt with by botanists. In the majority of Mastigophora, each individual possesses 1-4 flagella during the vegetative stage, although species of Polymasti- gina may possess up to 8 or more flagella and of Hypermastigina a greater number of flagella. The palmella stage (Fig. 88) is common among the Phytomastigina and the organism is capable in this stage not only of metabolic activity and growth, but also of reproduction. In this respect, this group shows also a close relationship to algae. All three types of nutrition, carried on separately or in combina- tion, are to be found among the members of Mastigophora. In holo- phytic forms, the chlorophyll is contained in the chromatophores which are of various forms among different species and which differ in colors, from green to red. The difference in color appears to be due to the pigments which envelop the chlorophyfl body (p. 78). Many forms adapt their mode of nutrition to changed environmental con- 198 MASTIGOPHORA, CHRYSOMONADINA 199 ditions ; for instance, from holophytic to saprozoic in the absence of the simhght. Holozoic, saprozoic and holophytic nutrition are said to be combined in such a form as Ochromonas. In association with chromatophores, there occurs refractile granules or bodies, the pyrenoids, which are connected with starch-formation. Reserve food substances are starch, oil, etc, (p. 98). In less complicated forms, the body is naked except for a slight cortical differentiation of the ectoplasm to delimit the body surface and is capable of forming pseudopodia. In others, there occurs a thin plastic pellicle produced by the cytoplasm, which covers the body surface closely. In still others, the body form is constant, being en- cased in a shell, test, or lorica, which is composed of chitin, pseudo- chitin, or cellulose. Not infrequently a gelatinous secretion envelops the body. In three families of Protomonadina there is a collar-like structure located at the anterior end, through which the flagellum protrudes. The great majority of Mastigophora possess a single nucleus, and only a few are multinucleated. The nucleus is vesicular and contains a conspicuous endosome. Contractile vacuoles are always present in the forms inhabiting fresh water. In simple forms, the contents of the vacuoles are discharged directly through the body surface to the exterior; in others there occurs a single contractile vacuole near a reservoir which opens to the exterior through the so-called cyto- pharynx. In the Dinoflagellata, there are apparently no contractile vacuoles, but non-contractile pusules (p. 246) occur in some forms. In chromatophore-bearing forms, there occurs usually a stigma which is located near the base of the flagellum and seems to be the center of phototactic activity of the organism which possesses it. Asexual reproduction is, as a rule, by longitudinal fission, but in some forms multiple fission also takes place under certain circum- stances, and in others budding may take place. Colony-formation (p. 145), due to incomplete separation of daughter individuals, is widely found among this group. Sexual reproduction has been re- ported in a number of species. The Mastigophora are free-living or parasitic. The free-living forms are found in fresh and salt waters of every description; many are free-swimming, others creep over the surface of submerged ob- jects, and still others are sessile. Together with algae, the Mastigoph- ora compose a major portion of plankton life which makes the foundation for the existence of all higher aquatic organisms. The parasitic forms are ecto- or endo-parasitic, and the latter inhabit either the digestive tract or the circulatory system of the host ani- 200 PROTOZOOLOGY mal. Trypanosoma, a representative genus of the latter group, in- cludes important disease-causing parasites of man and of domestic animals. The Mastigophora are divided into two subclasses as follows : With chroraatophores Subclass 1 Phytomastigina Without chromatophores Subclass 2 Zoomastigina (p. 263) Subclass 1 Phytomastigina Doflein The Phytomastigina possess the chromatophores and their usual method of nutrition is holophytic, though some are holozoic, sapro- zoic or mixotrophic; the majority are conspicuously colored; some that lack chromatophores are included in this group, since their structure and development resemble closely those of typical Phyto- mastigina. 1-4 flagella, either directed anteriorly or trailing Chromatophores yellow, brown or orange Anabolic products fat, leucosin Order 1 Chrysomonadina Anabolic products starch or similar carbohydrates Order 2 Cryptomonadina (p. 213) Chromatophores green Anabolic products starch and oil. Order 3 Phytomonadina (p. 217) Anabolic products paramylon Order 4 Euglenoidina (p. 232) Anabolic products oil Order 5 Chloromonadina (p. 243) 2 flagella, one of which transverse Order 6 Dinoflagellata (p. 245) Order 1 Chrysomonadina Stein The chrysomonads are minute organisms and are plastic, since the majority lack a definite cell-wall. Chromatophores are yellow to brown and usually discoid, though sometimes reticulated, in form. Metabolic products are leucosin and fats. Starches have not been found in them. 1-2 flagella are inserted at or near the anterior end of body where a stigma is present. Many chrysomonads are able to form pseudopodia for obtaining food materials which vary among different species. Nutrition, though chiefly holophytic, is also holozoic or saprozoic. Contractile vacuoles are invariably found in freshwater forms, and are ordinarily of simple structure. Under conditions not fully understood, the chrysomonads lose their flagella and undergo division with development of mucilaginous envelope and thus transform themselves often into large bodies known as the palmella phase and undertake metabolic activities as well as multiplication (Fig. 88). Asexual reproduction is, as a rule, by longitudinal division during either the motile or the palmella MASTIGOPHORA, CHRYSOMONADINA 201 stage. Incomplete separation of the daughter individuals followed by repeated fission, results in numerous colonial forms mentioned elsewhere (p. 146). Some resemble higher algae very closely. Sexual (^^ Fig. 88. The life-cycle of Chromulina, X about 200 (Kiihn). a, encyst- ment; b, fission; c, colony-formation; d, palmella-formation. reproduction is unknown in this group. Encystment occurs com- monly ; in this the fiagellum is lost and the cyst is often enveloped by a silicious wall possessing an opening with a plug. The chrysomonads inhabit both fresh and salt waters, often occur- ring abundantly in plankton. Motile stage dominant Suborder 1 Euchrysomonadina Palmella stage dominant Sarcodina-like; flagellate stage unknown Suborder 2 Rhizochrysidina (p. 209) With flagellate phase Suborder 3 Chrysocapsina (p. 210) Suborder 1 Euchrysomonadina Pascher With or without simple shell One flagellum Family 1 Chromulinidae 2 flagella Flagella equally long Family 2 Syncryptidae (p. 205) Flagella unequally long Family 3 Ochromonadidae (p. 206) With calcareous or silicious shell Bearing calcareous discs and rods. . . .Family 4 Coccolithidae (p. 208) Bearing silicious skeleton. Family 5 Silicoflagellidae (p. 209) Family 1 Chromulinidae Engler Minute forms, naked or with sculptured shell; with a single flagel- lum; often with rhizopodia; a few colonial; free-swimming or at- tached. 202 PROTOZOOLOGY Genus Chromulina Cienkowski. Oval; round in cross-section; amoeboid; 1-2 chromatophores ; palmella stage often large; in fresh water. Numerous species. The presence of a large number of these organisms gives a golden-brown color to the surface of the water. Fig. 89. a, b, Chromulina pascheri, X670 (Hofeneder); c, Chrysapsis sagene, XlOOO (Pascher); d, Chrysococcus ornatus, X600 (Pascher); e, Mallomonas litomosa, X400 (Stokes); f, Pyramidochrysis modesta, X670 (Pascher); g, Sphalero mantis ochracea, X600 (Pascher); h, Kephyrion ovum, X1600 (Pascher); i, Chrysopyxis cyathus, X600 (Pascher); j, Gyrtophora pedicellata, X400 (Pascher); k, Palaiinella cyrtophora, X400 (Lauterborn);!, Chrysosphaerellalongispina, X600 (Lauterborn). MASTIGOPHORA, CHRYSOMONADINA 203 C. pascheri Hofeneder (Fig. 89, a, b). 15-20)Lt in diameter. Genus Chrysamoeba Klebs. Body naked; flagellate stage ovoid, with 2 chromatophores, sometimes slender pseudopodia at the same time; flagellum may be lost and the organism becomes amoeboid, resembling Rhizochrysis (p. 210); standing fresh water. C. radians K. (Fig. 90, a, b). Flagellated form 16-20^ long; amoe- boid stage about 15/x with 10-20^ long radiating pseudopodia; fresh water. Genus Chrysapsis Pascher. Solitary; plastic or rigid; chromato- phore diffused or branching; with stigma; amoeboid movement; holophytic, holozoic; fresh water. Several species. C. sagene P. (Fig. 89, c). Anterior region actively plastic; stigma small; 8-14/n long; flagellum about SO/x long. Genus Chrysococcus Klebs. Shell spheroidal or ovoidal, smooth or sculptured and often brown-colored; through an opening a flagel- lum protrudes; 1-2 chromatophores; one of the daughter individuals formed by binary fission leaves the parent shell and forms a new one; fresh water. C. ornatus Pascher (Fig. 89, d). 14-16^ by 7-10m. Genus Mallomonas Perty (Pseudomallomonas Chodat). Body elongated; with silicious scales and often spines; 2 chromatophores rod-shaped; fresh water. Numerous species. M. litomosa Stokes (Fig. 89, e). Scales very delicate, needle-like projections at both ends; flagellum as long as body; 24-32^ by 8/x. Genus Pyramidochrysis Pascher. Body form constant; pyriform with 3 longitudinal ridges; flagellate end drawn out; a single chro- matophore; 2 contractile vacuoles; fresh water. P. modesta P. (Fig. 89,/). 11-13^ long. Genus Sphaleromantis Pascher. Triangular or heart-shaped; highly flattened; slightly plastic; 2 chromatophores; 2 contractile vacuoles; stigma large; long flagellum; fresh water. S. ochracea P. (Fig. 89, g). 6-13^ long. Genus Kephyrion Pascher. With oval or fusiform lorica; body fills posterior half of lorica; one chromatophore; a single short flagellum; small; fresh water. K. ovum P. (Fig. 89, h). Lorica up to 7n by 4^. Genus Chrysopyxis Stein. With lorica of various forms, more or less flattened; 1-2 chromatophores; a flagellum; attached to algae in fresh water. C. cyathus Pascher (Fig. 89, i). One chromatophore; flagellum twice body length; lorica 20-25^ by 12-15/x. Genus Cyrtophora Pascher. Body inverted pyramid with 6-8 204 PROTOZOOLOGY axopodia and a single flagellum; with a contractile stalk; a single chromatophore ; a contractile vacuole; fresh water. C. pedicellata P. (Fig. 89, j). Body 18-22^ long; axopodia 40-60m long ; stalk 50-80^ long. Genus Palatinella Lauterborn. Lorica tubular; body heartshaped; Fig. 90. a, flagellate and b, amoeboid phase of Chrysamoeba radians, X670 (Klebs); c, surface view and d, optical section of Porochrysis asper- gillus, X400 (Pascher); e-j, Myxochrysis paradoxa (Pascher). e, a medium large Plasmodium with characteristic envelop; the large food vacuole contains protophytan, Scenedesmus, X830; f, diagrammatic side view of a Plasmodium, engulfing a diatom; moniliform bodies are yellowish chromatophores, XlOOO; g-i, development of swarmer into Plasmodium (stippled bodies are chromatophores), X1200. anterior border with 16-20 axopodia; a single flagellum; a chromato- phore; several contractile vacuoles'; fresh water. P. cyrtophora L. (Fig. 89, k). Lorica 80-1 50/i long; body 20-25^ by 18-25/i; axopodia 50)u long. Genus Chrysosphaerella Lauterborn. In spherical colony, indivi- dual cell, oval or pyriform, with 2 chromatophores; imbedded in gelatinous mass ; fresh water. C. longispina L. (Fig. 89, I). Individuals up to 15/i by 9//; colony up to 250m in diameter; in standing water rich in vegetation. Genus Porochrysis Pascher. Shell with several pores through which rhizopodia are extended; a flagellum passes through an apical MASTIGOPHORA, CHRYSOMONADINA 205 pore; a single small chromatoi^hore; leucosin grain, contractile vacuole; fresh water. P. aspergillus P. (Fig. 90, c, d). Shell about 35/i long by 25m wide; chromatophore very small ; a large leucosin grain ; fresh water. Genus Myxochrysis Pascher. Body multinucleate, amoeboid; with yellowish moniliform chromatophores, many leucosin granules and contractile vacuoles; holozoic; surrounded by a brownish envelop which conforms with body form; flagellated swarmers develop into multinucleate Plasmodium; i)lasmotomy and plasmogamy; fresh water. M. paradoxa P. (Fig. 90, e-j). Plasmodium 15-18/i or more in diameter; in standing water. Family 2 Syncryptidae Poche Solitary or colonial chrysomonads with 2 equal flagella; with or without pellicle (when {)resent, often sculptured) ; some possess stalk. Genus Syncrypta Ehrcnbcrg. Spherical colonies; individuals with 2 lateral chromatophores, embedded in a gelatinous mass; 2 con- tractile vacuoles; without stigma; cysts unknown; fresh water. S. volvox E. (Fig. 91, a). 8-14^ by7-12M; colony 20-70ai in diam- eter; in standing water. Genus Synura Ehrenberg. Spherical or ellipsoidal colony com- posed of 2-50 ovoid individuals arranged radially; body usually covered by short bristles; 2 chromatophores lateral; no stigma; asexual reproduction of individuals is by longitudinal division ; that of colony by bipartition ; cysts spherical ; fresh water. S. uvella E. (Fig. 91, 6). Cells oval; bristles conspicuous; 20-40)u by 8-17/x; colony 100-400^ in diameter; if present in large numbers, the organism is said to be responsible for an odor of the water re- seml)ling that of ripe cucumber (Moore). aS. adamsi Smith (Fig. 91, c). Spherical colony with individuals radiating; individuals long spindle, 42-47^ by 6.5-7m; 2 flagella up to 17ai long; in fresh water i)ond. Genus Hymenomonas Stein. Solitary; ellipsoid to cylindrical; membrane brownish, often sculptured; 2 chromatophores; without stigma; a contractile vacuole anterior; fresh water. H. roseola S. (Fig. 91, d). 17-50m by 10-20^. Genus Derepyxis Stokes. With cellulose lorica, with or without a short stalk; body ellipsoid to spherical with 1-2 chromatophores; 2 equal flagella; fresh water. D. amphora S. (Fig. 91, c). Lorica 25-30m by 9-18^; on algae in standing water. 206 PROTOZOOLOGY D. ollula S. (Fig. 91,/). Lorica 20-25^ by 15^. Genus Stylochrysallis Stein. Body fusiform; with a gelatinous stalk attached to Volvocidae; 2 equal fiagella; 2 chromatophores; without stigma; fresh water. S. parasita S. (Fig. 91, g). Body 9-1 l^u long; stalk about 15/x long; on phytomonads. Fig. 91. a, Syncrypta volvox, X430 (Stein); b, Synura uvella, X500 (Stein); c, S. admnsi, X280 (Smith); d, Hymenomonas roseola, X400 (Klebs); e, Derepyxis amphora, X540 (Stokes); f, D. ollula, X600 (Stokes); g, Stylochrysallis parasitica, X430 (Stein). Family 3 Ochromonadidae Pascher With 2 unequal fiagella ; no pellicle and plastic ; contractile vacu- oles simple; with or without a delicate test; solitary or colonial; free-swimming or attached. Genus Ochromonas W3^ssotzki. Solitary or colonial; body surface delicate; posterior end often drawn out for attachment; 1-2 chro- matophores; usually with a stigma; encystment; fresh water. Many species. 0. muiahilis Klebs (Fig. 92, a). Ovoid to spherical; plastic, 15-30m by 8-22/i. 0. ludibunda Pascher (Fig. 92, b). Not plastic; 12-17m by 6-12^. Genus Uroglena Ehrenberg. Spherical or ovoidal colony, com- posed of ovoid or ellipsoidal individuals arranged on periphery of a MASTIGOPHORA, CHRYSOMONADINA 207 gelatinous mass; all individuals connected with one another by gelatinous processes running inward and meeting at a point; with a stigma and a plate-like chromatophore; asexual reproduction of Fig. 92. a, Ochromonas mutabilis, X670 (Senn); b, 0. ludihunda, X540 (Pascher); c, Uroglena volvox, X430 (Stein); d, Uroglenopsis americana, X470 (Lemmermann) ; e, Cyclonexis annularis, X540 (Stokes); f, Dino- bryon sertularia, X670 (Scherffel) ; g, Hyalohryonramosum, X540 (Lauter- born); h, Stylopyxis nmcicola, X470 (Bolochonzew). individuals by longitudinal fission, that of colony by bipartition; cysts spherical with spinous projections, and a long tubular process; fresh water. One species. U. volvox E. (Fig. 92, c). Cells 12-20m by 8-13m; colony 40-400/* in diameter; in standing water. Genus Uroglenopsis Lemmermann. Similar to Uroglena, but individuals without inner connecting processes. U. americana (Calkins) (Fig. 92, d). Each cell with one chro- matophore; 5-8^ long; fiagellum up to 32/i long; colony up to 300^ 208 PROTOZOOLOGY in diameter; when present in abundance, the organism gives an of- fensive odor to the water (Calkins). U. europaea Pascher. Similar to the last-named species; but chromatophores 2; cells up to 7/i long; colony 150-300^ in diameter. Genus Cyclonexis Stokes. Wheel-like colony, composed of 10-20 wedge-shaped individuals; young colony funnel-shaped; chromato- phores 2, lateral; no stigma; reproduction and encystment unknown; fresh water. C. annularis S. (Fig. 92, e). Cells 11-14^ long; colony 25-30^ in diameter; in marshy water with sphagnum. Genus Dinobryon Ehrenberg. Solitary or colonial; individuals with vase-like, hyaline, but sometimes, yellowish cellulose test, drawn out at its base; elongated and attached to the base of test with its attenuated posterior tip; 1-2 lateral chromatophores; usually with a stigma; asexual reproduction by binary fission; one of the daughter individuals leaving test as a swarmer, to form a new one; in colonial forms daughter individuals remain attached to the inner margin of aperture of parent tests and there secrete new tests; encystment common; the spherical cysts possess a short process; Ahlstrom (1937) studied variability of North American species and found the organisms occur more commonly in alkaline regions than elsewhere; fresh water. Numerous species. D. sertularia E. (Fig. 92,/). 30-44^ by 10-14^. D. divergens Imhof. 31-53)U long; great variation in different lo- calities (Ahlstrom). Genus Hyalobryon Lauterborn. Solitary or colonial; individual body structure similar to that of Dinobryon; lorica in some cases tubular, and those of 'young individuals are attached to the exterior of parent tests; fresh water. H. ramosum L. (Fig. 92, g). Lorica 50-70iu long by 5-9iu in diame- ter; body up to 30m by 5ix; on vegetation in standing fresh water. Genus Stylopyxis Bolochonzew. Solitary; body located at bottom of a delicate stalked lorica with a wide aperture; 2 lateral chromato- phores; fresh water. S. mucicola B. (Fig. 92, h). Lorica 17-18m long; stalk about 33/^ long; body 9-1 Iju long: fresh water. Family 4 Coccolithidae Lohmann The members of this family occur, with a few exceptions, in salt water only; with perforate (tremalith) or imperforate (discolith) discs, composed of calcium carbonate; 1-2 flagella; 2 yellowish MASTIGOPHORA, CHRYSOMONADINA 209 chromatophores ; a single nucleus; oil drops and leucosin; holophytic. Examples : Pontosphaera haeckeli Lohmann (Fig. 93, a). , Discosphaera tubifer Murray and Blackman (Fig. 93, h). Family 5 Silicoflagellidae Borgert Exclusively marine planktons; with siliceous skeleton which en- velops the body. Example: Distephanus speculum (Miiller) (Fig. 93, c). Fig. 93. a, Pontosphaera haeckeli, X1070 (Kiihn); b, Discosphaera tubi- fer, X670 (Klihn); c, Distephanus speculum, X530 (Kiihn); d, Rhizo- chrijsis scherffeli, X670 (Doflein); e-g, Hydrurus foetidus (e, entire colony; f, portion; g, cyst), e (Berthold), f, X330, g, X800 (Klebs); h, i, Chrysocapsa paludosa, X530 (West); j, k, Phaeosphaera gelatinosa (j, part of a mass, X70; k, three cells, X330) (West). Suborder 2 Rhizochrysidina Pascher No flagellate stage is known to occur; the organism possesses pseu- dopodia; highly provisional group, based wholly upon the absence of flagella; naked or with test; various forms; in some species chroma- 210 PROTOZOOLOGY tophores are entirely lacking, so that the organisms resemble some members of the Sarcodina. Several genera. Genus Rhizochrysis Pascher. Body naked and amoeboid; with 1-2 chromatophores : fresh water. R. scherffeli P. (Figs. 93, d; 94, a, h). 10-14/i in diameter; 1-2 chromatophores : branching rhizopods ; fresh water. Genus Chrysidiastrum Lauterborn. Naked; spherical; often sev- eral in linear association by pseudopodia; one j^ellow-brown chro- matophore; fresh water. C. catenaium L. Cells 12-14)u in diameter. Genus Chrysarachnion Pascher. Ameboid organism; with a chro- matophore, leucosin grain and contractile vacuole; many individuals arranged in a plane and connected by extremely fine rhizopods, the whole forming a cobweb network. Small animals are trapped by the net; chromatophores are small; nutrition both holophytic and holo- zoic; during division the chromatophore is often unevenly distrib- uted so that many individuals without any chromatophore are produced; fresh water. C. insidians P. (Fig. 94, c, d). Highly amoeboid individuals 3-4^ in diameter; chromatophore pale yellowish brown, but becomes blu- ish green upon death of organisms; a leucosin grain and a contractile vacuole; colony made up of 200 or more individuals. Genus Chrysothylakion Pascher. With retort-shaped calcareous shell with a bent neck and an opening; shell reddish brown (with iron) in old individuals ; through the aperture are extruded extremely fine anastomosing rhizopods; protoplasm which fills the shell is colorless; a single nucleus, two spindle-form brown chromatophores, several contractile vacuoles and leucosin body; marine water. C. vorax P. (Fig. 94, e,f). The shell measures 14-18/x long, 7-10/x broad, and 5-6/x high; on marine algae. Suborder 3 Chrysocapsina Pascher Palmella stage prominent; flagellate forms transient; colonial; individuals enclosed in a gelatinous mass; 1-2 flagella, one chromato- phore, and a contractile vacuole; one group of relatively minute forms and the other of large organisms. Genus Hydrurus Agardh. In a large (1-30 cm. long) branching gelatinous cylindrical mass; cells yellowish brown; spherical to ellipsoidal; with a chromatophore; individuals arranged loosely in gelatinous matrix; apical growth resembles much higher algae; mul- tiplication of individuals results in formation of pyrimidal forms MASTIGOPHORA, CHRYSOMONADINA 211 with a flagellum, a chromatophore, and a leucosin mass; cyst may show a wing-like rim; cold freshwater streams. Fig. 94. a, b, Rhizochrysis scherffeli, X500 (Scherffel). a, 4 chroma- tophore-bearmg individuals and an individual without chromatophore- b, the last-mentioned individual after 7 hours, c, d, Chrysarachnion insi- dians (Pascher). c, part of a colony composed of individuals with and without chromatophore, X1270; d, products of division, one individual lacks chromatophore, but with a leucosin body, X2530. e, f, Chrysothy- lakion vorax (Pascher). e, an individual with anastomosing rhizopodia and excretion granules," X870; f, optical section of an individual; the cyto- plasm contains two fusiform brownish chromatophores, a spheroid nucleus, a large leucosin body and contractile vacuole, X about 1200. 212 PROTOZOOLOGY H. foetidus Kirschner (Figs. 31, d-f; 93, e-g). Olive-green, feath- ery tufts, 1-30 cm. long, develops an offensive odor; sticky to touch; occasionally encrusted with calcium carbonate; in running fresh water. Genus Chrysocapsa Pascher. In a spherical to ellipsoidal gelati- nous mass; cells spherical to ellipsoid; 1-2 chromatophores; wither without stigma; freshwater. C. paludosa P. (Fig. 93, h, i). Spherical or ellipsoidal with cells distributed without order; with a stigma; 2 chromatophores; swarmer pyriform with 2 flagella; cells 11^ long; colony up to lOO/x in diameter. Genus Phaeosphaera West and West. In a simple or branching cylindrical gelatinous mass; cells spherical with a single chroma- tophore ; fresh water. P. gelatinosa W. and W. (Fig. 93, j, k). Cells 14-17. 5^ in diameter. References BtJTSCHLi, O. 1883-1887 Mastigophora. In: Bronn's Klassen und Ordnungen des Thierreichs. Vol. 1, part 2. DoFLEiN, F. and E. Reichenow. 1929 Lehrhuch der Protozoen- kunde. Jena. Kent, S. 1880-1882 A manual of Infusoria. London. Pascher, A. 1914 Flagellatae: Allgemeiner Teil. In: Die Siisswas- serflora Deutschlands. Part 1. Stein, F. 1878, 1883 Der Organismus der Infusionsthiere. 3 Abt. Der Organismus der Flagellate oder Geisselinfusorien. Parts 1 and 2. Leipzig. Ahlstrom, E. H. 1937 Studies on variability in the genus Dino- bryon (Mastigophora). Trans. Amer. Micr. Soc, Vol. 56. Fritsch, F. E. 1935 The structure and reproduction of the algae. Cambridge. Pascher, A. 1916 Studien iiber die rhizopodiale Entwicklung der Flagellaten. Arch. f. Protistenk., Vol. 36. 1917 Rhizopodialnetz als Fangvorrichtung bei einer plas- modialen Chxysomonade. Ibid., Vol. 37. 1917 Fusionsplasmodien bei Flagellaten und ihre Bedeut- ung fiir die Ableitung der Rhizopoden von den Flagellaten. Ibid. 1917 Flagellaten und Rhizopoden in ihren gegenseitigen Beziehungen. Ibid., Vol. 38. Scherffel, a. 1901 Kleiner Beitrag zur Phylogenie einiger Grup- pen niederer Organismen. Bot. Zeit., Vol. 59. Smith, G. M. 1933 The freshwater algae of the United States. New York. West, G. S. and F. E. Fritsch 1927 A treatise on the British fresh- water algae. Cambridge. Chapter 9 Order 2 Cryptomonadina Stein THE cryptomonads differ from the chrysomonads in having a constant body form. Pseudopodia are very rarely formed, as the body is covered by a pelHcle. The majority show dorso-ventral differentiation, with an oblique longitudinal furrow. 1-2 unequal flagella arise from the furrow or from the cytopharynx. In case 2 flagella are present, both may be directed anteriorly or one poster- iorly. These organisms are free-swimming or creeping. One or two chromatophores are usually present. They are discoid or band-form. The color of chromatophores varies: yellow, brown, red, olive-green. The nature of the pigment is not well understood, but it is said to be similar to that which is found in the Dinofiagel- lata (Pascher). One or more spherical pyrenoids which are enclosed within a starch envelope appear to occur outside the chromato- phores. Nutrition is mostly holophytic; a few are saprozoic or holo- zoic. Assimilation products are solid discoid carbohydrates which stain blue with iodine in Cryptomonas or which stain reddish violet by iodine as in Cryptochrysis; fat and starch are produced in holo- zoic forms which feed upon bacteria and small Protozoa. The stigma is usually associated with the insertion point of the flagella. Con- tractile vacuoles, one to several, are simple and are situated near the cj^topharynx. A single vesicular nucleus is ordinarily located near the middle of the body. Asexual reproduction, by longitudinal fission, takes place in either the active or the non-motile stage. Sexual reproduction is un- known. Some cryptomonads form palmella stage and others gelati- nous aggregates. In the suborder Phaeocapsina, the palmella stage is permanent. Cysts are spherical, and the cyst wall is composed of cellulose. The Cryptomonadina occur in fresh or sea water, living also often as symbionts in marine organisms. Flagellate forms predominant Suborder 1 Eucryptomonadina Palmella stage permanent Suborder 2 Phaeocapsina (p. 216) Suborder 1 Eucryptomonadina Pascher Truncate anteriorly; 2 anterior flagella; with an oblique furrow near anterior end Family 1 Oryptomonadidae (p. 214) Reniform; with 2 lateral flagella; furrow equatorial Family 2 Nephroselmidae (p. 215) 213 214 PROTOZOOLOGY Family 1 Cryptomonadidae Stein Genus Cryptomonas Ehrenberg. Body elliptical with a firm pel- licle; anterior end truncate; dorsal side convex, ventral side slightly so or flat; nucleus posterior; longitudinal furrow; tubular cavity extending to the middle of body, through which equally long flagella arise; 2 lateral chromatophores vary in color from green to blue- green, brown or rarely red; holophytic; with small starch-like bodies Fig. 95. a, Cryptomonas ovata, X800 (Pascher); b, Chilomonas Para- mecium, X1330 (Biitschli); c, d, ChrysideUa schaiidinni, X1330 (Winter); e, Cyathomonas truncata, X670 (Ulehla); f, Cryptochrysis commutata, X 670 (Pascher); g, Rhodomonas lens, X1330 (Ruttner); h, Nephroselmis olvacea, X670 (Pascher); i, Protochrysis phaeophycearum, X800 (Pascher); j, k, Phaeothamnion confer vicoluvi, X600 (Kiihn). which stain blue in iodine; 1-3 contractile vacuoles anterior; fresh water. Several species. C. ovata E. (Fig. 95, a). 20-30/x long; among vegetation. Genus Chilomonas Ehrenberg. Similar to Cryptomonas in general body form and structure, but colorless because of the absence of chromatophores; without pyrenoid; cytopharynx deep, lower half marked by "rudimentary trichocysts" ; 1-2 contractile vacuoles, anterior; nucleus in posterior half; endoplasm often filled with poly- gonal starch grains ; fresh water. C. Paramecium E. (Fig. 95, h). Posterior end narrowed, slightly bent "dorsally"; 20-40^^ long; saprozoic; widely distributed in stag- nant water and hay infusion. CRYPTOMONADINA 215 C. ohlonga Pascher. Oblong; posterior end broadly rounded; 20- 50/1 long. Genus Chrysidella Pascher. Somewhat similar to Cryptomonas, but much smaller; yellow chromatophores much shorter; those oc- curring in Foraminifera or Radiolaria as symbionts are known as Zooxanthellae. Several species. C. schaudinni (Winter) (Fig. 95, c, d). Body less than 10/x long; in the f oraminiferan Peneroplis pertusus. Genus Cyathomonas Fromentel. Body small, somewhat oval; without chromatophores; much flattened; anterior end obliquely truncate; with 2 equal or subequal anterior flagella; colorless; nu- cleus central; anabolic products, stained red or reddish violet by iodine; contractile vacuole usually anterior; a row of refractile granules, protrichocysts (p. 65), close and parallel to anterior margin of body; asexual reproduction by longitudinal fission; holozoic; in stagnant water and infusion. One species. C. truncata Ehrenberg (Fig. 95, e). 15-30iu long. Genus Cryptochrysis Pascher. Furrow indistinctly granulated; 2 or more chromatophores brownish, olive-green, or dark green, rarely red; pyrenoid central; 2 equal flagella; some lose flagella and may assume amoeboid form; fresh water. C. commutata P. (Fig. 95, /). Bean-shaped; 2 chromatophores; 19m by 10m. Genus Rhodomonas Karsten. Furrow granulated; chromatophore one, red (upon degeneration the coloring matter becomes dissolved in water) ; pyrenoid central ; fresh water. R. lens Pascher and Ruttner (Fig. 95, g). Spindle-form; about 16m long; in fresh water. Family 2 Nephroselmidae Pascher Body reniform; with lateral equatorial furrow; 2 flagella arising from furrow, one directed anteriorly and the other posteriorly. Genus Nephroselmis Stein. Reniform; flattened; furrow and c3^topharynx distinct; no stigma; 1-2 chromatophores, discoid, brownish green; nucleus dorsal; a central pyrenoid; 2 contractile vacuoles; with reddish globules; fresh water. N. olvacea S. (Fig. 95, h). 20-25m by 15m. Genus Protochrysis Pascher. Reniform; not flattened; with a dis- tinct furrow, but without cytopharynx; a stigma at base of flagella; 1-2 chromatophores, brownish yellow; p3^renoid central; 2 contrac- tile vacuoles; fission seems to take place during the resting stage; fresh water. 216 PROTOZOOLOGY P. phaeophycearum P. (Fig. 95, i). 15-17)u by 7-9 fi. Suborder 2 Phaeocapsina Pascher Palmella stage predominant; perhaps border-line forms between brown algae and cryptomonads. Example: Phaeothamnion confer- vicolum Lagerheim (Fig. 95, j, k) which is less than 10/z long. References Feitsch, F. E. 1935 The structure and reproduction of the algae. Cambridge. Pascher, A. 1913 Cryptomonadinae. Susswasserflora Deutschlands, etc. part 2. Jena. West, G. S, and F. E. Fritsch. 1927 A treatise on the British _ water algae. Cambridge. Chapter 10 Order 3 Phytomonadina Blochmann THE phytomonads are small, more or less rounded, green flagel- lates, with a close resemblance to the algae. They show a definite body form, and most of them possess a cellulose membrane, which is thick in some and thin in others. There is a distinct opening in the membrane at the anterior end, through which 1-2 (or seldom 4 or more) flagella protrude. The majority possess numerous grass-green chromatophores, each of which contains one or more pyrenoids. The method of nutrition is mostly holophytic or mixotrophic; some color- less forms are, however, saprozoic. The metabolic products are usually starch and oils. Some phytomonads are stained red, owing to the presence of haematochrome. The contractile vacuoles may be located in the anterior part or scattered throughout the body. The nucleus is ordinarily centrally located, and its division seems to be mitotic, chromosomes having been definitely noted in several species. Asexual reproduction is by longitudinal fission, and the daughter individuals remain within the parent membrane for some time. Sexual reproduction seems to occur widely. Colony formation also occurs, especially in the family Volvocidae. Encystment and forma- tion of the palmella stage are common among many forms. The phytomonads have a much wider distribution in fresh than in salt water. Solitary Membrane a single piece; rarely indistinct 2 flagella Family 1 Chlamydomonadidae 3 flagella Family 2 Trichlorididae (p. 222) 4 flagella Family 3 Carteriidae (p. 222) 5 flagella. Family 4 Chlorasteridae (p. 224) 6 or more flagella Family 5 Polyblepharididae (p. 224) Membrane bivalve Family 6 Phacotidae (p. 225) Colonial, of 4 or more individuals; 2 (1 or 4) flagella Family 7 Volvocidae (p. 225) Family 1 Chlamydomonadidae Butschli Solitary; spheroid, oval, or ellipsoid; with a cellulose membrane; 2 flagella; chromatophores, stigma, and pyrenoids usually present. Genus Chlamydomonas Ehrenberg. Spherical, ovoid or elongated; sometimes flattened; 2 flagella; membrane often thickened at an- terior end; a large chromatophore, containing one or more pyrenoids; stigma; a single nucleus; 2 contractile vacuoles anterior; asexual 217 218 PROTOZOOLOGY reproduction and palmella formation known; sexual reproduction isogamy or anisogamy ; fresh water. Numerous species. C. monadina Stein (Fig. 96, a-c). 15-30/x long; fresh water; Landacre noted that the organisms obstructed the sand filters used in connection with a septic tank, together with the diatom Navicula. C. angulosa Dill. About 20/i by 12-15^; fresh water. C. epiphytica Smith (Fig. 96, d). 8-9fi by 7-8m; in freshwater lakes. C. globosa Snow (Fig. 96, e). Spheroid or ellipsoid; 5-7/x in dia- meter; in freshwater lakes. C. gracilis S. (Fig. 96,/). 10-13m by 5-7/x; fresh water. Genus Haematococcus Agardh (Sphaerella Sommerfeldt). Sphe- roidal or ovoid with a gelatinous envelope; chromatophore peripheral and reticulate, with 2-8 scattered pyrenoids; several contractile vacuoles; haematochrome frequently abundant in both motile and encysted stages; asexual reproduction in motile form; sexual repro- duction isogamy ; fresh water. H. pluvialis (Flotow) (Figs. 40; 96, g). Spherical; with numerous radial cytoplasmic processes ; chromatophore U-shape in optical sec- tion; body 8-50/1, stigma fusiform, lateral; fresh water. Reichenow (1909) noticed the disappearance of haematochrome if the culture medium was rich in nitrogen and phosphorus. In bacteria-free cul- tures, Elliott (1934) observed 4 types of cells: large and small flagel- lates, palmella stage and haematocysts. Large flagellates predominate in liquid cultures, but when conditions become unfavorable, palmella stage and then haematocysts develop. When the cysts are placed in a favorable environment after exposure to freezing, desiccation, etc., they give rise to small flagellates which grow into palmella stage or large flagellates. No syngamy of small flagellates was noticed. Hae- matochrome appears during certain phases in sunlight and its ap- pearance is accelerated by sodium acetate under sunlight. Genus Sphaerellopsis Korschikoff (Chlamydococcus Stein). With gelatinous envelope which is usually ellipsoid with rounded ends; body elongate fusiform or pyriform, no protoplasmic processes to envelope; 2 equally long flagella; chromatophore large; a pyrenoid; with or without stigma; nucleus in anterior half; 2 contractile vacu- oles; fresh water. S. fluviaiilis (Stein) (Fig. 96, h). 14-30m by 10-20m; fresh water. Genus Brachiomonas Bohlin. Lobate; with horn-like processes, all directed posteriorly; contractile vacuoles; ill-defined chromato- phore; pyrenoids; with or without stigma; sexual and asexual re- production ; fresh, brackish or salt water. PHYTOMONADINA 219 Fig. 96. a-c, Chlamydomonas monadina, X470 (Goroschankin) ; d, C. epiphytica, X1030 (Smith); e, C. globosa, X2000 (Snow); f, C. gracilis, X770 (Snow); g, Haematococcus pluvialis, X500 (Reichenow); h. Sphaerel- lopsis fluviatilis, X490 (Korschikoff) ; i, Brachiomonas wesliana X960 (West); j, Lobomonas rostrata, X1335 (Hazen); k, Diplostauron penta- gonium, XlllO (Hazen); 1, Gigantochloris permaxima, X370 (Pascher); m, Gloeomonas ovalis, X330 (Pascher); n, Scourfieldia complanata, X1540 (West); o, Thorakomonas sabulosa, X670 (Korschikoff). B. wesliana Pascher (Fig. 96, i). 15-24/i by 13-23)u; brackish water. Genus Lobomonas Dangeard. Ovoid or irregularly angular; chro- matophore cup-shaped; pyrenoid; stigma; a contractile vacuole; fresh water. 220 PROTOZOOLOGY L. rostrata Hazen (Fig. 96, j). 5-1 2^ by 4-8^. Genus Diplostauron Korschikoff. Rectangular with raised cor- ners; 2 equally long fiagella; chromatophore; one pyrenoid; stigma; 2 contractile vacuoles anterior; fresh water. D. pentagonium (Hazen) (Fig. 96, k). lO-lSju by 9-lOAt. Genus Gigantochloris Pascher. Unusually large form, equalling in size a colony of Eudorina; flattened; oval in front view; elongate ellipsoid in profile; membrane radially striated; 2 fiagella widely apart, less than body length; chromatophore in network; numerous pyrenoids; often without stigma; in woodland pools. G. permaxima P. (Fig. 96, 1). 70-150m by 40-80m by 25-50/x. Genus Gloeomonas Klebs. Broadly ovoid, nearly subspherical; with a delicate membrane and a thin gelatinous envelope ; 2 fiagella widely apart; chromatophores numerous, circular or oval discs; pyrenoids (?); stigma; 2 contractile vacuoles anterior; freshwater. G. ovalis K. (Fig. 96, m). 38-42/^ by 23-33/1 ; gelatinous envelope over 2/t thick. Genus Scourfieldia West. Whole body flattened; ovoid in front view; membrane delicate; 2 fiagella 2-5 times body length; a chro- matophore; without pyrenoid or stigma; contractile vacuole anter- ior; nucleus central; fresh water. S. compla7iata W . (Fig. 96, n). 5.2-5.7/iby 4.4-4.6^; freshwater. Genus Thorakomonas Korschikoff. Flattened; somewhat irregu- larly shaped or ellipsoid in front view; membrane thick, enclustered with iron-bearing material, deep brown to black in color; proto- plasmic body similar to that of Chlamydomonas; a chromatophore with a pyrenoid; 2 contractile vacuoles; standing fresh water. T. sahulosa K. (Fig. 96, o). Up to 16m by 14m. Genus Coccomonas Stein. Shell smooth; globular; body not filling intracapsular space; stigma; contractile vacuole; asexual reproduc- tion into 4 individuals ; fresh water. C. orbicularis S. (Fig. 97, a). 18-25^ in diameter; fresh water. Genus Chlorogonium Ehrenberg. Fusiform; membrane thin and adheres closely to protoplasmic body; plate-like chromatophores usually present, sometimes ill-contoured; one or more pyrenoids; numerous scattered contractile vacuoles; usually a stigma; a central nucleus; asexual reproduction by 2 successive transverse fissions during the motile phase; isogamy reported; fresh water. C. euchlorum E. (Fig. 97, 6). 25-7 Om by 4-1 5m; in stagnant water. Genus Phyllomonas Korschikoff. Extremely flattened ; membrane delicate; 2 fiagella; chromatophore often faded or indistinct; numer- ous pyrenoids; with or without stigma; many contractile vacuoles; fresh water. PHYTOMONADINA 221 P. phacoides K. (Fig. 97, c). Leaf -like; rotation movement; up to IOOm long; in standing fresh water. Genus Sphaenochloris Pascher. Body truncate or concave at flagel- late end in front view; sharply pointed in profile; 2 flagella widely apart; chromatophore large; pyrenoid; stigma; contractile vacuole anterior; fresh water. S. printzi P. (Fig. 97, d). Up to 18m by 9/x. Genus Korschikoffia Pascher. Elongate pyriform with an undu- lating outline; anterior end narrow, posterior end more bluntly Fig. 97. a, Coccomonas orbicularis, X500 (Stein); b, Chlorogonium euchlorum, X430 (Jacobsen); c, Phyllomonas -phacoides, X200 (Kor- schikoff); d, Sphaenochloris printzi, X600 (Printz); e, Korschikoffia guttula, X1670 (Pascher); f, Furcilla lobosa, X670 (Stokes); g, Hyalo- gonium klebsi, X470 (Klebs); h, Polytoma uvella, X670 (Dangeard); i, Parapolytoma satura, X1600 (Jameson); j, Trichloris paradoxa, X990 (Pascher). rounded; plastic; chromatophores in posterior half; stigma absent; contractile vacuole anterior; 2 equally long flagella; nucleus nearly central ; salt water. K. guttula P. (Fig. 97, e). 6-1 0/z by 5m ; brackish water. Genus Furcilla Stokes. U-shape, with 2 posterior processes; in side view somewhat flattened ; anterior end with a papilla ; 2 flagella equally long; 1-2 contractile vacuoles anterior; oil droplets; fresh water. F. lobosa S. (Fig. 97,/). 11-14^ long; fresh water. Genus Hyalogonium Pascher. Elongate spindle-form; anterior end bluntly rounded; posterior end more pointed; 2 flagella; protoplasm 222 PROTOZOOLOGY colorless; with starch granules; a stigma; asexual reproduction re- sults in up to 8 daughter cells ; fresh water. H. klebsi P. (Fig. 97, g). 30-80m by up to 10m; stagnant water. Genus Polytoma Ehrenberg (Chlamydohlepharis France; Tussetia Pascher). Ovoid; no chromatophores ; membrane yellowish to brown; pyrenoid unobserved; 2 contractile vacuoles; 2 flagella about body length; stigma if present, red or pale-colored; many starch bodies and oil droplets in posterior half of body; asexual re- production in motile stage; isogamy; saprozoic; in stagnant fresh water. P. uvella E. (Figs. 8, e; 97, h). Oval to pyriform; stigma may be absent; 15-30^ by 9-20m. Genus Paxapolytoma Jameson. Anterior margin obliquely trun- cate, resembling a cryptomonad, but without chromatophores; with- out stigma and starch; division into 4 individuals within envelope; fresh water. P. saturaJ. (Fig. 97, i). About 15ai by lO/x; fresh water. Family 2 Trichlorididae With three flagella. Genus Trichloris Scherffel and Pascher. Bean-shape; flagellate side flattened or concave; opposite side convex; chromatophore large, covering convex side; 2 pyrenoids surrounded by starch granules; a stigma near posterior end of chromatophore; nucleus central; numerous contractile vacuoles scattered; 3 flagella near anterior end. T. paradoxa S and P. (Fig. 97, j). 12-15m broad by 10-12^ high; flagella up to 30m long. Family 3 Carteriidae With four flagella arising from anterior pole. Genus Carteria Diesing {Corhierea, Pithiscus Dangeard; Tetra- mastix Korschikoff). Ovoid, chromatophore cup-shaped; pyrenoid; stigma; 2 contractile vacuoles; fresh water. Numerous species. C. cordiformis (Carter) (Fig. 98, a). Heart-shaped in front view; ovoid in profile; chromatophore large; 18-23m by 16-20^. C. ellipsoidalis Bold. Ellipsoid; chromatophore; a small stigma; division into 2, 4, or 8 individuals in encysted stage; 6-24m long; fresh water, Maryland (Bold, 1938). Genus Pyramimonas Schmarda (Pyramidomonas Stein). Small pyramidal or heart-shaped body; with bluntly drawn-out posterior end; usually 4 ridges in anterior region; 4 flagella; green chromato- PHYTOMONADINA 223 phores cup-shaped ; with or without stigma ; with a large pyrenoid in the posterior part; contractile vacuoles in the anterior portion; fresh water. Several species. P. tetrarhynchus S. (Fig. 98, h). 20-28^ by 12-18^; fresh water; Wisconsin (Smith, 1933). P. montana Geitler. Bluntly conical; anterior end 4-lobed or truncate ; posterior end narrowly rounded ; plastic ; pyrif orm nucleus Fig. 98. a, Carteria cordiformis, X600 (Dill); b, Pyraviimonas tetra- rhynchus, X400 (Dill); c, d, Polyiomella agilis, XlOOO (Doflein); e, Spirogonium chlorogonioides, X670 (Pascher); f, Tetrablepharis multifilis, X670 (Pascher); g, Spermatozopsis exultans, X1630 (Pascher); h, Chlo- r aster gyrans, X670 (Stein); i, Polyhlepharides singularis, X870 (Dan- geard); j, k, Pocillomonas flos aquae, X920 (Steinecke); 1, m, Phacotus lenticularis, X430 (Stein); n, Pteromonas angulosa, X670 (West); o, p, Dysmorphococcus variabilis, XlOOO (Bold). anterior, closely associated with 4 flagella; stigma; 2 contractile vacuoles anterior; chromatophore cup-shaped, granular, with scat- tered starch grains and oil droplets; a pyrenoid with a ring of small starch grains; 17-22.5m long (Geitler); 12-20m by 8-16m (Bold); flagella about body length; fresh water, Maryland (Bold, 1938). 224 PROTOZOOLOGY Genus Polytomella Aragao. Ellipsoid, or oval, with a small papilla at anterior end, where 4 equally long flagella arise; with or without stigma ; starch ; fresh water. P. agilis A. (Fig. 98, c, d). Numerous starch grains; S-lS^t by 5-9/x; flagella 12-17/x long; fresh water; hay infusion. P. caeca Pringsheim. Ovoid with bluntly pointed posterior end; 12-20m by 10-12^; membrane delicate; a small papilla at anterior end; no stigma; two contractile vacuoles below papilla; cytoplasm ordinarily filled with starch grains ; fresh water. Genus Medusochloris Pascher. Hollowed hemisphere with 4 proc- esses, each bearing a flagellum at its lower edge; a lobed plate- shaped chromatophore; without pyrenoid below convex surface. One species. M. phiale P. In salt water pools with decaying algae in the Baltic. Genus Spirogonium Pascher. Body spindle-form; membrane deli- cate; flagella a little longer than body; chromatophore conspicuous; a pyrenoid; stigma anterior; 2 contractile vacuoles; fresh water. One species. S. chlorogonioides (P). (Fig. 98, e). Body up to 25m by 15m. Genus Tetrablepharis Senn. Ellipsoid to ovoid; pyrenoid present; fresh water. T. multifilis (Klebs) (Fig. 98,/). 12-20m by 8-15m ; stagnant water. Genus Spermatozopsis Korschikoff. Sickle-form; bent easily, oc- casionally plastic; chromatophore mostly on convex side; a distinct stigma at more rounded anterior end; flagella equally long; 2 con- tractile vacuoles anterior; fresh water infusion. S. exultans K. (Fig. 98, g). 7-9m long; also biflagellate; in fresh water with algae, leaves, etc. Family 4 Chlorasteridae With 5 flagella arising from anterior pole. Genus Chloraster Ehrenberg. Similar to Pyramimonas, but an- terior half with a conical envelope drawn out at four corners; with 5 flagella ; fresh or salt water. C. gyrans E. (Fig. 98, h). Up to 18m long; standing water; also re- ported from salt water. Family 5 Polyblepharididae Dangeard With 6 or more flagella arising from anterior end. Genus Polyblepharides Dangeard. Ellipsoid or ovoid; flagella 6-8, shorter than body length; chromatophore; a pyrenoid; a central nucleus; 2 contractile vacuoles anterior; cysts; a questionable genus; fresh water. PHYTOMONADINA 225 P. singularis D. (Fig. 98, i). lO-lifx by 8-9/^. Genus Pocillomonas Steinecke. Ovoid with broadly concave an- terior end; covered with gelatinous substance with numerous small projections; 6 flagella; chromatophores disc-shaped; 2 contractile vacuoles anterior; nucleus central; starch bodies; without pyrenoid. P.flos aquae S. (Fig. 98, j, k). 13m by lOyu; fresh water pools. Family 6 Phacotidae Poche The shell typically composed of 2 valves; 2 flagella protrude from anterior end; with stigma and chromatophores ; asexual reproduction within the shell; valves may become separated from each other ow- ing to an increase in gelatinous contents. Genus Phacotus Perty. Oval to circular in front view; lenticular in profile; protoplasmic body does not fill dark-colored shell com- pletely; flagella protrude through a foramen; asexual reproduction into 2 to 8 individuals ; fresh water. P. lenticularis (Ehrenberg) (Fig. 98, I, m). 13-20/x in diameter; in stagnant water. Genus Pteromonas Seligo. Body broadly winged in plane of suture of 2 valves;. protoplasmic body fills shell; chromatophore cup- shaped; one or more pyrenoids; stigma; 2 contractile vacuoles; asexual reproduction into 2-4 individuals; sexual reproduction by isogamy ; zygotes usually brown; fresh water. Several species. P. angulosa (Lemmermann) (Fig. 98, n). With a rounded wing and 4 protoplasmic projections in profile; 13-17iu by 9-20ai; fresh water. Genus Dysmorphococcus Takeda. Circular in front view; anterior region narrowed; posterior end broad; shell distinctl,y flattened pos- teriorly, ornamented by numerous pores; sutural ridge without pores; 2 flagella; 2 contractile vacuoles; stigma, pyrenoid, cup-shaped chromatophore; nucleus; multiplication by binary fission; fresh water. D. variahilis T. (Fig. 98, o, p). Shell 14-1 9m by 13-17m; older shells dark brown; fresh water; Maryland (Bold, 1938). Family 7 Volvocidae Ehrenberg An interesting group of colonial flagellates; individual similar to Chlamydomonadidae, with 2 equally long flagella (one in Mastigo- sphaera; 4 in Spondylomorum) , green chromatophores, pyrenoids, stigma, and contractile vacuoles; body covered by a cellulose mem- brane and not plastic; colony or coenobium is discoid or spherical; exclusively freshwater inhabitants. 226 PROTOZOOLOGY Genus Volvox Linnaeus. Often large spherical or subspherical colonies, consisting of a large number of cells which are differen- tiated into somatic and reproductive cells; somatic cells numerous, embedded in gelatinous matrix, and contains a chromatophore, one or more pyrenoids, a stigma and several contractile vacuoles; in some species protoplasmic connections occur between adjacent cells; generative cells few and large. Both mono- and bi-sexual re- productions occur; monosexual gametes usually fewer and larger in size than bisexual ones, each producing a young colony by repeated division; bisexual reproduction anisogamy; zygotes usually brown- ish red in color, with smooth, undulating, or spinous envelopes; fresh water. Many species. Smith (1944) made a comprehensive study of the known species. V. globator L. (Fig. 99, a). Monoecious. Sexual colonies 350-500m in diameter; 5000-15,000 cells, with cytoplasmic connections; 3-7 microgametocytes, each of which develops into over 250 microgam- etes; 10-40 macrogametes; zygotes 35-45m in diameter, covered with many sharply pointed spines. Partheno genetic colonies 400-600/x in diameter; 4-10 gonidia, 10-13ju in diameter; young colonies up to 250m. Europe and North America. V. aureus Ehrenberg (Figs. 72; 99, h). Dioecious. Male colonies 300-350ju in diameter; 1000-1500 cells, with cytoplasmic connec- tions; numerous microgametocytes; clusters of some 32 microgam- etes, 15-18m in diameter. Female colonies 300-400^; 2000-3000 cells; 10-14 macrogametes; zygotes 40-60ju with smooth surface. Parthenogenetic colonies up to 500/x; 4-12 gonidia; young colonies 150)u in diameter. Europe and North America. V. tertius Meyer. Dioecious. Male colonies up to 170/x in diameter; 180-500 cells, without cytoplasmic connections; about 50 micro- gametocytes. Female colonies up to 500jLt; 500-2000 cells; 2-12 macrogametes; zygotes 60-65^ with smooth wall. Parthenogenetic colonies up to 600m in diameter; 500-2000 cells; 2-12 gonidia. Europe and North America. V. spermatosphaera Powers. Dioecious, Male colonies up to 100m in diameter; cells, without connection, up to 128, each becoming microgametocyte. Female colonies up to 500m in diameter; 6-16 macrogametes; zygotes 35-45m; with smooth membrane. Partheno- genetic colonies up to 650m in diameter; 8-10 gonidia; young colonies ellipsoid, up to 100m in diameter. North America. V . weismannia P. Dioecious. Male colonies 100-150m in diameter; 250-500 cells; 6-50 microgametocytes; clusters of microgametes (up to 128) discoid, 12-15m in diameter. Female colonies up to 400m; PHYTOMONADINA 227 Fig. 99. a, Volvox globator, X200 (Janet); b, V. aureus, XllO (Klein); c, Gonium. sociale, X270 (Chlordat); d, G. pedorale, X670 (Hartmann); e, G.formosum, X600 (Pascher). 228 PROTOZOOLOGY 2000-3000 cells; 8-24 macrogametes; zygotes 30-50/i in diameter, with reticulate ridges on shell. Partheno genetic colonies up to 400/x; 1500-3000 cells; 8 or 10 gonidia, 40-60ju in diameter; young colonies 100-200/i in diameter. North America. V. perglohator P. Dioecious. Male colonies 300-450^; 5000-10,000 cells, with delicate cytoplasmic connections; 60-80 microgameto- cytes. Female colonies 300-550/x; 9000-13,000 cells; 50-120 macro- gametes; zygotes 30-34)Lt, covered with bluntly pointed spines. Parthenogenetic colonies up to 1.1 mm; 3-9 gonidia; young colonies 250-275M. North America. Genus Gonium Miiller. 4 or 16 individuals arranged in one plane; cell ovoid or slightly polygonal; with 2 flagella arranged in the plane of coenobium; with or without a gelatinous envelope; protoplasmic connections among individuals occur occasionally; asexual reproduc- tion through simultaneous divisions of component cells; sexual re- production isogamy; zygotes reddish; fresh water. G. socials (Dujardin) (Fig. 99, c). 4 individuals form a discoid colony; cells 10-22^ by 6-16ai wide; in open waters of ponds and lakes. G. pedorale M. (Fig. 99, d). 16 (rarel}^ 4 or 8) individuals form a colony; 4 cells in center; 12 peripheral, closely arranged; cells 5-14/^ by lOyu; colony up to 90/x in diameter; fresh water. G. formosum Pascher (Fig. 99, e) 16 cells in a colony further apart; peripheral gelatinous envelope reduced; cells similar in size to those of G. sociale but colony somewhat larger; freshwater lakes. Genus Stephanoon Schewiakoff. Spherical or ellipsoidal colony, surrounded by gelatinous envelope, and composed of 8 or 16 bi- flagellate cells, arranged in 2 alternating rows on equatorial plane; fresh water. S. askenasii S. (Fig. 100, a). 16 individuals in ellipsoidal colony; cells 9/x in diameter; flagella up to 30At long; colony 78/i by 60m- Genus Platydorina Kofoid. 32 cells arranged in a slightly twisted plane; flagella directed alternately to both sides; fresh water. P. caudata K. (Fig. 100, 6). Individual cells 10-15^ long; colony up to 165/x long, 145/i wide, and 25)U thick; rivers and lakes. Genus Spondylomorum Ehrenberg. 16 cells in a compact group in 4 transverse rings; each with 4 flagella; asexual reproduction by simultaneous division of component cells; fresh water. One species. S. quaternarium E. (Fig. 100, c). Cells 12-26/x by 8-15/i; colony up to 60/i long. Genus Chlamydobotrys Korschikoff. Colony composed of 8 or 16 individuals; cells with 2 flagella; chromatophore ; stigma ; no pyrenoid ; fresh water. PHYTOMONADINA 229 C. stellata K. (Fig. 100, d). Colony composed of 8 individuals arranged in 2 rings; individuals 14-15^ long; colony 30-40m in diameter; Maryland (Bold, 1933). Genus Stephanosphaera Cohn. Spherical or subspherical colony, with 8 (rarely 4 or 16) cells arranged in a ring; cells pyriform, but Fig. 100. a, Stephanoon askenasii, X440 (Schewiakoff) ; b, Platydorina caudata, X280 (Kofoid); c, Spondylomorum qiiaternarium, X330 (Stein); d, Chlamydobotrys stellata, X430 (Korschikoff); e, Stephanosphaera plu- vialis, X250 (Hieronymus); f, Pandorina niorum, X300 (Smith); g, Mastigosphaera gobii, X520 (Schewiakoff); h, Eiidorina elegans, X310 (Goebel); i, Pleodorina illinoisensis, X200 (Kofoid). with several processes; 2 flagella on one face; asexual reproduction and isogamy (p. 152) ; fresh water. S. pluvialis C. (Figs. 74; 100, e). Cells 7-13m long; colony 30-60/i in diameter. Genus Pandorina Bory. Spherical or subspherical colony of usu- ally 16 (sometimes 8 or 32) biflagellate individuals, closely packed 230 PROTOZOOLOGY within a gelatinous, but firm and thick matrix; individuals often angular; with stigma and chromatophores; asexual reproduction through simultaneous division of component individuals; anisogamy preceded by division of each cell into 16 to 32 gametes; zygotes colored and covered by a smooth wall; fresh water. One species. P. morum (Miiller) (Figs. 75; 100, /). Cells 8-17m long; colony 20-40/x, up to 250/i in diameter; ponds and ditches. Genus Mastigosphaera Schewiakoff. Similar to Pandorina; but individuals with a single flagellum which is 3.5 times the body length; fresh water. M. gohii S. (Fig. 100, g). Individual 9m long; colony 30-33^. Genus Eudorina Ehrenberg. Spherical or ellipsoidal colony of usually 32 or sometimes 16 spherical cells; asexual reproduction similar to that of Pandorina; sexual reproduction with 32-64 spheri- cal green macrogametes and numerous clustered microgametes; red- dish zygote with a smooth wall; fresh water. E. elegans E. (Figs. 76; 100, h). Cells 10-24^ in diameter; colony 40-1 50^1 in diameter; in ponds, ditches and lakes. Genus Pleodorina Shaw. Somewhat similar to Eudorina, being composed of 32, 64, or 128 ovoid or spherical cells of 2 types: small somatic and large generative, located within a gelatinous matrix; fresh water. P. illinoisensis Kofoid (Figs. 31, h, c; 100, i). 32 cells in ellipsoid colony, 4 vegetative and 28 reproductive individuals; arranged in 5 circles, 4 in each polar circle, 8 at equator and 8 on either side of equator; 4 small vegetative cells at anterior pole; vegetative cells 10-16m in diameter; reproductive cells 19-25/x in diameter; colony up to 160/x by 130m. P. californica S. Spherical colony with 64 or 128 cells, of which 1/2-2/3 are reproductive cells; vegetative cells 13-15/i; reproductive cells up to 27m; colony up to 450m, both in diameter. References Bold, H. C. 1938 Notes on Maryland Algae. Bull. Torrey Bot. Club., Vol. 65. Crow, W. B. 1918 The classification of some colonial chlamy- domonads. New Phytologist, Vol. 17. Dangeard, p. 1900 Observations sur la structure and le developpe- ment du Pandorina morum. Le Botaniste, Vol. 7. Elliott, A. M. 1934 Morphology and life history of Haematococcus pluvialis. Arch. f. Protistenk., Vol. 82. Entz, G. Jr. 1913 Cytologische Beobachtungen an Polytoma uvella. Verb. Deutsch. Zool. Ges. Ver., Vol. 23. PHYTOMONADINA 231 Fritsch, F. E. 1935 The structure and reproduction of the algae. Cambridge. Gerloff, J. 1940 Beitrage zur Kenntnis der Variabilitat und Sys- tematik der Gattung Chlamydomonas. Arch. f. Protistenk., Vol. 94. Janet, C. 1912, 1922, 1923 Le Volvox. I, II, and III Memoires. Paris. KoFOiD, C. A. 1900 Plankton Studies, Nos. 2 and 3. Ann. Mag. Nat. Hist., Ser. 7, Vol. 6. Mast, S. 0. 1928 Structure and function of the eye-spot in unicel- lular and colonial organisms. Arch. f. Protistenk., Vol. 60. Pascher, a. 1927 Volvocales — Phytomonadinae. In: Die Siisswasser- flora Deutschlands, Part 4. Pringsheim, E. G. 1937 Zur Kenntnis saprotropher Algen und Flagellaten. II. Arch. f. Protistenk., Vol. 88. Shaw, W. R. 1894 Pleodorina, a new genus of the Volvocideae. Bot. Gaz., Vol. 19. Smith, G. M. 1933 The freshwater algae of the United States. New York. — '■ 1944 A comparative study of the species of Volvox. Trans. Amer. Micr. Soc, Vol. 63. West, G. S. and F. E. Fritsch 1927 A treatise on the British fresh- water algae. Cambridge. Chapter 11 Order 4 Euglenoidina Blochmann THE body is as a rule elongated; some are plastic, others have a definite body form with a well-developed, striated or variously sculptured pellicle. At the anterior end, there is an opening through which a flagellum protrudes. In holophytic forms the so-called cyto- stome and cytopharynx, if present, are apparently not concerned with the food-taking, but seem to give a passage-way for the flagellum and also to excrete the waste fluid matters which become collected in one or more contractile vacuoles located around the reservoir. In holozoic forms, a well-developed cytostome and cytopharynx are present. Ordinarily there is only one flagellum, but some possess two or three. Chromatophores are present in the majority of the Eu- glenidae, but absent in two families. They are green, vary in shape, such as spheroidal, band-form, cup-form, discoidal, or fusiform, and usually possess pyrenoids. Some forms may contain haematochrome. A small but conspicuous stigma is invariably pres- ent near the anterior end of the body in chromatophore-bearing forms. Reserve food material is the paramylon body, fat, and oil, the presence of which depends naturally on the metabolic condition of the organism. The paramylon body assumes diverse forms in dif- ferent species, but is, as a rule, constant in each species, and this facilitates specific identification to a certain extent. Nutrition is holophytic in chromatophore-possessing forms, which, however, may be saprozoic, depending on the amount of light and organic sub- stances present in the water. The holozoic forms feed upon bacteria, algae, and smaller Protozoa. The nucleus is, as a rule, large and distinct and contains almcJst always a large endosome. Asexual reproduction is by longitudinal fission; sexual reproduction has been observed in a few species. En- cystment is common. The majority inhabit fresh water, but some live in brackish or salt water, and a few are parasitic in animals. With stigma Family 1 Euglenidae Without stigma With 1 flagellum Family 2 Astasiidae (p. 239) With 2 flagella Family 3 Anisonemidae (p. 241) Family 1 Euglenidae Stein Body plastic ("euglenoid"), but, as a rule, more or less spindle- 232 EUGLENOIDINA 233 shaped during movement; the majority possess a single anterior flagellum (with the exception of Eutreptia and Euglenamorpha) ; green chromatophores (except one genus) and stigma occur, though in some cases absent; haematochrome often coexists; metaboHc products oil and paramylon; asexual reproduction by longitudinal fission in either active or resting stage; mostly freshwater inhabit- ants. Genus Euglena Ehrenberg. Short or elongated spindle, cylindrical, or band-form; pellicle usually marked by longitudinal or spiral striae; some highly plastic with a thin pellicle; others regularly spi- rally twisted; stigma usually anterior; chromatophores numerous and discoid, band-form, or fusiform; pyrenoids may or may not be sur- rounded by starch envelope; metabolic products paramylon bodies which may be two in number, one being located on either side of nucleus, and rod-like to ovoid in shape or numerous and scattered throughout; contractile vacuole small, near reservoir; asexual repro- duction by longitudinal fission; sexual reproduction reported in Euglena sanguinea; common in stagnant water, especially where algae occur; when present in large numbers, the active organisms may form a green film on the surface of water and resting or en- C3'sted stages may produce conspicuous green spots on the bottom of pond or pool; in fresh water. Numerous species. E. pisciformis Klebs (Fig. 101, a). 25-30^ by 7-10m; spindle-form with bluntly pointed anterior and sharply attenuated posterior end; slightly plastic; highly active; paramylon indistinct; a few chroma- tophores lateral and discoidal ; each with 2 pyrenoids ; flagellum fairly long. Johnson observed that division into 2 or 4 individuals occurs in enc3^sted forms. E. viridis Ehrenberg (Fig. lOl, h). 50-60/x by 14-18/x; anterior end rounded, posterior end pointed; spindle-shaped during motion, highly plastic when stationary; pellicle obliquely striated; chromato- phores more or less band-form, arranged in a stellate form; nucleus posterior; nutrition holophytic, but also able to carry on saprozoic nutrition, during which period chromatophores degenerate. Multi- plication in thin-walled cysts (Johnson). E. acus E. (Figs. 24, h; 101, c). 50-200ai long; long spindle-form; posterior end sharply pointed; flagellum short; spiral striation on pellicle very delicate; paramylon bodies several, rod-form; nucleus central; stigma distinct; numerous disc-like chromatophores; slug- gish. E. spirogyra E. (Figs. 24, c; 101, d). 80-125m by 10-20^; cylin- drical; with spiral striae, consisting of small knobs; numerous disc- 234 PROTOZOOLOGY like chromatophores ; 2 ovoidal paramylon bodies, one on either side of centrally located nucleus; flagellum short; stigma prominent; sluggish. E. oxyuris Schmarda (Fig. 101, e). 150-500m by 30-45ju; almost always spirally twisted, somewhat flattened; pellicle with spirally arranged striae; numerous chromatophores; 2 ovoid paramylon bodies conspicuous, one on either side of nucleus; flagellum short. Fig. 101. a, Euglena pisciformis, X270 (Klebs); b, E. viridis, X370 (Lemmermann) ; c, E. acus, X270 (Klebs); d, E. spirogyra, X430 (Stein); e, E. oxyuris, X430 (Stein); f, E. sanguinea, X130 (Klebs); g, E. deses, X230 (Lemmermann); h, E. gracilis, X270 (Klebs). E. sanguinea E. (Figs. 39, e-h; 101, /). 80-200ai by 25-45/x; flagellum long; pelUcular striation conspicuous; haematochrome granular, scattered in sun light; often found in crust on the surface or half-dry bed of a pool. E. deses E. (Figs. 24, a; 101, g). 85-1 55m by 15-22)u; elongate, highly plastic; body striation faintly visible; stigma distinct; nucleus EUGLENOIDINA 235 central, numerous chromatophores hemi-lenticular; several small rod-shaped paramylon bodies scattered; flagellum short. E. gracilis Klebs (Figs. 39, a-d; 101, h). 35-55m by 6-23m; cylin- drical to elongated oval; highly plastic; flagellum less than body length; fusiform chromatophores 10-20, discoid; nucleus central; pyrenoids. E. rubra Hardy. 70-170^ by 25-36//; cylindrical, rounded anteri- orly and drawn out posteriorly; pellicle spirally striated; nucleus posterior; flagellum longer than body; the base of flagellum ar- ranged as in E. acus (Fig. 24) ; stigma about 7)U in diameter, lateral to the reservoir, near which a contractile vacuole is formed; chro- matophores, many, spindle-shaped, with 3 longitudinal grooves; when taken out of body disc-shaped; haematochrome granules red, numerous, measure 0.3-0.5iu in diameter; paramylon bodies, numer- ous, ellipsoid; reproductive and temporary cysts and protective cysts (34-47/i in diameter), with gelatinous coat; multiplication noted only in encysted forms (Johnson). Johnson (1939) found that the color of this Euglena was red in the morning and dull green in the late afternoon, due to the dif- ference in the distribution of haematochrome within the body. When haematochrome granules are distributed throughout the body, the organism is bright-red, but when they are condensed in the center of the body, the organism is dull green. When part of the area of the pond was shaded with a board early in the morning, shortly after sunrise all the scum became red except the shaded area. When the board was removed, the red color appeared in 1 1 minutes while the temperature of the water remained 21°C. In the evening the change was reversed. Johnson and Jahn (1942) later found that green-red color change could be induced by raising the temperature of the water to 30-40°C. and by irradiation with infrared rays or visible light. The two workers hold that the function of haematochrome may be protective, since it migrates to a position which shields the chromatophores from very bright light. If this is true, it is easy to find the species thriving in hot weather in shallow ponds where temperature of the water rises to 35-45°C. In colder weather, it is supposed that this Euglena is less abundant and it exists in a green phase, containing a few haematochrome granules. E. vermiformis Carter. 45ju by 5/x; without flagellum; a slow spiral movement; retains cylindrical form during locomotion; among de- bris; stigma conspicuous; delicate pellicle not striated; about 8 pe- ripheral chromatophores; many small paramylon bodies in the form of flattened elliptical rings; brackish water. 236 PROTOZOOLOGY Genus Khawkinea Jahn and McKibben. Similar to Genus Eu- glena, but without chromatophores and thus permanently colorless; fresh water. K. halli J. and Mc. 40-45^ (30-65m) by 12-14^; fusiform; pellicle spirally striated; plastic; flagellum slightly longer than body; stigma 2-3/i in diameter, yellow-orange to reddish-orange, composed of many granules; numerous (25-100) paramylon bodies elliptical or polyhedral; cysts 20-30/i in diameter; putrid leaf infusion; saprozoic. K. ocellata (Khawkine). Similar to above; flagellum 1.5-2 times body length; fresh water. Genus Phacus Nitzsch. Highly flattened; asymmetrical; body- form constant; pellicle often with prominent longitudinal or oblique striae; a flagellum and a stigma; nucleus posterior; a short "cyto- pharynx"; green chromatophores rounded discoid; with or without paramylon bodies around a pja-enoid; in fresh water. Numerous species. Allegre and Jahn (1943) surveyed species of this genus in Iowa. P. pleuronectes (Miiller) (Fig. 102, a). 45-lOOju by 30-70/i; short posterior prolongation slightly curved; a prominent fold on convex side, extending to middle of body; longitudinally striated; one or more circular paramylon bodies; colorless forms sometimes appear; flagellum as long as body. P. longicaudus (Ehrenberg) (Fig, 102, 6). 120-170^ by 45-70/x; usually twisted with a long caudal prolongation; stigma prom- inent; discoidal paramylon body central; pellicle longitudinally striated. P. pyrum (E.) (Fig. 102, c). About 30-50iu long; pyriform, with a short caudal prolongation; pellicle obliquely striated. P. triqueter (E.) (Fig. 102, d). 50-55m by 30-35^; ovate; with a longitudinal ridge; posterior end acuminate; oblique striation dis- tinct; 1-2 paramylon bodies, P. anacoelus Stokes (Fig. 102, e). About 42^ long; oval or round; with flagellum as long as body. P. acuminata S. (Fig. 102, /). About 30-40/z by 20-30/x; nearly circular in outline; longitudinally striated; fold long; flagellum as long as bod}^; 2 small paramylon bodies. Genus Crumenula Dujardin {Lepocinclis Perty). Body more or less ovo-cylindrical; rigid with spirally striated pellicle; often with a short posterior spinous projection; stigma sometimes present; nu- merous discoidal chromatophores marginal; paramylon bodies usu- ally large and ring-shaped, laterally disposed; without pyrenoids; fresh water. Several species. EUGLENOIDINA 237 C. ova (Ehrenberg) (Fig. 102, g). 20-40/1 long; in fresh water with Euglena. Genus Trachelomonas Ehrenberg. With a lorica which often pos- sesses numerous spinous projections; sometimes yellowish to dark brown; a single flagellum protrudes from anterior aperture, the rim of which is frequently thickened to form a collar; chromatophores either 2 curved plates or numerous discs; paramylon bodies small grains; stigma and pyrenoids; multiplication by longitudinal fis- sion; one daughter individual retains lorica and flagellum, while the other escapes through flagellar aperture, forms a new flagellum and secretes a lorica; cysts common; specific differentiation is based upon the lorica; fresh water. Numerous species. T. hispida (Perty) (Figs. 31, a; 102, h). Lorica oval, with numer- ous minute spines; brownish; 8-10 chromatophores; 20-42/z by 15-26iu; many varieties. T. urceolata Stokes (Fig. 102, i). Lorica vasiform, smooth with a short neck; about 45/x long. T. piscatoris (Fisher) (Fig. 102, j). Lorica cylindrical with a short neck and with numerous short, conical spines; 25-40/i long; flagel- lum 1-2 times body length. T. verrucosa Stokes (Fig. 102, k). Lorica spherical, with numerous knob-like attachments; no neck; 24-25/i in diameter. T. vermiculosa Palmer (Fig. 102, I). Lorica spherical; with many sausage-form markings; 23/i in diameter. Genus Cryptoglena Ehrenberg. Body rigid, flattened; 2 band-form chromatophores lateral; a single flagellum; nucleus posterior; among freshwater algae. One species. C. pigra E. (Fig, 102, m). Ovoid, pointed posteriorly; flagellum short; stigma prominent; 10-15)U by 6-10/x; standing water. Genus Ascoglena Stein. Encased in a flexible, colorless to brown lorica, attached with its base to foreign object; solitary; without stalk; body ovoidal, plastic; attached to test with its posterior end; a single flagellum; a stigma; numerous chromatophores discoid; with or without pyrenoids; reproduction as in Trachelomonas fresh water. A. vaginicola S. (Fig, 102, n). Lorica about 43;u b}^ 15ju. Genus Colacium Ehrenberg. Stalked individuals form colony; frequently attached to animals such as copepods, rotifers, etc; stalk mucilaginous; individual cells pyriform, ellipsoidal or cylindrical; without flagellum; a single flagellum only in free-swimming stage; discoidal chromatophores numerous; with pyrenoids; multiplication by longitudinal fission; also by swarmers, possessing a flagellum and a stigma; fresh water. Several species. 238 PROTOZOOLOGY C. vesiculosum E, (Fig. 102, o). Colony of 2-8 cells; also solitary; 20-30/i by 9-18m; attached to freshwater copepods. Genus Eutreptia Perty {Eutreptiella da Cunha). With 2 flagella at Fig. 102. a, Phacus pleuronedes, X670 (Lemmermann); b, P. longi- caudus, X430 (Stein); c, P. pynim, X400 (Lemmermann); d, P. triqueter, X430 (Stein); e, P. anacoelus, X330 (Stokes); f, P. acuminata X560 (Stokes); g, Crumenula ova, X430 (Stein); h, Trachelomonas hispida, X430 (Stein); i, T. urceolata, X430 (Stokes); j, T. piscatoris, X520 (Fischer); k, T. verrucosa, X550 (Stokes); I, T. verndculosa, X800 (Pal- mer); m, Cryptoglena pigra, X430 (Stein); n, Ascoglena vaginicola, X390 (Stein); o, Colaciuvi vesictilosum, X390 (Stein); p, Eutreptia viridis, X270 (Klebs); q. E. marina, X670 (da Cunha); r, Euglenamorpha hegneri, X730 (Wenrich). anterior end; pellicle distinctly striated; plastic; spindle-shaped dur- ing movement; stigma; numerous discoid chromatophores; pyren- oids absent; paramylon bodies spherical or subcylindrical; multipli- EUGLENOIDINA 239 cation as in Euglena; cyst with a thick stratified wall; fresh or salt water. E. viridis P. (Fig. 102, p). 50-70/1 by 5-13m; in fresh water; a variety was reported from brackish water ponds. E. marina (da Cunha) (Fig. 102, q). Flagella unequal in length; longer one as long as body, shorter one f ; body 40-50/^ by S-lO/x; salt water. Genus Euglenamorpha Wenrich, Body form and structure similar to those of Euglena, but with 3 flagella; in gut of frog tadpoles. One species. E. hegneri W. (Fig. 102, r). 40-50^ long. Family 2 Astasiidae Biitschli Similar to Euglenidae in body form and general structure, but without chromatophores; body is plastic, although it assumes usually an elongated form; there is a cyto pharynx and cytostome, the former being connected with the reservoir near which contractile vacuole occurs; without stigma; fiagellum usually straight and its free end vibrates in a characteristic manner; asexual reproduction by longitudinal fission. Genus Astasia Dujardin. Body plastic, although ordinarily elongate; fresh water or endoparasitic (?) in Cyclops, etc. Several species. A. klebsi Lemmermann (Fig. 103, a). Spindle-form; posterior portion drawn out; fiagellum as long as body; plastic; paramylon bodies oval; 50-60)u by 13-20^; stagnant water. Genus Urceolus Mereschkowsky {Phialonema Stein). Body color- less; plastic; flask-shaped; striated; a funnel-like neck; posterior region stout; a single fiagellum protrudes from funnel and reaches in- ward the posterior third of body; fresh or salt water. U. cyclostomus (Stein) (Figs. 8, /; 103, h). 25-50/x long; fresh water. U. sahulosus (Stokes) (Fig. 103, c). Spindle-form; covered with minute sand-grains; about 58iu long; fresh water. Genus Peranema Dujardin. Elongate with a broad, rounded or truncate posterior end during locomotion; highly plastic when sta- tionary; delicate pellicle shows a fine striation; fiagellum long, tapers toward free end and vibrates; nucleus central; contractile vacuole; holozoic and saprozoic; in stagnant water; often in hay infusion. P. trichophorum (Ehrenberg) (Figs. 26; 103, d). 20-70jti long; body ordinarily filled with paramylon or starch grains derived from Astasia, Menoidium, etc.; very common. 240 PROTOZOOLOGY Fig. 103. a, Astasia klebsi, X500 (Klebs); b, Urceolus cyclostomus, X430 (Stein); c, U. sabulosus, X430 (Stokes); d, Peranema tricho- phoruni, X530 (Kudo); e, Petalmonas mediocanellata, XlOOO (Klebs); f, Menoidiwm incurvum, X1400 (Hall); g, Scytomonas jnisilla, X430 (Stein); h, Anisonema acinus, X400 (Klebs); i, A. truncatum, X430 (Stein); j, A. emerginatum, X530 (Stokes); k, Heteronema acus, X430 (Stein); 1, H. mutabile, Xl20 (Stokes); m, Tropidoscyphus octocostatus, X290 (Lemraermann); n, Distigma proteus, X430 (Stein); o, Entosi- phon sulcatum, X430 (Stein); p. Notosolenus apocaviptus, X1200 (Stokes): q, N. sinatus, X600 (Stokes); r, Marsupiogaster striata, X590 (Schewia- koff); s, M. picta (Faria, da Cunha and Pinto). EUGLENOIDINA 241 P. granuUfera Penard. Much smaller, 8-15/i long; spherical or elongate; pelhcle granulated; standing water. Genus Petalomonas Stein, Colorless; constant in form; pellicle often with longitudinal keels on one side; a single flagellum; ho lo zoic or saprozoic; cytostome at anterior end; cytopharynx fairly deep; in fresh water, rich in vegetable matter. Many species. P. medio canellata S. (Fig. 103, e). Ovoid with longitudinal furrow; flagellum about as long as body; 22-23 /x long. Genus Menoidium Perty. Rigid body, more or less curved; pellicle striated ; a single flagellum ; fresh water. M. incurvum (Fresenius) (Figs. 24, d; 67; 103, /). Crescentic cyl- inder; flagellum as long as body; nucleus central or terminal; 15- 25/i by 7-8^; in standing fresh water. Hall (1923) made a careful cytological study of the organism (p. 144). M. tortuosum Stokes. S-form; posterior end drawn out to a sharp point; elongate paramylon bodies; 42-78/1 long; in infusion. Genus Scytomonas Stein. Oval or pyrrform, with a delicate pel- licle; a single flagellum; a contractile vacuole with a reservoir; ho lo zoic on bacteria; longitudinal fission in motile stage; stagnant water and coprozoic. S. pusilla S. (Fig. 103, g). About 15/x long. Genus Copromonas Dobell. Elongate ovoid; with a single flagel- lum; a small cytostome at anterior end; ho lo zoic on bacteria; per- manent fusion followed by encystment (p. 151); coprozoic in faecal matters of frog, toad, and man; several authors hold that this genus is probably identical with Scytomonas which was incompletely de- scribed by Stein. C. subtilis D. (Fig. 73). 7-20^ long. Family 3 Anisonemidae Schewiakoff Colorless body plastic or rigid with a variously marked pellicle; 2 flagella, one directed anteriorly and the other usually posteriorly; contractile vacuoles and reservoir; stigma absent; paramylon bodies usually present; free-swimming or creeping. Genus Anisonema Dujardin. Generally ovoid; more or less flat- tened; asymmetrical; plastic or rigid; a slit-like ventral furrow; flagella at anterior end; cytopharynx long; contractile vacuole an- terior; nucleus posterior; in fresh water. Several species. A. acinus D. (Fig. 103, h). Rigid; oval; somewhat flattened; pel- licle slightly striated; 25-40^ by 16-22^. A. truncatum Stein (Fig. 103, i). Rigid; ovoid; 60/i by 20/x. A. emarginatum Stokes (Fig. 103, j). Rigid; 14/i long; flagella long. 242 PROTOZOOLOGY Genus Heteronema Dujardin. Plastic; rounded or elongate; flagella arise from anterior end, one directed forward and the other trailing; cytostome near base of flagella; ho lo zoic; fresh water. Sev- eral species. H. acus (Ehrenberg) (Fig. 103, k). Extended body tapers towards both ends; anterior flagellum as long as body, trailing one about 1/2; contractile vacuole anterior ; nucleus central ; 45-50/x long ; fresh water. H. mutahile (Stokes) (Fig. 103, I). Elongate; highly plastic; longi- tudinally striated; about 254;u long; in cypress swamp. Genus Tropidoscyphus Stein. Slightly plastic; pellicle with 8 longitudinal ridges; 2 unequal flagella at anterior pole; holozoic or saprozoic; fresh or salt water. T. octocostatus S. (Fig. 103, m). 35-63m long; fresh water, rich in vegetation. Genus Distigma Ehrenberg. Plastic; elongate when extended; body surface without any marking; 2 flagella unequal in length, di- rected forward; cytostome and cytopharynx located at anterior end; endoplasm usually transparent; holozoic. One species. D. proteus E. (Fig. 103, n). 50-1 lO/x long when extended; nucleus central; stagnant water; infusion. Genus Entosiphon Stein. Oval, flattened; more or less rigid; flagella arise from a cytostome, one flagellum trailing; protrusible cytophar3^nx a long conical tubule almost reaching posterior end; nucleus centro -lateral; fresh water. E. sulcatum (Dujardin) (Fig. 103, o). About 20/i long. E. ovatum Stokes. Anterior end rounded; 10-12 longitudinal striae; about 25-28/i long. Genus Notosolenus Stokes. Free-swimming; rigid oval; ventral surface convex, dorsal surface with a broad longitudinal groove; flagella anterior; one long, directed anteriorly and vibratile; the other shorter and trailing; fresh water with vegetation. N. apocamptus S. (Fig. 103, p). Oval with broad posterior end; 6-1 Iju long. N. sinuatus S. (Fig. 103, q). Posterior end truncate or concave; about 22/i long. Genus Marsupiogaster Schewiakoff. Oval; flattened; asymme- trical; cytostome occupies entire anterior end; cytopharynx con- spicuous, 1/2 body length; body longitudinally striated; 2 flagella, one directed anteriorly, the other posteriorly; spherical nucleus; contractile vacuole anterior; fresh or salt water. M. striata Schewiakoff (Fig. 103, r). About 27m by 15^; fresh water; Hawaii. EUGLENOIDINA, CHLOROMONADINA 243 M. picta Faria, da Cunha and Pinto (Fig. 103, s). In salt water; Rio de Janeiro. Order 5 Chloromonadina Klebs The chloromonads are of rare occurrence and consequently not well known. The majority possess small discoidal grass-green chro- FiG. 104. a, Gonyostomum semen, X540 (Stein); b, Vacuolaria virescens, X460 (Senn);c, Trentonia flagellata, X330 (Stokes); d, Thaumatomastix setifera, X830 (Lauterborn). matophores with a large amount of xanthophyll which on addition of an acid become blue-green. No pyrenoids occur. The metabolic products are fatty oil. Starch or allied carbohydrates are absent. Stigma is also not present. Genus Gonyostomum Diesing (Rhaphidomonas Stein). With grass-green chromatophores; highly refractile trichocyst-like struc- tures in cytoplasm; in fresh water. A few species. G. semen D. (Fig. 104, a). Sluggish animal; about 45-60^ long; in marshy water among decaying vegetation. Genus Vacuolaria Cienkowski. Highly plastic ; without trichocyst- like structures; anterior end narrow; with 2 flagella; cysts with a gelatinous envelope. One species. V. virescens. C. (Fig. 104, h). About 50-150)u long; fresh water. Genus Trentonia Stokes. Bi-flagellate as in the last genus; but flattened; anterior margin slightly bilobed. One species. T. flagellata S. (Fig. 104, c). Slow-moving organism; encystment followed by binary fission; about 60/i long; fresh water. 244 PROTOZOOLOGY Genus Thaumatomastix Lauterborn. Colorless; pseudopodia formed; 2 flagella, one extended anteriorly, the other trailmg; holo- zoic; perhaps a transitional form between the Mastigophora and the Sarcodina. One species. T. setifera L. (Fig. 104, d). About 20-35m by 15-28^; fresh water. References Allegre, C. F. and T. L. Jahn 1943 A survey of the genus Phacus Dujardin (Protozoa; Euglenoidina). Trans. Amer. Micr. Soc, Vol. 62. Dangeard, p. 1901 Recherches sur les Eugl^niens. Le Botaniste. P. 97. Fritsch, F. E. 1935 The structure and reproduction of the algae. Vol. 1. Cambridge. Hall, R. P. 1923 Morphology and binary fission of Menoidium incurvum (Fres.) Klebs. Uni. Cal. Publ. Zool., Vol. 20. Johnson, L. P. 1939 A study of Euglena rubra Hardy 1911. Trans. Amer. Micr. Soc, Vol. 58. 1944 Euglenae of Iowa. Ibid. Vol. 63. and T. L. Jahn 1942 Cause of the green-red color change in Euglena rubra. Physiol. Zool., Vol. 15. Lemmermann, E. 1913 Eugleninae. In: Siisswasserfl. Deutschlands, Part 2. Pascher, a. 1913 Chloromonadinae. Ibid. Part 2. Smith, G. M. 1933 The freshwater algae of the United States. New York. Wenrich, D. H. 1924 Studies on Euglenamorpha hegneri n. g., n. sp., a euglenoid flagellate found in tadpoles. Biol. Bull., Vol. 47. West, G. S. and F. E. Fritsch. 1927 A treatise on the British fresh- water algae. Cambridge. Chapter 12 Order 6 Dinoflagellata BUtschli THE dino flagellates make one of the most distinct groups of the Mastigophora, inhabiting mostly marine water, and to a lesser extent fresh water. In the general appearance, the arrangement of the two flagella, the characteristic furrows, and the possession of brown chromatophores, they are closely related to the Crypto- monadina. The body is covered by an envelope composed of cellulose which may be a simple smooth piece, or may be composed of two valves or of numerous plates, that are variously sculptured and possess Anterior flagellar pore Annulus or girdle Hypocone Longitudinal flagellum ( Posterior flagellar pore Fig. 105. Diagram of a typical naked dinoflagellate (Lebour). manifold projections. Differences in the position and course of the furrows and in the projections of the envelope produce numerous asymmetrical forms. The furrows, or grooves, are a transverse an- nulus and a longitudinal sulcus. The annulus is a girdle around the middle or toward one end of the body. It may be a complete, incomplete or sometimes spiral ring. While the majority show a single transverse furrow, a few may possess several. The part of the shell anterior to the annulus is called the epitheca and that posterior to the annulus the hypotheca. In case the envelope is not developed, the terms epicene and hypocone are used (Fig. 105). The sulcus may run from end to end or from one end to the annulus. The two flagella arise typically from the furrows, one being transverse and the other longitudinal. The transverse flagellum which is often band-form, encircles the body and undergoes undulating movements, which in former years were looked upon as ciliary movements (hence the name Cilioflagel- lata). In the suborder Prorocentrinea, this flagellum vibrates freely 245 246 PROTOZOOLOGY in a circle near the anterior end. The longitudinal flagellum often projects beyond the body and vibrates. Combination of the move- ments of these flagella produces whirHng movements characteristic of the organisms. The majority of dinofiagellates possess a single somewhat massive nucleus with evenly scattered chromatin, and usually several endo- somes. There are two kinds of vacuoles. One is often surrounded by a ring of smaller vacuoles, while the other is large, contains pink- colored fluid and connected with the exterior by a canal opening into a flagellar pore. The latter is known as the pusule which functions as a digestive organella (Kofoid and Swezy). In many freshwater forms a stigma is present, and in Pouchetiidae there is an ocellus composed of an amyloid lens and a dark pigment-ball. The majority of planktonic forms possess a large number of small chromatophores which are usually dark yellow, brown or sometimes slightly greenish and are located in the periphery of the body, while bottom-dwelling and parasitic forms are, as a rule, colorless, because of the absence of chromatophores. A few forms contain haematochrome. The method of nutrition is holophytic, holozoic, saprozoic, or mixotrophic. In holophytic forms, anabolic products are starch, oil, or fats. Asexual reproduction is bj^ binary or multiple fission or budding in either the active or the resting stage and differs among different groups. Encystment is of common occurrence. In some forms the cyst wall is formed within the test. The cysts remain alive for many years; for example, Ceratium cysts w^ere found to retain their vital- ity in one instance for six and one-half years. Conjugation and sexual fusion have been reported in certain forms, but definite knowledge on sexual reproduction awaits further investigation. The dinofiagellates are abundant in the plankton of the sea and play an important part in the economy of marine life as a whole. A number of parasitic forms are also known. Their hosts include vari- ous diatoms, copepods and several pelagic animals. Bivalve shell without furrows Suborder 1 Prorocentrinea Naked or with shell showing furrows. .Suborder 2 Peridiniinea (p. 248) Naked; without furrows; no transverse flagellum Suborder 3 Cystoflagellata (p. 261) Suborder 1 Prorocentrinea Poche Test bivalve; without any groove; with yellow chromatophores; 2 flagella anterior, one directed anteriorly, the other vibrates in a circle; fresh or salt water. DINOFLAGELLATA 247 Family Prorocentridae Kofoid Genus Prorocentrum Ehrenberg. Elongate oval; anterior end bluntly pointed, with a spinous projection at pole; chromatophores small, yellowish brown; salt water. P. micans E. (Fig. 106, a). 36-52jli long; a cause of "red water." P. triangulatum Martin. Triangular with rounded posterior end; shell-valves flattened; one valve with a delicate tooth; surface cov- ered with minute pores; margin striated; chromatophores yellow- FiG. 106. a, Prorocentrum micans, X420 (Schiitt); b, c, Exuviaella marina, X420 (Schiitt); d, e, Cystodinium steini, X370 (Klebs); f, Gleno- dinium cinctum, X590 (Schilling); g, G. pulvisculum, X420 (Schilling); h, G. uliginosuvi, X590 (Schilling); i, G. edax, X490 (Schilling); j, G. neglectum, X650 (Schilling). brown, irregular, broken up in small masses; 17-22/i (excluding tooth); Martin found it extremely abundant in brackish water in New Jersey. Genus Exuviaella Cienkowski. Subspherical or oval; no anterior projection, except 2 flagella; 2 lateral chromatophores, large, brown, each with a pyrenoid and a starch body; nucleus posterior; salt water. Several species. E. marina C. (Fig. 106, b, c). 36-50^ long. 248 PROTOZOOLOGY E. apora Schiller. Compressed, oval; striae on margin of valves; chromatophores numerous yellow-brown, irregular in form; S0-32fx by 21-26)u (Schiller); 17-22m by 14-19^ (Lebour; Martin); common in brackish water, New Jersey. Suborder 2 Peridiniinea Poche Typical dinoflagellates with one to many transverse annuli and a sulcus; 2 flagella, one of which undergoes a typical undulating movement, while the other usually directed posteriorly. According to Kofoid and Swezy, this suborder is divided into two tribes. Body naked or covered by a thin shell Tribe 1 Gymnodinioidae Body covered by a thick shell Tribe 2 Peridinioidae (p. 257) Tribe 1 Gymnodinioidae Poche Naked or covered by a single piece cellulose membrane with an- nulus and sulcus, and 2 flagella; chromatophores abundant, yellow or greenish platelets or bands; stigma sometimes present; asexual reproduction, binary or multiple division; holophytic, holozoic, or saprozoic; the majority are deep-sea forms; a few coastal or fresh water forms also occur. With a cellulose membrane Family 1 Cystodiniidae Without shell Furrows rudimentary Family 2 Pronoctilucidae (p. 249) Annulus and sulcus distinct Solitary With ocellus Family 3 Pouchetiidae (p. 249) Without ocellus With tentacles Family 4 Noctilucidae (p. 251) Without tentacles Free-living Family 5 Gymnodiniidae (p. 251) Parasitic Family 6 Blastodiniidae (p. 254) Permanently colonial Family 7 Polykrikidae (p. 257) Family 1 Cystodiniidae Kofoid and Swezy Genus Cystodinium Klebs. In swimming phase, oval, with ex- tremely delicate envelope; annulus somewhat acyclic; cyst-mem- brane drawn out into 2 horns. C. steini K. (Fig. 106, d, e). Stigma beneath sulcus; chromato- phores brown; swarmer about 45)u long; freshwater ponds. Genus Glenodinium Ehrenberg. (Glenodiniopsis, Stasziecella Woloszynska). Spherical; ellipsoidal or reniform in end-view; an- nulus a circle; several discoidal, yellow to brown chromatophores; horseshoe- or rod-shaped stigma in some; often with gelatinous en- velope; fresh water. Many species. DINOFLAGELLATA 249 G. cinctum E. (Fig. 106,/). Spherical to ovoid; annulus equatorial; stigma horseshoe-shaped; 43/i by 40/i. G. pulvisculum Stein (Fig. 106, g). No stigma; 38/x by 30/x. G. uUginosum Schilling (Fig. 106, h). 36-48m by 30/x. G. edax S. (Fig. 106, i). 34m by 33/x. G. neglectum S. (Fig. 106, j)- 30-32/x by 29m. Family 2 Pronoctilucidae Lebour Genus Pronoctiluca Fabre-Domergue. Body with an antero- ventral tentacle and sulcus; annulus poorly marked; salt water. P. tentaculatum (Kofoid and Swezy) (Fig. 107, a). About 54m long; off California coast. Genus Oxyrrhis Dujardin. Subovoidal, asymmetrical posteriorly; annulus incomplete; salt water. 0. marina D. (Fig. 107, b). 10-37m long. Family 3 Pouchetiidae Kofoid and Swezy Ocellus consists of lens and melanosome (pigment mass); sulcus and annulus somewhat twisted; pusules usually present; cytoplasm colored; salt water (pelagic). Genus Pouchetia Schiitt. Nucleus anterior to ocellus; ocellus with red or black pigment mass with a red, brown, yellow, or colorless central core; lens hyaline; body surface usually smooth; ho lo zoic; encystment common; salt water. Many species. P.fusus S. (Fig. 107, c). About 94m by 41m; ocellus 27m long. P. maxima Kofoid and Swezy (Fig. 107, d). 145m by 92m; ocellus 20m; off California coast. Genus Protopsis Kofoid and Swezy. Annulus and sulcus similar to those of Gymnodinium or Gyrodinium; with a simple or compound ocellus; no tentacles; body not twisted; salt water. A few species. P. ochrea (Wright) (Fig. 107, e). 55m by 45m; ocellus 22m long; Nova Scotia. Genus Nematodinium Kofoid and Swezy. With nematocysts; girdle more than 1 turn; ocellus distributed or concentrated, pos- terior; ho lo zoic; salt water. A'', partitum K. and S. (Fig. 107,/). 91m long; off California coast. Genus Proterythropsis Kofoid and Swezy. Annulus median; ocel- lus posterior; a stout rudimentary tentacle; salt water. One species, P. crassicaudata K. and S. (Fig. 107, g). 70m long; off California. Genus Erythropsis Hertwig. Epicone flattened, less than 1/4 hypocone; ocellus very large, composed of one or several hyaline lenses attached to or imbedded in a red, brownish or black pigment 250 PROTOZOOLOGY Fig. 107. a, Pronoctiluca tentaculatum, X730 (Kofoid and Swezy); b, Oxyrrhis marina, X840 (Senn); c. Pouchetia fusus, X340 (Schiitt); d, P. maxima, X330 (Kofoid and Swezy); e, Protopsis ochrea, X340 (Wright); f, Nematodinium partitum, X560 (Kofoid and Swezy); g, Pro- tenjthropsis crassicaudata, x740 (Kofoid and Swezy); h, Erythropsis cormita, X340 (Kofoid and Swezy); i, j , Nodiluca scintillans (i, side view; j, budding process), Xl40 (Robin), DINOFLAGELLATA 251 body with a red, brown or yellow core, located at left of sulcus; sulcus expands posteriorly into ventro-posterior tentacle; salt water. Several species. E. cornuta (Schiitt) (Fig. 107, h). 104/x long; off California coast (Kofoid and Swezy). Family 4 Noctilucidae Kent Contractile tentacle arises from sulcal area and extends poste- riorly; a flagellum; this group has formerly been included in the Cystoflagellata; studies by recent investigators, particularly by Kofoid, show its affinity with the present suborder ; ho lozoic; saltwater. Genus Noctiluca Suriray. Spherical, bilaterally symmetrical; peri- stome marks the median line of body; cytostome at the bottom of peristome; with a conspicuous tentacle; cytoplasm greatly vacuo- lated, and cytoplasmic strands connect the central mass with peri- phery; specific gravity is less than that of sea water, due to the pre- sence of an osmotically active substance with a lower specific gravity than sodium chloride, which appears to be ammonium chloride (Goethard and Heinsius); peripheral granules luminescent (p. 100); cytoplasm colorless or blue-green; sometimes tinged with j^ellow coloration in center; swarmers formed by budding, and each posses- ses one flagellum, annulus, and tentacle; widely distributed in salt w^ater; ho lozoic. One species. A^. scintillans (Macartney) (A^. miliaris S.) (Fig. 107, i, j). Usu- ally 500-1000/i in diameter, with extremes of 200;u and 3 mm. Gross (1934) observed that complete fusion of two swarmers (isoga- metes) results in cyst formation from which trophozoites develop. Acid content of the body fluid is said to be about pH 3. Genus Pavillardia Kofoid and SwezJ^ Annulus and sulcus similar to those of Gyimiodinium; longitudinal flagellum absent; stout finger-like mobile tentacle directed posteriori}^; salt water. One species. P. tentaculifera K. and S. 58ju by 27 /x; pale yellow; off California. Family 5 Gymnodiniidae Kofoid Naked forms with simple but distinct 1/2-4 turns of annulus; with or without chromatophores; fresh or salt water. Genus Gymnodinium Stein. Pellicle delicate; subcircular; bi- laterally symmetrical; numerous discoid chromatophores vari- colored (yellow to deep brown, green, or blue) or sometimes absent; stigma present in few; many with mucilaginous envelope; salt, brackish, or fresh water. Numerous species. 252 PROTOZOOLOGY Fig. 108. a, Gymnodinium aeruginosum, X500 (Schilling); b, G. ro- tundatum, X360 (Klebs); c, G. poZwsire, X 360 (Schilling); d, G. agile, X740 (Kofoid and Swezy); e, Hemidinium nasuttiin, X670 (Stein); f, Aviphidinium lacustre, X440 (Stein); g, A. scissiim, X880 (Kofoid and Swezy); h, Gyrodinium hiconicxim, X340 (Kofoid and Swezy); i, G. hyalinum, X670 (Kofoid and Swezy); j, Cochlodinium atromacu- latum, X340 (Kofoid and Swezy); k, Torodinium robustum, X670 (Kofoid and Swezy); 1, Massartia nieuportensis, X670 (Conrad); m, Chilodinium cruciatum, X900 (Conrad); n, o, Trochodinium prismaticum, X1270 (Conrad); p, Ceratodinium asymmetricum, X670 (Conrad). DINOFLAGELLATA 253 G. aeruginosum S. (Fig. 108, a). Chromatophores green; 33-35^ by 22/x; ponds and lakes. G. rotundatum Klebs (Fig. 108, b). 32-35^ by 22-25^; fresh water. G. palustre Schilling (Fig. 108, c). 45/Lt by 38/x; fresh water. G. agile Kofoid and Swezy (Fig. 108, d). About 28m long; along sandy beaches. Genus Hemidinium Stein. Asymmetrical; oval; annulus about half a turn, only on left half. One species. H. nasutum S. (Fig. 108, e). Sulcus posterior; chromatophores yellow to brown; with a reddish brown oil drop; nucleus posterior; transverse fission; 24-28m by 16-17^; fresh water. Genus Amphidinium Claparede and Lachmann. Form variable; epicone small; annulus anterior; sulcus straight on hypocone or also on part of epicone; with or without chromatophores; mainly holo- phytic, some holozoic; coastal or fresh water. Numerous species. A. lacustre Stein (Fig. 108, /). 30/i by 18^; in fresh and salt (?) water, A. scissum Kofoid and Swezy (Fig. 108, g). 50-60/x long; along sandy beaches. A. fusiforme Martin. Fusiform, twice as long as broad: circular in cross-section; epicone rounded conical; annulus anterior; hypo- cone 2-2.5 times as long as epicone; sulcus obscure; body filled with yellowish green chromatophores except at posterior end; stigma dull orange, below girdle; nucleus ellipsoid, posterior to annulus; pellicle delicate; 17-22^ by 8-1 1/x in diameter. Martin (1929) found that it was extremely abundant in parts of Delaware Bay and gave rise to red coloration of the water ("Red water"). Genus Gyrodinium Kofoid and Swezy. Annulus descending left spiral; sulcus extending from end to end; nucleus central; pusules; surface smooth or striated; chromatophores rarely present; cyto- plasm colored; holozoic; salt or fresh water. Many species. G. biconicum K. and S. (Fig. 108, h). 68ju long; salt water; off Cali- fornia. G. hyalinum (Schilling) (Fig. 108, i). About 24/u long ; fresh water. Genus Cochlodinium Schutt. Twisted at least 1.5 turns; annulus descending left spiral; pusules; cytoplasm colorless to highly colored; chromatophores rarely present; holozoic; surface smooth or striated; salt water. Numerous species. C. atromaculatum Kofoid and Swezy (Fig. 108, j). 183-185ju by 72m; longitudinal flagellum 45m long; off California. Genus Torodinium Kofoid and Swezy. Elongate; epicone several times longer than hypocone; annulus and hypocone form augur- 254 PROTOZOOLOGY shaped cone; sulcus long; nucleus greatly elongate; salt water. 2 species. T. rohustum K. and S. (Fig. 108, k). 67-75m long; off California. Genus Massartia Conrad. Cylindrical; epicone larger (9-10 times longer and 3 times wider) than hypocone; no sulcus; with or without yellowish discoid chromatophore. M. niewportensis C. (Fig. 108, I). 28-37iu long; brackish water. Genus Chilodinium Conrad. Ellipsoid; posterior end broadly rounded, anterior end narrowed and drawn out into a digitform process closely adhering to body; sulcus, apex to 1/5 from posterior end; annulus oblique, in anterior 1/3. C. cruciatum C. (Fig. 108, m). 40-50iu by 30-40)li; with trichocysts; brackish water. Genus Trochodinium Conrad. Somewhat similar to Amphidi- nium; epicone small, button-like; hypocone with 4 longitudinal rounded ridges; stigma; without chromatophores. T. prismaticum C. (Fig. 108, n, o). 18-22/i by 9-12/i; epicone 5-7ju in diameter; brackish water. Genus Ceratodinium Conrad. Cuneiform; asymmetrical, color- less, more or less flattened; annulus complete, oblique; sulcus on half of epicone and full length of hypocone; stigma. C. asymmetricum C. (Fig.l08,p). 68-80^ by about lO^t; brackish water. Family 6 Blastodiniidae Kofoid and Swezy All parasitic in or on plants and animals; in colony forming genera, there occur trophocyte (Chatton) by which organism is attached to host and more or less numerous gonocytes (Chatton). Genus Blastodinium Chatton. In the gut of copepods; spindle- shaped, arched, ends attenuated; envelope (not cellulose) often with 2 spiral rows of bristles; young forms binucleate; when present, chromatophores in yellowish brown network; swarmers similar to those of Gymnodinium; in salt water. Many species. B. spinulosum C. (Fig. 109, a). About 235m by 33-39)li; swarmers 5-10^; in Palacalanus parvus, Clausocalanus arcm'corm's and C. furcatus. Genus Oodinium Chatton. Spherical or pyriform; with a short stalk; nucleus large; often with yellowish pigment; on Salpa, Anne- lida, Siphonophora, etc. 0. poucheti (Lemmermann) (Fig. 109, b, c). Fully grown indivi- duals up to 170m long; bright yellow ochre; mature forms become detached and free, dividing into numerous gymnodinium-like swarmers; on the tunicate, Oikopleura dioica. DINOFLAGELLATA 255 Genus Apodinium Chatton. Young individuals elongate, spherical or pyriform; binucleate; adult colorless; formation of numerous swarmers in adult stage is peculiar in that lower of the 2 individuals formed at each division secretes a new envelope, and delays its Fig. 109. a, Blastodinium spinulosiwi, X240 (Chatton); b, c, Oodi- nium poucheti (c, a swarmer) (Chatton); d, e, Apodinium inycetoides (d, swarmer-formation, X450; e, a younger stage, X 640) (Chatton); f, Chytriodiniiim parasiticum in a copepod egg (Dogiel) ; g, Trypanodinium ovicola, X1070 (Chatton); h, Duboscqella tintinnicola (Duboscq and Collin); i, j, Haplozoon clymenellae (i, mature colony, X300; j, a swarmer, X1340) (Shumway); k, Syndinium turbo, X1340 (Chatton); 1, Paradi- niuni poucheti, X800 (Chatton); m, Ellobiopsis chattoni on Calanus fin- mar chicus (CauUery); n, Paraellobiopsis coutieri (Collin). 256 PROTOZOOLOGY further division until the upper one has divided for the second time, leaving several open cups; on tunicates. A. mycetoides C. (Fig. 109, d, e). On gill-slits of Fritillaria pel- lucida. Genus Chytriodinium Chatton. In eggs of planktonic copepods; young individuals grow at the expense of host egg and when fully formed, body divides into many parts, each producing 4 swarmers. Several species. C. parasiticum (Dogiel) (Fig. 109, /). In copepod eggs; Naples. Genus Trypanodinium Chatton. In copepod eggs; swarmer-stage only known. T. ovicola C. (Fig. 109, g). Swarmers biflagellate; about 15/x long. Genus Duboscqella Chatton. Rounded cell with a large nucleus; parasitic in Tintinnidae. One species. D. tintinnicola (Lohmann) (Fig. 109, h). Intracellular stage oval, about 100/x in diameter with a large nucleus; swarmers biflagellate. Genus Haplozoon Dogiel. In gut of polychaetes; mature forms composed of variable number of cells arranged in line or in pyramid; salt water. Many species. H. clymenellae (Calkins) (Fig. 109, i, j). In Clymenella torquata; colonial forms consist of 250 or more cells; Woods Hole. Genus Syndiniiim Chatton. In gut and body cavity of marine copepods; multinucleate round cysts in gut considered as young forms; multinucleate body in host body cavity with numerous needle-like inclusions. S. turbo C. (Fig. 109, k). In Paracalanus parvus, Corycaeus ven- ustus, Calanus finmarchicus; swarmers about 15/i long. Genus Paradinium Chatton. In body-cavity of copepods; mul- tinucleate body without inclusions; swarmers formed outside the host body. P. poucheti C. (Fig. 109, I). In the copepod, Acartia clausi; swarm- ers about 25/n long, amoeboid. Genus EUobiopsis Caullery. Pyriform; with stalk; often a septum near stalked end; attached to anterior appendages of marine cope- pods. E. chattoni C. (Fig. 109, m). Up to 700/i long; on antennae and oral appendages of Calanus finmarchicus, Pseudocalanus elongatus and Acartia clausi. Genus Paraellobiopsis Collin. Young forms stalkless; spherical; mature individuals in chain-form; on Malacostraca. P. coutieri C. (Fig. 109, n). On appendages of Nebalia hipes. DINOFLAGELLATA 257 Family 7 Polykrikidae Kofoid and Swezey Two, 4, 8, or 16 individuals permanently joined; individuals similar to Gymnodinium; sulcus however extending entire body length; with nematocysts (Fig. 110, b); greenish to pink; nuclei about 1/2 the number of individuals; holozoic; salt water. Genus Polykrikos Biitschli. With the above-mentioned char- acters; salt or brackish water, P. kofoidi (Chatton) (Fig. 110, a, b). Greenish grey to rose; com- posed of 2, 4, 8, or 16 individuals; with nematocysts; each nemato- cyst possesses presumably a hollow thread, and discharges under suitable stimulation its content; a binucleate colony composed of 4 individuals about UOm long; off California, P. barnegatensis Martin, Ovate, nearly circular in cross-section, slightly concave ventrally; composed of 2 individuals; constriction slight; beaded nucleus in center; annuli descending left spiral, dis- placed twice their width; sulcus ends near anterior end; cytoplasm colorless, with numerous oval, yellow-brown chromatophores; nem- atocysts absent; 46^ by 31.5)u; in brackish water of Barnegat Bay. Tribe 2 Peridinioidae Poche The shell composed of epitheca, annulus and hypotheca, which may be divided into numerous plates; body form variable. With annulus and sulcus Shell composed of plates; but no suture Family 1 Peridiniidae Breast plate divided by sagittal suture . Family 2 Dinophysidae (p. 261) Without annulus or sulcus Family 3 Phytodiniidae (p. 261) Family 1 Peridiniidae Kent Shell composed of numerous plates; annulus usually at equator, covered by a plate known as cingulum; variously sculptured and finely perforated plates vary in shape and number among different species; in many species certain plates drawn out into various proc- esses, varying greatly in different seasons and localities even among one and the same species; these processes seem to retard descending movement of organisms from upper to lower level in water when flagellar activity ceases; chromatophores numerous small platelets, yellow or green; some deep-sea forms without chromatophores; chain formation in some forms; mostly surface and pelagic inhabitants in fresh or salt water. Genus Peridinium Ehrenberg. Subspherical to ovoid; reniform in cross-section; annulus slightly spiral with projecting rims; hypotheca often with short horns and epitheca drawn out; colorless, green, or 258 PROTOZOOLOGY brown; stigma usually present; cysts spherical; salt or fresh water. Numerous species. P. tahulatum Claparede and Lachmann (Fig. 110, c). 48m by 44^; fresh water. Fig. 110. a, b, Pohjkrikos kofoidi (a, colony of four individuals, X340; b, a nematocyst, X1040) (Kofoid and Swezy); c, Peridinium tahulatum, X460 (Schilling); d, P. divergens, X340 (Calkins); e, Ceratiuni hirundi- nella, X540 (Stein); f. C. longipes, XlOO (Wailes); g, C. tripos, Xl40 (Wailes); h, C. fusus, XlOO (Wailes); i, Heterodinium scrippsi, X570 (Kofoid and Adamson). DINOFLAGELLATA 259 P. divergens (E.) (Fig. 110, d). About 45ju in diameter; yellowish, salt water. Genus Ceratium Schrank. Body flattened; with one anterior and 1-4 posterior horn-Uke processes; often large; chromatophores yel- low, brown, or greenish; color variation conspicuous; fission is said to take place at night and in the early morning; fresh or salt water. Numerous species; specific identification is difficult due to a great variation (p. 176). C. hirundinella (Miiller) (Figs. 85; 110, e). 1 apical and 2-3 anta- pical horns; seasonal and geographical variations (p. 177); chain- formation frequent; 95-700iu long; fresh and salt water. Numerous varieties. C. longipes (Bailey) (Fig. 110, /). About 210m by 51-57^; salt water. C. tripos (Muller) (Fig. 110, g). About 225/x by 75m; salt water. Wailes (1928) observed var. atlantica in British Columbia; Martin (1929) in Barnegat Inlet, New Jersey. C.fusus (Ehrenberg) (Fig. 110, h). 300-600m by 15-30m; salt water; widely distributed; British Columbia (Wailes), New Jersey (Martin), etc. Genus Heterodinium Kofoid. Flattened or spheroidal; 2 large antapical horns; annulus submedian; with post-cingular ridge; sulcus short, narrow; shell hyaline, reticulate, porulate; salt water. Numer- ous species. H. scrippsi K. (Fig. 110, i). 130-155m long; Pacific and Atlantic (tropical). Genus Dolichodinium Kofoid and Adamson. Subconical, elongate; without apical or antapical horns; sulcus not indenting epitheca; plate porulate; salt water. D. lineatum (Kofoid and Michener) (Fig. Ill, a). 58m long; eastern tropical Pacific. Genus Goniodoma Stein. Polyhedral with a deep annulus; epi- theca and hypotheca slightly unequal in size, composed of regularly arranged armored plates; chromatophores small brown platelets; fresh or salt water. G. acuminata (Ehrenberg) (Fig. 111,6). About 50m long; salt water. Genus Gonyaulax Diesing. Spherical, polyhedral, fusiform, elongated with stout apical and antapical prolongations, or dorso- ventrally flattened; apex never sharply attenuated; annulus equa- torial; sulcus from apex to antapex, broadened posteriorly; plates 1-6 apical, 0-3 anterior intercalaries, 6 precingulars, 6 annular plates, 6 postincingulars, 1 posterior intercalary and 1 antapical; 260 PROTOZOOLOGY porulate; chromatophores yellow to dark brown, often dense; with- out stigma; fresh, brackish or salt water. Numerous species. G. polyedra Stein (Fig. Ill, c). Angular, polyhedral; ridges along sutures, annulus displaced 1-2 annulus widths, regularly pitted; salt water. "Very abundant in the San Diego region in the summer Fig. 111. a, Dolichodinium lineatum, X670 (Kofoid and Adamson); b, Goniodoma acuminata, X340 (Stein); c, Gonaulax polyedra, X670 (Kofoid); d, G. apiculata, X670 (Lindemann).; e, Spiraulax jolliffei, right side of theca, X340 (Kofoid); f, Dinophxjsis acuta, X580 (Schutt); g, h, Oxyphysis oxytoxoides, X780 (Kofoid); i, Phytodinium simplex, X340 (Klebs); j, k, Dissodinium lunula: ], primary cyst (Dogiel); k, secondary cyst with 4 swarmers (Wailes), X220. plankton, July-September, when it causes local outbreaks of 'red water,' which extend along the coast of southern and lower Cali- fornia" (Kofoid, 1911). DINOFLAGELLATA 261 G. apiculata (Penard) (Fig. Ill, d). Ovate, chromatophores yel- lowish brown; 30-60;* long; fresh water. Genus Spiraulax Kofoid. Biconical; apices pointed; sulcus not reaching apex; no ventral pore; surface heavily pitted; salt water. S. jolliffei (Murray and Whitting) (Fig. Ill, e). 132^ by 92^; California. Family 2 Dinophysidae Kofoid Genus Dinophysis Ehrenberg. Highly compressed ; annulus wid- ened, funnel-like, surrounding small epitheca; chromatophores yel- low; salt water. Several species. D. acuta E. (Fig. 111,/). Oval ; attenuated posteriorly; 54-94/i long; widely distributed; British Columbia (Wailes). Genus Oxyphysis Kofoid. Epitheca developed; sulcus short; sulcal lists feebly developed; sagittal suture conspicuous; annulus im- pressed; salt water. 0. oxytoxoides K. (Fig. Ill, g,h). 63-68/1 by 15m; off Alaska. Fig. 112. a, Leptodiscus medusoides, X50 (Hertwig); b, Craspedotella pileolus, XllO (Kofoid). Family 3 Phytodiniidae Klebs Genus Phytodinium Klebs. Spherical or ellipsoidal; without fur- rows; chromatophores discoidal, yellowish brown. P. simplex K. (Fig. Ill, i). Spherical or oval; 42-50/i by 30-45/i fresh water. Genus Dissodinium Klebs {Pyrocystis Paulsen). Primary cyst, spherical, uninucleate; contents divide into 8-16 crescentic second- ary cysts which become set free; in them are formed 2, 4, 6, or 8 Gymnodinium-like swarmers; salt water. D. lunula (Schutt) (Fig. Ill, j, k). Primary cysts 80-155/1 in diameter; secondary cysts 104-130/i long; swarmers 22/i long; widely distributed; British Coumbia (Wailes). Suborder 3 Cystoflagellata Haeckel Since Noctiluca which had for many years been placed in this suborder, has been removed, according to Kofoid, to the second sub- order, the Cystoflagellata becomes a highly ill-defined group and 262 PROTOZOOLOGY includes two peculiar marine forms: Leptodiscus medusoides Hertwig (Fig. 112, a), and Craspedotella pileolus Kofoid (Fig. 112, h), both of which are medusoid in general body form. References Chatton, E. 1920 Les P^ridieniens parasites ; morphologie, repro- duction, ethologie. Arch. zool. exp. et g^n. Vol. 59. DiwALD, K. 1939 Ein Beitrag zur VariabiUtat und Systematik der Gattung Peridinium. Arch. f. Protistenk., Vol. 93. Fritsch, F. E. 1935 The structure and reproduction of the algae. Vol. 1. Cambridge. Gross, F, 1934 Zur Biologie und Entwicklungsgeschichte von Noctiluca miliaris. Archiv. f. Protistenk., Vol. 83. Kofoid, C. A. 1906 On the significance of the symmetry of the Dinoflagellata. Uni. Calif. Publ. Zool., Vol. 3. — 1920 A new morphological interpretation of Noctiluca and its bearing on the status of Cysto^agellata. Ibid., Vol. 19. and A. M. Adamson 1933 The Dinoflagellata: The family Heterodiniidae of the Peridinioidae. Mem. Mus. Comp. Zool., Harvard, Vol. 54. - and Olive Swezy 1921 The free-living unarmored Dino- flagellata. Mem. Uni. Calif., Vol. 5. Lebour, Marie V. 1925 The dinoflagellates of northern seas. Lon- don. Martin, G. W. 1929 Dinoflagellates from marine and brackish waters of New Jersey. Uni. Iowa Studies in Nat. Hist., Vol. 12, Reichenow, E. 1930 Parasitische Peridinea. Grimpe's Die Tierwelt der Nord- und Ostsee. Part 19. Schilling, A. 1913 Dinojiagellatae (Peridineae). Siisswasserflora Deutschlands, etc. H. 3. Wailes, G. H. 1928 Dinoflagellates and Protozoa from British Columbia. Vancouver Mus. Notes. Vol. 3. Chapter 13 Subclass 2 Zoomastigina Doflein THE Zoomastigina lack chromatophores and their body organ- izations vary greatly from a simple to a very complex type. The majority possess a single nucleus which is, as a rule, vesicular in structure. A characteristic organella, the parabasal body (p. 66) is present in numerous forms and myonemes are found in some species. Nutrition is holozoic or saprozoic (parasitic). Asexual reproduction is by longitudinal fission; sexual reproduction is unknown. Encystment occurs commonly. The Zoomastigina are free-living or parasitic in various animals. With pseudopodia besides flagella Order 1 Rhizomastigina With flagella only With 1-2 flagella Order 2 Protomonadina (p. 268) With 3-8 flagella Order 3 Polymastigina (p. 293) With more than 8 flagella Order 4 Hypermastigina (p. 318) Order 1 Rhizomastigina Biitschli A number of borderline forms between the Sarcodina and the Mastigophora are placed here. Flagella vary in number from one to several and pseudopods also vary greatly in number and in appear- ance. With many flagella Family 1 Multiciliidae With 1-3 rarely 4 flagella Family 2 Mastigamoebidae Family 1 Multiciliidae Poche Genus Multicilia Cienkowski. Generally spheroidal, but amoeboid; with 40-50 flagella, long and evenly distributed; one or more nuclei; holozoic; food obtained by means of pseudopodia; multiplication by fission; fresh or salt water. M. marina C. (Fig. 113, a). 20-30/i in diameter; uninucleate ; salt water. M. lacustris Lauterborn (Fig. 113, 6). Multinucleate; 30-40/ii in diameter; fresh water. Family 2 Mastigamoebidae With 1-3 or rarely 4 flagella and axopodia or lobo podia; uninucle- ate; flagellum arises from a basal granule which is connected with the nucleus by a rhizoplast; binary fission in both trophic and encysted stages; sexual reproduction has been reported in one spe- 263 264 PROTOZOOLOGY cies; holozoic or saprozoic; the majority are free-living, though a few parasitic. Genus Mastigamoeba Schulze {Mastigina Frenzel). Monomasti- gote, uninucleate, with finger-like pseudopodia; flagellum long and connected with nucleus; fresh water, soil or endocommensal. Fig. 113. a, MuUicilia marina, X400 (Cienkowski) ; b, M. lacustris, X400 (Lauterborn) ; c, Mastigamoeba aspera, X200 (Schulze); d, M, longifilum, X340 (Stokes); e, M. setosa, X370 (Goldschmidt); f, Masti- gellavitrea, X 370 (Goldschmidt). M. aspera S. (Fig. 113, c). Subspherical or oval; during locomotion elongate and narrowed anteriorly, while posterior end rounded or lobed; numerous pseudopods slender, straight; nucleus near flagel- late end; 2 contractile vacuoles; 150-200^ by about 50/x; in ooze of pond. M. longifilum Stokes (Fig. 113, d). Elongate, transparent; flagel- lum twice body length; pseudopods few, short; contractile vacuole anterior; body 28/i long when extended, contracted about 10/x; stag- nant water. ZOOMASTIGINA, RHIZOMASTIGINA 265 M. setosa (Goldschmidt) (Fig. 113, e). Up to 140^ long. M. hylae (Frenzel) (Fig. 114, a). In hind gut of frogs and tadpoles; 80-100m by 20)u; flagellum about 10m long. Genus Mastigella Frenzel. Flagellum apparently not connected with nucleus; pseudopods numerous, digitate; body form changes actively and continuously; contractile vacuole. M. vitrea Goldschmidt (Fig. 113,/). 150/x long; sexual reproduction (Goldschmidt). Fig. 114. a, Mastigamoeha hylae, X690 (Becker); b, Adinomonas mirabilis, X1140 (Griessmann) ; c. Dimorpha mutans, X940 (Blochmann); d, Pteridomonas pulex, X540 (Penard); e, Histomonas meleagridis, X940 (Tyzzer); f, Rhizomastix gracilis, X1340 (Mackinnon). Genus Actinomonas Kent. Generally spheroidal, with a single flagellum and radiating pseudopods; ordinarily attached to foreign object with a cytoplasmic process, but swims freely by withdrawing it; nucleus central; several contractile vacuoles; ho lo zoic. A. mirabilis K. (Fig. 114, 6). Numerous simple filopodia; about lOju in diameter; flagellum 20/i long; fresh water. Geuus Dimorpha Gruber. Ovoid or subspherical ; with 2 flagella and radiating axopodia, all arising from an eccentric centriole; nu- cleus eccentric; pseudopods sometimes withdrawn; fresh water. D. mutans G. (Fig. 114, c). 15-20^ in diameter; flagella about 20- 30m long. Genus Pteridomonas Pen