: D ; rr : r- i lo o o o m o PROTOZOOLOGY K ; PROTOZOOLOGY By RICHARD R. KUDO, D.Sc. Professor of Zoology The University of Illinois Urbana, Illinois With three hundred and seventy-six illustrations Fourth Edition CHARLES C THOMAS • PUBLISHER Springfield, Illinois • U.S.A. Charles C Thomas • Publisher Bannerstone House 301-327 East Lawrence Avenue, Springfield, Illinois Published simultaneously in the British Commonwealth oj Nations by Blackwell Scientific Publications, Ltd., Oxford, England Published simultaneously in Canada by The Ryerson Press. Toronto This monograph is protected by copyright. No part of it may be reproduced in any manner without written permission from the publisher. Copyright 1931, 1939, 1946, and 1954 by Charles C Thomas • Publisher First Edition, January, 1931 Second Edition, September, 1939 Third Edition, January, 1946 Third Edition, Second Printing, November, 1947 Third Edition, Third Printing, August, 1950 Fourth Edition, September, 1954 Library of Congress Catalog Card Number: 54-6567 Printed in the United States oj America "The revelations of the Microscope are perhaps not excelled in importance by those of the telescope. While exciting our curiosity, our wonder and admiration, they have proved of infinite service in advancing our knowledge of things around us." Leidy LIBRfcfcYl^ /^' Preface THE fourth edition of Protozoology maintains its original aim in setting forth "introductory information on the common and rep- resentative genera of all groups of both free-living and parasitic Protozoa" for seniors and graduates in zoology in colleges and uni- versities. It has been noted in recent years that students frequently wished to obtain a fuller knowledge on certain topics, organisms, processes, etc., than that which was found in the former edition. In order to meet this need without too great an expansion, references have been given to various items in the text and a list of a much larger number of literature has been appended to each chapter. Furthermore, this enlargement of references increases the usefulness of this work to advanced students, teachers of biology, field workers in various areas of biological science, veterinarians, physicians, pub- lic health workers, laboratory diagnosticians and technicians, etc. While the chapter arrangement remains the same as before, a thorough revision has been carried on throughout the text in the light of many recently published contributions to protozoology. Good illustrations are indispensable in this kind of work, since they are far more easily comprehended than lengthy statements. There- fore, old illustrations were replaced by more suitable ones and many new illustrations have been added, bringing up the total number of the text figures now to 376. Except diagrams, all figures are accom- panied by the scales of magnification. For illustrations that have been adopted from published papers, the indebtedness of the author is expressed by mentioning the authors' names. R. R. Kudo Urbana, Illinois 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 20 Free-living Protozoa, p. 20; parasitic Proto- zoa, p. 28. 3 Morphology 39 The nucleus, p. 40; the cytoplasm, p. 45; loco- motor organellae, p. 49; fibrillar structures, p. 60; protective or supportive organellae, p. 70; hold-fast organellae, p. 76; parabasal appa- ratus, p. 77; Golgi apparatus, p. 78; chondri- osomes, p. 80; contractile and other vacuoles, p. 83; chromatophore and associated organellae, p. 89. 4 Physiology 97 Nutrition, p. 97; reserve food matter, p. 112; respiration, p. 116; excretion and secretion, p. 118; movements, p. 122; irritability, p. 130. 5 Reproduction 145 Nuclear division, p. 145; cytoplasmic division, p. 166; colony formation, p. 173; asexual repro- duction, p. 175; sexual reproduction and life- cycles, p. 180; regeneration, p. 212. 6 Variation and heredity 223 Part II: Taxonomy and special biology 247 CHAPTER 7 Major groups and phylogeny of Protozoa 249 8 Phylum Protozoa 254 Subphylum 1 Plasmodroma 254 Class 1 Mastigophora 254 Subclass 1 Phytomastigina 256 Order 1 Chrysomonadina 256 IX V "C30 CONTENTS 9 Order 2 Cryptomonadina 272 10 Order 3 Phytomonadina 276 11 Order 4 Euglenoidina 293 Order 5 Chloromonadina 306 12 Order 6 Dinoaagellata 310 13 Subclass 2 Zoomastigina 333 Order 1 Rhizomastigina 333 14 Order 2 Protomonadina 339 15 Order 3 Polymastigina 369 16 Order 4 Hypermastigina 404 J 7 Class 2 Sarcodina 417 Subclass 1 Rhizopoda 418 Order 1 Proteomyxa 418 18 Order 2 Mycetozoa 427 19 Order 3 Amoebina 435 20 Order 4 Testacea 472 21 Order 5 Foraminifera 493 22 Subclass 2 Actinopoda 505 Order 1 Heliozoa 505 23 Order 2 Radiolaria 516 24 Class 3 Sporozoa 526 Subclass 1 Telosporidia 526 Order 1 Gregarinida 527 25 Order 2 Coccidia 570 26 Order 3 Haemosporidia 599 27 Subclass 2 AcnidQsporidia 635 Order 1 Haplosporidia 635 Order 2 Sarcosporidia 638 28 Subclass 3 Cnidosporidia 643 Order 1 Myxosporidia 643 Order 2 Actinorayxidia 660 29 Order 3 Microsporidia 668 Order 4 Helicosporidia 678 30 Subphylum 2 Ciliophora 683 Class 1 Ciliata 683 Subclass 1 Protociliata 685 31 Subclass 2 Euciliata 690 Order 1 Holotricha 690 Suborder 1 Astomata 691 32 Suborder 2 Gymnostomata 700 Tribe 1 Prostomata 700 33 Tribe 2 Pleurostomata 723 CONTENTS xi Tribe 3 Hypostomata 728 34 Suborder 3 Trichostomata 737 35 Suborder 4 Hymenostomata 758 36 Suborder 5 Thigmotricha 774 37 Suborder 6 Apostomea 789 38 Order 2 Spirotricha 796 Suborder 1 Heterotricha 796 39 Suborder 2 Oligotricha 814 40 Suborder 3 Ctenostomata 829 41 Suborder 4 Hypotricha 832 42 Order 3 Chonotricha 847 43 Order 4 Peritricha 850 44 Class Suctoria 863 45 Collection, cultivation, and observation of Protozoa 879 Author index 905 Subject index 919 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 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 protozoology maintain that the Protozoa are non-cellular, and not unicellular, 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 organism (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 organism, and further considered that the protozoan is a non-cellular but complete organ- ism, differently organized as compared with cellular organisms, the Metazoa and Metaphyta. Although some writers (Hyman, 1940; Lwoff, 1951) follow this view, the great majority of protozoologists continue to consider the Protozoa as unicellular animals. Through the processes of organic evolution, they have undergone cytological differentiation and the Metazoa histological differentiation. In being unicellular, the Protozoa and the Protophyta are alike. The majority of Protozoa may be distinguished from the majority of Protophyta on the basis of dimensions, methods of nutrition, direc- tion of division-plane, etc. While many Protophyta possess nuclear material, it is not easy to detect it in many forms; on the other hand, all Protozoa contain at least one easily observable nucleus. The binary fission of Protozoa and Protophyta is longitudinal and trans- verse respectively. Most of Ciliata, however, multiply by transverse division. In general the nutrition of Protozoa is holozoic and of Protophyta, holophytic or saprophytic; but there are large numbers of Protozoa which nourish themselves by the latter methods. Thus an absolute and clean-cut separation of the two groups of unicellular organisms is not possible. Haeckel (1866) coined the name Protista to include these organisms in a single group, but this is not generally 6 PROTOZOOLOGY adopted, since it includes undoubted animals and plants, thus creat- ing an equal amount of confusion between it and the animal or the plant. Calkins (1933) excluded chromatophore-bearing Mastigoph- ora from his treatment of Protozoa, thus placing organisms similar in every way, except the presence or absence of chromatophores, in two different (animal and plant) groups. This intermingling of char- acteristics between the two groups of microorganisms shows clearly their close interrelationship and suggests 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. 173), 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 so dependent upon other cells as are the cells in Metazoa; therefore, they do not form any true 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 reaching the gastrula stage. Thus, colonial Protozoa are only cell-aggregates without histological differentiation and may thus be distinguished from the Metazoa. An enormous number of 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- covering the nature and mechanism of various phenomena, the INTRODUCTION 7 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 discussion of any 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 re- mote past and which probably were somewhat similar to the primi- tive 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 and physiological variations are encountered commonly in all forms. Whether variation is due to germinal or environmental conditions, is often difficult to determine. Studies on conjugation in Paramecium by utilizing the mating types first noted by Sonneborn (1937, 1938) not only brought to light a wealth of important information regarding the genetics of Protozoa, but also are revealing a close insight concerning the relationship between the nuclear and cytoplasmic factors of heredity in the animal. Parasitic Protozoa are confined 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 legeri 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 (Kudo, 1924, 1925). By observing certain intestinal Protozoa in some mon- keys, Hegner (1928) obtained 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 (1934), gives further proof that the Blattidae and the Isoptera are closely related. Study of a particular group of parasitic Protozoa and their hosts may throw light on the geographic condition of the earth which existed in the remote past. The members of the genus Zelleriella are usually found in the colon of the frogs belonging to the family Lepto- dactylidae. Through an extensive study of these amphibians from South America and Australia, Metcalf (1920, 1929) found that the species of Zelleriella occurring 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 some 25 species of Protozoa occur in man. Entamoeba histolytica, Balantidium coli, and four 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 Leish- mania. 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 these 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. Some of the forms found in domestic animals are morphologically indistinguishable from those occurring in man. Balantidium coli is 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 of animals, such as southern cattle fever, dourine, nagana, blackhead, coccidio- sis, etc., are caused by Protozoa. 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. 114). 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 the polluted water, but that upon reaching a certain population, they would cease to multi- ply and would allow the excess organic substances to undergo de- composition. Numerous holozoic Protozoa, however, feed on the bac- teria and prevent them from reaching the saturation population. Protozoa thus seem to help indirectly in the purification of the water. Protozoology therefore must be considered as part of modern sani- tary 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 (Kudo, 1920) (p. 648). Success in fish- culture, therefore, requires among other things a thorough knowl- edge of Protozoa. Since Russel and Hutchinson (1909) suggested some forty 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 decrease 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 climate various sarcodinans, flagellates and less frequently ciliates, are present and active throughout the year. The exact relation between specific Protozoa and bacteria in 10 PROTOZOOLOGY the soil is not yet clear in spite of the numerous experiments and observations. All soil investigators should be acquainted with the biology and taxonomy of free-living Protozoa. It is a matter of common knowledge that the silkworm and the honey bee suffer from microsporidian infections (p. 670). Sericulture in south-western Europe suffered great damages in the middle of the nineteenth century because of the "pebrine" disease, caused by the microsporidian, Nosema bombycis. 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 biological control of them by means of Protozoa and Protophyta. While the majority of Protozoa lack permanent skeletal structures and their fossil forms are little known, 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 one 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 400 simple lenses, including one which, it is said, had a magnification of 270 times (Harting). Among the many things he discovered were various Protozoa. According to Dobell (1932), Leeuwenhoek saw in 1674 for the first time free- living fresh- water Protozoa. Between 1674 and 1716, he observed many Protozoa which he reported to the Royal Society of Lon- don and which, as Dobell interpreted, were Euglena ("green in the middle, and before and behind white"), Vorticella, Stylonychia, Carchesium, Volvox, Coleps, Kerona, Anthophysis, Elphidium, etc. Huygens gave in 1678 "unmistakable descriptions of Chilodon(-ella), Paramecium, Astasia and Vorticella, all found in infusions" (Dobell). Colpoda was seen by Buonanni (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 40 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 (1744) studied division in some ciliates, including probably Para- mecium, which generic name was coined by Hill in 1752. Noctiluca was first described by Baker (1753). Rosel von Rosenhof (1755) observed an organism, which he called "der kleine Proteus," and also Vorticella, Stentor, and Volvox. The "Proteus" which Linnaeus named Volvox chaos (1758) and later re- named Chaos protheus (1767), cannot be identified with any of the known amoeboid organisms (Kudo, 1946). Wrisberg (1764) coined the term "Infusoria" (Dujardin; Woodruff). By using the juice of geranium, Ellis (1769) caused the extrusion of the "fins" (trichocysts) in Paramecium. Eichhorn (1783) observed the heliozoan, Actino- sphaerium, which now bears his name. O. F. Miiller described Ceratium a little later and published two works on the Infusoria (1773, 1786) although he included unavoidably some Metazoa 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. Lamarck (1816) named Folliculina. At the beginning of the nineteenth century the cylcosis in Para- mecium was brought to light by Gruithuisen. Goldfuss (1817) coined the term Protozoa, including in it the coelenterates. Nine years later there appeared d'Orbigny's systematic study of the Foramini- 12 PROTOZOOLOGY fera, which he considered "microscopical cephalopods." In 1828 Ehrenberg began publishing his observations on Protozoa and in 1838 he summarized his contributions in Die Infusionsthicrchen als vollkommene Organismen, in which he diagnosed genera and species so well that many of them still hold good. Ehrenberg excluded Rota- toria and Cercaria 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 employed by Purkinje (1840) in the same sense as it is used today. The Protozoa was given a distinct definition by Siebold in 1845, as follows: "Die Thiere, in welchen die verschiedenen Systeme der Organe nicht scharf ausgeschieden sind, und deren unregelmassige Form und ein- fache Organization sich auf eine Zelle reduzieren lassen." Siebold subdivided Protozoa into Infusoria and Rhizopoda. The sharp differ- entiation of Protozoa as a group certainly inspired numerous micros- copists. As a result, several students brought forward various 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 Suctoria, Stein failed to see the real nature (1849), but his two monographs on Ciliata and Mastigophora (1854, 1859-1883) contain concise descriptions and excellent illustrations of numerous species. Haeckel who went a step further than Siebold by distinguish- ing between Protozoa and Metazoa, devoted 10 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 and interesting forms. This work was followed by Stokes' The Fresh- water Infusoria of the United States, which appeared in 1888. INTRODUCTION 13 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 30 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 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 Dofiein in Germany wrote modern textbooks of protozo- ology dealing with the biology as well as the taxonomy. Jennings de- voted his time for nearly 40 years to the study of genetics of Pro- tozoa. Recent development of bacteria-free culture technique in cer- tain flagellates and ciliates, has brought to light important informa- tion regarding the nutritional requirements and metabolism 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 (1932), in the fall of 1674, the oocysts of the coccidian Eimeria stiedae, in the contents of the gall bladder of an old rabbit; in 1681, Giardia intes- tinalis in his own diarrhceic stools; and in 1683, Opalina and Nycto- therus in the gut contents of frogs. The oral Trichomonas of man was observed by O. F. Miiller (1773) who named it Cercaria tenax (Do- bell, 1939). There is no record of anyone having seen Protozoa living in other organisms, until 1828, when Dufour's account of the grega- rine from the intestine of coleopterous insects appeared. Some ten years later, Hake rediscovered the oocysts of Eimeria stiedae. A 14 PROTOZOOLOGY flagellate was observed in the blood of salmon by Valentin in 1841, and the frog trypanosome was discovered by Gluge (1842) and Gruby (1843), the latter author creating the genus Trypanosoma for it. The gregarines were a little later given attention by Kolliker (1848) and Stein (1848). The year 1849 marks the first record of an amoeba being found in man, for Gros then observed Entamoeba 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 coli was discovered by Malmsten in 1857. Lewis in 1870 observed Entamoeba coli in India, and Losch in 1875 found Entamoeba histo- lytica in Russia. During the early part of the last century, an epi- demic disease, pebrine, of the silkworm appeared in Italy and France, and a number of biologists became engaged in its investigation. Fore- most 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 scien- tific study of a parasitic protozoan which resulted 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 (Butschli, 1881), Microsporidia and Sarcosporidia (Balbiani, 1882). Parasitic protozoology received a far-reaching stimulus when Laveran (November, 1880) discovered the microgamete formation ("flagellation") of a malaria parasite in the human blood. Smith and Kilborne (1893) demonstrated that Babesia of the Texas fever of cattle in the southern United States was transmitted by the cattle tick from host to host, and thus revealed for the first time the close relationship which exists between an arthropod and a parasitic proto- zoan. Two years later Bruce discovered Trypanosoma brucei in the blood of domestic animals suffering from "nagana" disease in Africa and later (1897) demonstrated by experiments that the tsetse fly transmits the trypanosome. Studies of malaria organisms continued and several important contributions appeared. Golgi (1886, 1889) studied the schizogony and its relation to the occurrence of fever, and was able to distinguish the types of fever. MacCallum (1897) INTRODUCTION 15 observed the microgamete formation in Haemoproteus of birds and suggested that the "flagella" observed by Laveran were micro- gametes of Plasmodium. In fact, he later observed the formation of the zygote through fusion of a microgamete and a macrogamete of Plasmodium falciparum. 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 ob- servations on the life-cycle of Coccidia (1898, 1899). Ross (1898, 1898a) revealed the development of Plasmodium r dictum (P. praecox) in Culex fatigans and established the fact that the host birds become infected by this protozoan through the bites of the infected mosquitoes. Since that time, investigators too numer- ous to mention here (p. 600), studied the biology and development of the malarial organisms. Among the more recent findings is the exo-erythrocytic development, fuller information on which is now being sought. In 1902, Dutton found that the sleeping sickness in equatorial Africa was caused by an infection by Trypanosoma gam- biense. In 1903, Leishman and Donovan discovered simultaneously Leishmania donovani, the causative organism of "kala-azar" in India. 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 MacNeal 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. During and since World War I, it became known that numer- ous intestinal Protozoa of man are widely present throughout the tropical, subtropical and temperate zones. Taxonomic, morphologi- cal and developmental studies on these forms have therefore ap- peared in an enormous number. Cutler (1918) seems to have suc- ceeded in cultivating Entamoeba histolytica, though his experiment was not repeated by others. Barret and Yarborough (1921) culti- 1G PROTOZOOLOGY vated Balantidium coli and Boeck (1921) cultivated Chilomastix mesnili. Boeck and Drbohlav (1925) succeeded in cultivating Enta- moeba histolytica, and their work was repeated and improved upon by many investigators. While the in-vitro cultivation has not thrown much light on metabolic activities of this and other parasitic amoebae, as no one of them would grow in culture without some other organisms, it has increased our knowledge on the biology of these parasites. References Allman, G. J.: (1855) On the occurrence among the Infusoria of peculiar organs resembling thread-cells. Quart J. Micr. Sc., 3: 177. Baker, H.: (1753) Employment for the microscope. London. Balbiani, G.: (1882) Sur les microsporidies ou psorospermies des articules. C. R. Acad. Sc, 95:1168. Barret, H. P. and Yarbrough, N. : (1921) A method for the culti- vation of Balantidium coli. Am. J. Trop. Med., 1:161. Boeck, W. C. : (1921) Chilomastix mesnili and a method for its cul- ture. J. Exper. Med., 33:147. -and Drbohlav, J.: (1925) The cultivation of Endamoeba histolytica. Am. J. Hyg., 5:371. Bruce, D. : (1895) Preliminary report on the tsetse fly disease or nagana in Zululand. Umbobo. (1897) Further report, etc. Umbobo. Butschli, 0.: (1880-1889) Protozoa. Bronn's Klassen und Ord- nungen des Thierreichs. Vols. 1-3. (1881) Myxosporidia. Zool. Jahrb. 1880, 1 : 162. Buonanni, F.: (1691) Observationes circa Viventia, etc. Rome. Calkins, G. N.: (1901) The Protozoa. Philadelphia. (1933) The biology of the Protozoa. 2 ed. Philadelphia. Claparede, J. L. R. A. E. and Lachmann, J.: (1858-59) Etudes sur les Infusoires et les Rhizopodes. Vol. 1. Geneva. Cleveland, L. R., Hall, S. R. and Sanders, E. P.: (1934) The woodfeeding roach, Cryptocercus, its Protozoa, and the sym- biosis between Protozoa and roach. Mem. Am. Acad. Arts & Sc, 17:185. Cohn, F. J.: (1853) Beitrage zur Entwickelungsgeschichte der In- fusorien. Zeitschr. wiss. Zool., 6:253. Cole, F. J.: (1926) The history of protozoology. London. Cutler, D. W. : (1918) A method for the cultivation of Entamoeba histolytica. J. Path. Bact., 22:22. Davaine, C. : (1854) Sur des animalcules infusoires, etc. C. R. Soc Biol., 1:129. Dobell, C: (1911) The principles of protistology. Arch. Protist., 23:269. (1932) Antony van Leeuwenhoek and his "little animals." New York. (1939) The common flagellate of the human mouth, Tri- INTRODUCTION 17 chomonas tenax (O.F.M.) : its discovery and its nomenclature. Parasit., 31:138. Doflein, F. : (1901) Die Protozoen als Parasiten und Krankheitser- reger. Jena. and Reichenow, E.: (1929) Lehrbuch der Protozoenkunde. 5 ed. Jena. Donovan, C. : (1903) The etiology of one of the heterogeneous fevers in India. Brit. M. J., 2:1401. d'Orbigny, A. : (1826) Tableau methodique de la Classe des Cephal- opodes. Ann. Sci. Nat., 7:245. Dufour, L.: (1828) Note sur la gregarine, etc. Ibid., 13:366. Dujardin, F. : (1835) Sur les pretendus estomacs des animalcules infusoires et sur une substance appelee sarcode. Ann. Sci. Nat. Zool., 4:343. (1841) Histoire naturelle des zoophytes. Infusoires. Paris. Dutton, J. E. : (1902) Preliminary note upon a trypanosome occur- ring in the blood of man. Rep. Thomson Yates Lab., 4:455. Ehrenberg, C. G.: (1838) Die Infusionsthierchen als vollkommene Organismen. Leipzig. Eichhorn, J. C: (1783) Zugabe zu meinen Beytragen, etc. Danzig. Eimer, T. : (1870) Ueber die ei- und kugelformigen sogenannten Psorospermien der Wirbelthiere. Wurzburg. Ellis, J.: (1769) Observations on a particular manner of increase in the animalcula, etc. Phil. Trans., 59:138. Gluge, G. : (1842) Ueber ein eigenthumliches Entozoon im Blute des Frosches. Arch. Anat. Phys. wiss. Med., 148. Goldfuss, G. A.: (1817) Ueber die Entwicklungsstufen des Thieres. Nurnberg. Golgi, C.: (1886) Sulla infezione malarica. Arch. Sci. Med., 10:109. — (1889) Sul ciclo evolutio dei parassiti malarici nella febbre terzana, etc. Ibid., 13:173. Gros, G.: (1849) Fragments d'helminthologie et de physiologie mi- croscopique. Bull. Soc. Imp. Nat. Moscou, 22:549. Gruby, D.: (1843) Recherches et observations sur une nouvelle espece d'hematozoaire, Trypanosoma sanguinis. C. R. Acad. Sc, 17:1134. Haeckel, E. H.: (1862) Betrachtungen ueber die Grenzen und Ver- wandschaft der Radiolarien und ueber die Systematik der Rhizopoden im Allgemeinen. Berlin. (1866) Generelle Morphologie der Organismen. Berlin. Hake, T. G.: (1839) A treatise on varicose capillaries, as constitut- ing the structure of carcinoma of the hepatic ducts, etc. Lon- don. Harris, J.: (1696) Some microscopical observations of vast num- bers of animalcula seen in water. Phil. Trans., 19:254. Hegner, R. : (1928) The evolutionary significance of the protozoan parasites of monkeys and man. Quart. Rev. Biol., 3:225. Hill, J. : (1752) An history of animals, etc. London. Hyman, Libbie H. : (1940) The invertebrates: Protozoa through Ctenophora. New York. IS PROTOZOOLOGY Jennings, H. S. : (1909) Heredity and variation in the simplest or- ganisms. Am. Nat., 43:322. Joblot, L. : (1718) Descriptions et usages de plusieurs nouveaux mi- croscopes, etc. Paris. Kloss, H.: (1855) Ueber Parasiten in der Niere von Helix. Abh. Senckenb. Naturf. Ges., 1:189. Kolliker, A.: (1848) Beitrage zur Kenntnis niederer Thiere. Zeitschr. wiss. Zool., 1:34. Kudo, R. R. : (1920) Studies on Myxosporidia. Illinois Biol. Monogr. 5:nos. 3, 4. (1924) Studies on Microsporidia parasitic in mosquitoes. III. Arch. Protist., 49:147. - (1925) IV. Centralbl. Bakt. I. Orig., 96:428. (1946) Pelomyxa carolinensis Wilson. I. Jour. Morph., 78: 317. Laveran, A.: (1880) Note sur un nouveau parasite trouve dans le sang de plusieurs malades atteints de fievre palustre. Bull Acad. Med., 9:1235, 1268, 1346. (1880a) Un nouveau parasite trouve dans le sang des malades atteints de fievre palustre. Bull. Mem. Soc. Med. Hopit. Paris, 17:158. Leidy, J.: (1879) Freshwater Rhizopods of North America. Rep. U. S. Geol. Survey, 12. Leishman, W. B.: (1903) On the possibility of the occurrence of trypanosomiasis in India. British Med. Jour., 1:1252. Leuckart, R. : (1879) Die Parasiten des Menschen. 2 ed. Leipzig. Lewis, T. R. (1870) A report on the microscopic objects found in cholera evacuations, etc. Ann. Rep. San. Comm. Gov. India (1869) 6:126. (1878) The microscopic organisms found in the blood of man and animals, etc. Ibid. (1877) 14:157. Linnaeus, C.: (1758) Systema Naturae. 10 ed. 1:820. — ■ (1767) Systema Naturae. 12 ed. 1:1324. Losch, F. : (1875) Massenhafte Entwickelung von Amoben im Dick- darm. Arch. path. Anat., 65:196. Lwoff, A.: (1951) Biochemistry and physiology of Protozoa. New York. MacCallum, W. G.: (1897) On the flagellated form of the malarial parasite. Lancet, 2:1240. Malmsten, P. H.: (1857) Infusorien als Intestinal-Thiere beim Menschen. Arch. path. Anat., 12:302. Metcalf, M. M.: (1920) Upon an important method of studying problems of relationship and of geographical distribution. Proc. Nat. Acad. Sc, 6:432. (1929) Parasites and the aid they give in problems of taxon- omy, geographical distribution, and paleogeography. Smith. Misc. Coll., 81: no. 8. Musgrave, W. E. and Clegg, M. T. : (1904) Amebas: their cultiva- tion and aetiologic significance. Dep. Inter., Biol. Lab. Bull., Manila, no. 18:1. INTRODUCTION 19 Novy, F. G. and MacNeal, W. J.: (1905) On the trypanosomes of birds. J. Inf. Dis., 2:256. Pasteur, L. : (1870) Etudes sur la maladie des vers a soie. Paris. Perty, M.: (1852) Zur Kenntnis kleinster Lebensformen, etc. Bern. Rosel von Rosenhof, A. J.: (1755) Der kleine Proteus. Der Monat.-herausgeg. Insect. -Belust., 3:622. Ross, R.: (1898) Report on the cultivation of Proteosoma Labbe in grey mosquitoes. Gov. Print. Calcutta. (1898a) Preliminary report on the infection of birds with Proteosoma by the bites of mosquitoes. Ibid. Russell, E. J. and Hutchinson, H. B.: (1909) The effect of partial sterilization of soil on the production of plant food. J. Agr. Sc, 3:111. Schaudinn, F. and Siedlecki, M.: (1897) Beitrage zur Kenntnis der Coccidien. Verhandl. deut. zool. Ges., p. 192. Siebold, C. T. v.: (1845) Bericht ueber die Leistungen in der Na- turgeschichte der Wiirmer, etc. Arch. Naturg., 11:256. Siedlecki, M.: (1898) Etude cytologique et cycle evolutif de la coc- cidie de la seiche. Ann. Inst. Pasteur, 12:799. Etude cytologique et cycle evolutif de Adelea ovata Schneider. Ibid., 13:169. Smith, T. and Kilborne, F. L. : (1893) Investigations into the na- ture, causation, and prevention of Texas or southern cattle fever. Bull. Bur. Animal Ind., U. S. Dep. Agr., No. 1. Sonneborn, T. M. : (1937) Sex, sex inheritance and sex determina- tion in Paramecium aurelia. Proc. Nat. Acad. Sc, 23:378. (1938) Mating types in Paramecium aurelia, etc. Proc. Am. Phil. Soc, 79:411. Stein, S. F. N. v.: (1854) Die Infusionsthiere auf ihre Entwickel- ungsgeschichte untersucht. Leipzig. (1859-83) Der Organismus der Infusionsthiere. Leipzig. Stokes, A. C: (1888) A preliminary contribution toward a history of the fresh-water Infusoria of the United States. J. Trenton Nat. Hist. Soc, 1:71. Trembley, A.: (1744) Observations upon several newly discovered species of freshwater polypi. Phil. Trans., 43:169. Valentin: (1841) Ueber ein Entozoon im Blute von Salmo fario. Arch. Anat. Phys. wiss. Med., p. 435. Woodruff, L. L. : (1937) Louis Joblot and the Protozoa. Sc Monthly, 44:41. (1939) Some pioneers in microscopy, with special reference to protozoology. Tr. N. Y. Acad. Sc, Ser. 2, 1:74. Wrisberg, H. A.: (1765) Observationum de Animalculis infusoriis Satura. Gottingen. Chapter 2 Ecology WITH regard to their habitats, the Protozoa may be divided into free-living forms and those living on or in other organisms. Mastigophora, Sarcodina, Ciliata, and Suctoria include both free- living and parasitic Protozoa, but Sporozoa are exclusively parasi- tic. 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 ca- pacity to remain viable in the 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 20 ECOLOGY 21 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' (1935) obtained from the thermal springs, of Virginia, several species of Mastigoph- ora, Ciliata, and an amoeba which were living in the water, the tem- perature of which was 34-36°C, but did not notice any protozoan in the water which showed 39-41°C. Uyemura (1936, 1937) 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, Paramecium 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. Gay lord (1908) exposed Trypanosoma gambiense to liquid air for 20 minutes without apparent injury, but the organisms were killed after 40 min- utes' immersion, 22 PROTOZOOLOGY Kiihne (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 (1902), when Stentor coeruleus was slowly subjected 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 process and resumed its normal activity. If the lowering of tempera- ture is rapid and the medium becomes solidly frozen, Stentor per- ishes. 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 Spirostomum ambiguum, 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 knowlcsi 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 1 hawed rapidly. Low temperature on Protozoa (Luyet and Gehenio, 1940). Light. In the Phytomastigina which include chromatophore-bear- ECOLOGY 23 ing flagellates, the sun light is essential to photosynthesis (p. 107). The 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 Protozoa. 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, Trepomonas agilis, Hexamita inflata, Rhynchomonas nasuta, Hetero- nema acus, Bodo, Cercomonas, Dactylochlamys, Ctenostomata, etc. The so-called "sewage organisms" abound in such habitat (Lackey, 1925). Certain free-living Protozoa which inhabit waters rich in decom- 24 PROTOZOOLOGY posing organic matter are frequently found in the faecal matter of various animals. Their cysts either pass through the alimentary 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 is 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- pomonas agilis, Naegleria gruberi, Acanthamoeba hyalina, Chlamy- dophrys stercorea and Tillina magna. As a rule, the presence of sodium chloride in the sea water prevents the occurrence of numerous species 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 lacustre, Cerat- ium hirundinella; Sarcodina: Lieberkiihnia wagneri; Ciliata: Meso- dinium pidex, 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 folliculata; Sxictoria, : 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 marinum, 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, Stylonychia, 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 ECOLOGY 25 morphological changes in the experimental protozoans which might be attributed to the presence of the salt in the water, except Amoeba 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 Ewplotes 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 days 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 (1939) 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. Some Protozoa appear to tolerate a wide range of pH. The interesting pro- teomyxan, Leptomyxa reticulata, occurs in soil ranging in pH 4.3 to 7.8, and grows very well in non-nutrient agar between pH 4.2 and 8.7, provided a suitable bacterial strain is supplied as food (Singh, 1948) ; and according to Loefer and Guido (1950), a strain of Euglena gracilis (var. bacillaris) grows between pH 3.2 and 8.3. However, the majority of Protozoa seem to prefer a certain range of pH for the maximum metabolic activity. The hydrogen-ion concentration of freshwater bodies varies 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, Hyalobryon, etc., occur. In standing deep fresh water, the bottom region is often acid because of the decom- posing organic matter, while the surface water is less acid or slightly alkaline due to the photosynthesis of green plants which utilize car- bon dioxide. In some cases different pH may bring about morpho- 26 PROTOZOOLOGY logical differences. For example, in bacteria-free cultures of Para- mecium bursaria in a tryptone medium, Loefer (1938) found that at pH 7.6-8.0 the length averaged 86 or 87/x, but at 6.0-6.3 the length was about 129/z. 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 44m wide) were found in culture at pH 5.7-7.4. Many workers have made observations on the pH range of the water or medium in which certain protozoans live, grow, and multiply, some of which data are collected in Table 1 . Table 1 . — Protozoa and hydrogen-ion concentration Protozoa pH range of medium in which Optimum range Observers growth occurs 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 . — 5.0-6.5 Schoenborn E. deses 6.5-8.0 7.0 Dusi 5.3-8.0 7.0 Hall E. piscijormis 6.0-8.0 6.5-7.5 Dusi 5.4-7.5 6.8 Hall E. viridis — 5.0 Schoenborn Chilomonas Paramecium 4.8-8.0 6.8 Mast and Pace 4.1-8.4 4.9;7.0 Loefer Chlorogonium euchlorum 4.8-8.7 7.1-7.5 " C. elongatum 4.8-8.7 7.1-7.5 " C. teragamum 4.2-8.6 6.7-8.3 " Colpidmm campylum — 5.4 Kidder Glaucoma scintillans — 5.6-6.8 a G. ficara 4.0-9.5 5.1;6.7 Johnson Tetrahymena pyriformis — 5.6-8.0 Kidder T. vorax — 6.2-7.6 u Paramecium bursaria 4.9-8.0 6.7-6.8 Loefer B. In cultures containing bacteria Carteria obtusa — 3.5-4.5 Wermel Trichomonas vaginalis 6.4-8.4 — Bland et al. Actinosphaerium eichhorni — 7.2-7.6 Howland 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 — 6.9-7.1 Wichterman P. aurelia 5.7-7.8 6.7 Morea ECOLOGY 27 Table 1. — Continued Protozoa pH range of medium in whicl Optimum Observers growth occurs range 5.9-8.2 5.9-7.7 Phelps — 7.0-7.2 Wichterman P. multim icronucleatum 4.8-8.3 7.0 Jones — 6.5-7.0 Wichterman P. trichium — 6.7-7.1 " P. bursaria — 7.1-7.3 a P. poly car yum — 6.9-7.3 " P. calkinsi — 6.5-7.8 " P. woodruffi — 7.0-7.5 " 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 — " Amphileptus sp. 6.8-7.5 7.1-7.3 « Spirostomvm ambiguum 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 pustulata 6.0-8.0 6.7;8.0 Darby 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 protozoa. Noland (1925) studied more than 65 species of fresh-water ciliates with respect to various factors and came to the conclusion that the nature and amount of available food has more to do with the distribution of these organisms than any other one factor. Di- dinium nasutum feeds almost exclusively on paramecia; therefore, it cannot live in the absence of the latter ciliate. As a rule, euryphagous Protozoa which feed on a variety of food organisms are widely dis- tributed, while stenophagous forms that feed on a few species of food organisms are limited in their distribution. In nature, Protozoa live in association with diverse organisms. The interrelationships which exist among them are not understood in most cases. For example, the relationship between Entamoeba histolytica and certain bacteria in successful in-vitro cultivation has 28 PROTOZOOLOGY not yet been comprehended. Certain strains of bacteria were found by Hardin (1944) to be toxic for Paramecium multimicronucleatum, but if Oikomonas termo was present in the culture, the ciliate was maintained indefinitely. This worker suggested that the flagellate may be able to "detoxify" the metabolic products produced by the bacteria. Food relation in ciliates (Faure-Fremiet, 1950, 1951a). The adaptability of Protozoa to varied environmental conditions influences their distribution. The degree of adaptability varies a great deal, not only among different species, but also among the individuals of the same species. Stentor coeruleus which grows ordi- narily under nearly anaerobic conditions, is obviously not influenced by alkalinity, pH, temperature or free carbon dioxide in the water (Sprugel, 1951). 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. 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 bod}' of water, as shown by various species of Chonotricha, Peritricha, and Suctoria. In other cases, there is a definite relationship between the commensal and the host. For example, Kerona polyporum is found ECOLOGY 29 on various species of Hydra, and many ciliates placed in Thigmo- tricha (p. 774) are inseparably associated with certain species of 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 alimentary 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: Endamoeba blattae, Lophomonas blattarum, L. striata, Nyctotherus ovalis, etc., of the cockroach; Entamoeba coli, Iodamoeba biitschlii, Endolimax nana, Dientamoeba fragilis, Chilomastix 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 insects 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 perish (Cleveland, 1924, 1925). Recently, Cleveland (1949-1950c) found that the molting hormone produced by Crypto- cercus induces sexual reproduction in several flagellates inhabiting its hind-gut (p. 185). Thus the association here may be said to be an absolute 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. 464) feeds on the body cells of .30 PROTOZOOLOGY Hydra which, according to Reynolds and Looper (1928), die on an average in 6.8 days as a result of the infection and the amoebae dis- appear in from 4 to 10 days if removed from a host Hydra. Costia necatrix (p. 372) 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 func- tions of the host tissue. Ichthyophthirius multifiliis (p. 709), another ectoparasite of freshwater and marine fishes, goes further by com- pletely burying themselves in the epidermis and feeds on the host's tissue cells and, not infrequently, 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 cytoplasm 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 gambiense, 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 first slightly raised nodules 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 may also occur 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. 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 ECOLOGY 31 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. 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- fEssssxgsss «m .&?: %: %m ¥■ '■■■■■■' 'i**£i?< ■'•:■: -X^^<: ; . $ ?-: 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. pie, the circular muscle fibers of the small iniestine 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 "epithelial cells" (Fig. 1, b), 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), 32 PROTOZOOLOGY Practically all Microsporidia are cytozoic, and the infected cells become hypertrophied enormously, producing in one genus the so- called Glugea cysts (Figs. 287, 290). In many cases, the hypertrophy of the nucleus of the infected cell is far more conspicuous than that of the cytoplasm (Figs. 287, 291) (Kudo, 1924). When the gonads are parasitized heavify, the germ cells of the host animal often do not develop, thus resulting in parasitic castra- tion. For example, the ciliate, Orchitophrya steUarum, a parasite in the male reproductive organ of Asterias rubens, was found by Vevers (1951) to break down completely all germinal tissues of the testes in the majority of the host starfish. In other cases, the protozoan does not invade the gonads, but there is no development of the germ cells. The microsporidian, Nose ma apis, attacks solely the gut epithelium of the honey bee, but the ovary of an infected queen bee degenerates to varying degrees (Hassanein, 1951). Still in other instances, the Protozoa invade developing ova of the host, but do not hinder their development, though the parasites multiply, as in Nosema bombycis in the silkworm (Stempell, 1909) and Babesia bigemina in the cattle tick (Dennis, 1932). 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 hwisi 1 a considerable number of ECOLOGY 33 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- 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 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 and trypanosomes (Taliaferro, 1926; Maegraith, 1948; Culbertson, 1951). Immunity (Taliaferro, 1941). 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 34 PROTOZOOLOGY 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 (Tetrahymena) pyriformis, a free-living ciliate, was found to occur in the body cavity of the larvae of Theobaldia 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. 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 endoparasitjsm. When these endocommensals began ECOLOGY 35 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 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. Hyperparasitism. Certain parasitic Protozoa have been found to parasitize other protozoan or metazoan parasites. This association is named hyperparasitism. The microsporidian Nosema notabilis (p. 672) is an exclusive parasite of the myxosporidian Sphaerospora polymorpha, which is a very common inhabitant of the urinary blad- der of the toad fish along the Atlantic and Gulf coasts. A heavy in- fection of the microsporidian results in the degeneration and death of the host myxosporidian trophozoite (Kudo, 1944). Thus Nosema notabilis is a hyperparasite. Organisms living on and in Protozoa (Duboscq and Grasse, 1927, 1929; Georgevitch, 1936; Grasse, 1936; Kirby, 1932, 1938, 1941, 1941a, 1942, 1942a, 1942b, 1944, 1946) References Bland, P. B., Goldstein, L., Wenrich, D. H. and Weiner, Elea- nor: (1932) Studies on the biology of Trichomonas vaginalis. Am. J. Hyg., 56:492. 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. Pub. Health, Rep., 45:481. Chambers, R. and Hale, H. P.: (1932) The formation of ice in pro- toplasm. Proc. Roy. Soc. London, Ser. B, 110:336. Cleveland, L. R. : (1924) The physiological and symbiotic relation- ships between the intestinal protozoa of termites and their host, with special reference to Reticulitermes flavipes Kollar. Biol. Bull., 46:177. (1925) The effects of oxygenation and starvation on the sym- biosis between the termite, Termopsis, and its intestinal flagel- lates. Ibid., 48:309. — (1926) Symbiosis among animals with special reference to termites and their intestinal flagellates. Gen. Rev. Biol., 1:51. — (1928) Tritrichomonas fecalis nov. sp. of man, etc. Amer. J. Hyg., 8:232. (1949) Hormone-induced sexual cycles of flagellates. I. Jour. Morph., 85:197. - (1950) II. Ibid., 86:185. - (1950a) III. Ibid., 86:215. - (1950b) IV. Ibid, 87:317. — (1950c) V. Ibid, 87:349. 36 PROTOZOOLOGY Coggeshall, L. T. : (1939) Preservation of viable malaria parasites in the frozen state. Proc. Soc. Exp. Biol., 42:499. Oulbertson, J. T. : (1951) Immunological mechanisms in parasitic. infections. In: Most's Parasitic infections in man. New York. Dallinger, W. H.: (1887) The president's address. J. Roy. Micro. Soc, London, 7:185. Darby, H. H.: (1929) The effect of the hydrogen-ion concentration on the sequence of protozoan forms. Arch. Protist., 65:1. Dennis, E. W. : (1932) The life-cycle of Babesia bigemina, etc., Univ. Cal. Publ. Zoology, 36:263. Deschiens, R. : (1934) Influence du froid sur les formes vegetatives de ramibe dysenterique. C. R. Soc. Biol., 115:793. Dobell, C. : (1918) Are Entamoeba histolytica and E. ranarum the same species? Parasit., 10:294. Doudoroff, M.: (1936) Studies in thermal death in Paramecium. J. Exper. Zool., 72:369. Drew, G. H.: (1911) Experimental metaphasia. I. J. Exper. Zool., 10:349. Duboscq, O. and Grasse, P.: (1927) Flagelles et Schizophytes de Calotermes (Glyptotermes) iridipennis. Arch. zool. exp. gen., 66: 451. — (1929) Sur quelques protistes d'un Calotermes, etc. Ibid., 68:8. Efimoff, W. W. : (1924) Ueber Ausfrieren und Ueberkaeltung der Protozoen. Arch. Protist., 49: 431. Fatjre-Fremiet, E.: (1950) Ecology of ciliate infusoria. Endeavour 9, 3 pp. (1951) The marine sand-dwelling ciliates of Cape Cod. Biol. Bull., 100:59. (1951a) Ecologie des Protistes littoraux. Ann. Biol., 27:205. Finley, H. E. : (1930) Toleration of freshwater Protozoa to increased salinity. Ecology, 11:337. Frisch, J. A.: (1939) The experimental adaptation of Paramecium to sea water. Arch. Protist., 93:38. Gaylord, H. R.: (1908) The resistance of embryonic epithelium, etc. J. Infect. Dis., 5:443. Georgevitch, J.: (1936) Ein neuer Hyperparasit, Leishmania esocis n. sp. Arch. Protist., 88:90. Glaser, R. W. and Coria, N. A.: (1933) The culture of Paramecium caudatum free from living microorganisms. Jour. Parasit., 20: 33. — (1935) The culture and reactions of purified Protozoa. Am. J. Hyg, 21:111. Grasse, P. P.: (1938) La veture schizophytique des flagelles ter- miticoles, etc. Bull. Soc. zool. France, 63:110. Greeley, A. W. : (1902) On the analogy between the effects of loss of water and lowering of temperature. Amer. Jour. Physiol., 6: 122. Hardin, G. : (1944) Symbiosis of Paramecium and Oikomonas. Ecol- ogy, 25:304. ECOLOGY 37 Hassanein, M. H. : (1951) Studies on the effect of infection with Nosema apis on the physiology of the queen honey-bee. Quart. J. Micr. Sc, 92:225. Howland, Ruth: (1930) Micrurgical studies on the contractile vac- uole. III. J. Exper. Zool., 55:53. Jahn, T. L. : (1933) Studies on the physiology of the euglenoid flag- ellates. IV. Arch. Protist., 79:249. Janda, V. and Jirovec, O. : (1937) Ueber kiinstlich hervorgerufenen Parasitismus eines freilebenden Ciliaten Glaucoma piriformis, etc. Mem. Soc. Zool. Tehee. Prague, 5:34. Kidder, G. W.: (1941) Growth studies on ciliates. VII. Biol. Bull, 80:50. Kirby, H. Jr.: (1932) Flagellates of the genus Trichonympha in termites. Univ. Cal. Publ. Zool., 37:349. — (1938) The devescovinid flagellates, etc. Ibid., 43:1. — (1941) Devescovinid flagellates of termites. I. Ibid., 45:1. — (1941a) Organisms living on and in Protozoa. Calkins and Summers' Protozoa in biological research. — (1942) Devescovinid flagellates of termites. II. Uni. Cal. Publ. Zool., 45:93. — (1942a) III. Ibid., 45:167. — (1942b) A parasite of the macronucleus of Vorticella. Jour. Parasit., 28:311. (1944) The structural characteristics and nuclear parasites of some species of Trichonympha in termites. Uni. Cal. Publ. Zool., 49:185. (1946) Gigayitomonas herculea, etc. Ibid., 53:163. Klebs, G.: (1893) Flagellatenstudien. Zeitschr. wiss. Zool, 55: 265. Kolkwitz, R. and Marsson, M.: (1909) Oekologie der tierischen Sabrobien. Intern. Rev. Ges. Hydrobiol. u. Hydrogr., 2:126. Kudo, R. R. : (1924) A biologic and taxonomic study of the Micro- sporidia. Illinois Biol. Monogr., 9: nos. 3 and 4. (1929) Histozoic Myxosporidia found in freshwater fishes of Illinois, U. S. A. Arch. Protist., 65:364. — (1944) Morphology and development of Nosema notabilis Kudo, parasitic in Sphaerospora polymorpha Davis, a parasite of Opsanus tau and 0. beta. Illinois Biol. Monogr., 20:1. Kuhne, W. : (1864) Untersuchungen ueber das Protoplasma und die Contractilitat. Leipzig. Lackey, J. B.: (1925) The fauna of Imhof tanks. Bull. N. J. Agr. Ex. St., No. 417. Lauterborn, R. : (1901) Die "sapropelische" Lebewelt. Zool. Anz., 24:50. Loefer, J. B.: (1935) Relation of hydrogen-ion concentration to growth of Chilomonas and Chlorogonium. Arch. Protist., 85: 209. (1938) Effect of hydrogen-ion concentration on the growth and morphology of Paramecium bursaria. Ibid., 90:185. (1939) Acclimatization of fresh- water ciliates and flagellates to media of higher osmotic pressure. Physiol. Zool., 12:161, 38 PROTOZOOLOGY and Guido, Virginia M.: (1950) Growth and survival of Euglena gracilis, etc. Texas J. Sc, 2:225. Luyet, B. J. and Gehenio, P. M.: (1940) The mechanism of injury and death by low temperature. A review. Biodynamica, 3: no. 60. Maegraith, B. : (1948) Pathological processes in malaria and black- water fever. Springfield, Illinois. Molisch, H. : (1897) Untersuchungen ueber das Erfrieren der Pflan- zen. Jena. Needhum, J. G., Galtsoff, P. S., Lutz, F. E. and Welch, P. S.: (1937) Culture methods for invertebrate animals. Ithaca, N. Y. Noland, L. E.: (1925) Factors influencing the distribution of fresh- water ciliates. Ecology, 6:437. Phelps, A.: (1934) Studies on the nutrition of Paramecium. Arch. Protist., 82:134. Reynolds, B. D. : (1936) Colpoda steini, a facultative parasite of the land slug, Agriolimax agrestis. J. Parasit., 22:48. — and Looper, J. B. : (1928) Infection experiments with Hy- dramoeba hydroxena nov. gen. Ibid., 15:23. Rosenberg, L. E.: (1936) On the viability of Trichomonas augusta. Tr. Am. Micr. Soc, 55:313. Sandon, H. : (1927) The composition and distribution of the pro- tozoan fauna of soil. Edinburgh. Schoenborn, H. W. : (1950) Nutritional requirements and the ef- fect of pH on growth of Euglena viridis in pure culture. Tr. Am. Micr. Soc, 69:217. Singh, B. N. : (1948) Studies on giant amoeboid organisms. I. J. Gen. Microbiol., 2:7. Sprugel, G., Jr.: (1951) Vertical distribution of Stentor coeruleus in relation to dissolved oxygen levels in an Iowa pond. Ecology, 32:147. Stempell, W. : (1909) Ueber Nosema bombycis. Arch. Protist., 16: 281. Taliaferro, W. H.: (1926) Host resistance and types of infections in trypanosomiasis and malaria. Quart. Rev. Biol., 1:246. (1941) The immunology of the parasitic Protozoa. In: Calk- ins and Summers' Protozoa in biological research. Uyemura, M.: (1936) Biological studies of thermal waters in Japan. IV. Ecolog. St., 2:171. — (1937) V. Rep. Japan. Sc. A., 12:264. Vevers, H. G.: (1951) The biology of Asterias rubens. II. J. Mar. Biol. A. Un. Kingd. 29:619. Wichterman, R. : (1948) The hydrogen-ion concentration in the cultivation and growth of 8 species of Paramecium. Biol. Bull., 95:271. Wolfson, C. : (1935) Observations on Paramecium during exposure to sub-zero temperatures. Ecology, 16:630. Yocom, H. B.: (1934) Observations on the experimental adaptation of certain freshwater ciliates to sea water. Biol. Bull, 67:273. Chapter 3 Morphology PROTOZOA range in size from submicroscopic 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 (n) 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. 109). 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. 392); radial symmetry in Gonium, Cyclonexis, etc.; and universal symmetry, in certain Heliozoa, Vol vox, etc. The fundamental component of the protozoan body is the pro- toplasm which is without exception differentiated into the nucleus and the cytoplasm. Haeckel's (1868, 1870) monera are now considered as nonexistent, since improved microscopic technique has failed in re- cent years to reveal any anucleated protozoans. The nucleus and the cytoplasm are inseparably important to the well-being of a proto- zoan, as has been shown by numerous investigators since Verworn's pioneer work. In all cases, successful regeneration of the body is ac- complished 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 cytoplasm degener- ate, which indicates their intimate association in carrying on the activities of the body. It appears certain that the nucleus controls the assimilative phase of metabolism which takes place in the cyto- plasm in normal animals, while the cytoplasm is capable of carrying on the catabolic phase of the metabolism. Aside from the importance 39 40 PROTOZOOLOGY as the controlling center of metabolism, evidences point to the con- clusion that the nucleus contains the genes or hereditary factors which characterize each species of Protozoa from generation to gen- eration, 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 morphological components of the protozoan nucleus are the nuclear membrane, chromatin, plastin and nucleoplasm or nuclear sap. Their interrelationship varies sometimes from one de- velopmental stage to another, 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, c, e) consists of a nuclear mem- brane which is sometimes very delicate but distinct, nucleoplasm, achromatin 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 differ- ent 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 solely upon the difference in affinity to nuclear stains which cannot be standardized and hence do not give uniformly the same result. Following Minchin (1912), the term endosome is advocated here to designate one or more conspicuous bodies other than the chromatin granules, present within the nuclear membrane (Fig, 2, b, d). Fig. 2. a-f, vesicular nuclei; g-j, compact nuclei, X980. a, b, nuclei of Entamoeba invadens (a, in life; b, in stained organism); c, d, nuclei of Amoeba spumosa (c, in life, showing a large endosome; d, stained); e, f, nuclei of A. proteus (e, in life; f, a nucleus subjected to Feulgen's nucleal reaction) ; g, h, nuclei of Paramecium aurelia (g, in life under phase micro- scope, snowing two vesicular micronuclei and compact macronucleus; h, Feulgen-stained nuclei); i, j, nuclei of Frontonia leucas, showing a micro- nucleus and macronucleus, both of which are compact ($ in life, showing many endosomes imbedded among the granules; j, nuclei stained with acidified methyl green), 42 PROTOZOOLOGY When viewed in life, the nucleoplasm is ordinarily homogeneous and structureless. But, upon fixation, there appear invariably achro- matic strands or networks which seem to connect the endosome and the nuclear membrane (Fig. 2, b, d). Some investigators hold that these strands or networks exist naturally in life, but due to the simi- larity of refractive indices of the strands and of the nucleoplasm, they are not visible and that, when fixed, they become readily recog- nizable 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. 174, c), according to Cleveland and his associates (1934). Others maintain that the achro- matic structures prominent in fixed vesicular nuclei are mere arti- facts brought about by fixation and do not exist in life and that the nucleoplasm is a homogeneous liquid matrix of the nucleus in which the chromatin is usually distributed as small granules. Frequently larger granules of various sizes and forms may occur along the inner surface of the nuclear membrane. These so-called peripheral granules that occur in Amoeba, Entamoeba, Pelomyxa, etc., are apparently not chromatinic (Fig. 2, a, e). The vesicular nucleus is most com- monly present in various orders of Sarcodina and Mastigophora. The compact nucleus (Fig. 2, g-j), 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 Ciliophora 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 distinct, and the chromatin substance is usually of spheroidal form, varying in size among different species and often even in the same species. In the majority of species, the chromatin granules are small and compact (Fig. 2, h, i), though in some forms, such as Nyctotheru-s ovalis (Fig. 3), they may reach 20/x or more in diameter in some indi- viduals and while the smaller chromatin granules seem to be homo- geneous, larger forms contain alveoli of different sizes in which smaller chromatin granules are suspended (Kudo, 1936). Precise knowledge of chromatin (thymo- or desoxyribose-nucleic acid) 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 nu- cleus 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, MORPHOLOGY 43 while nuclear membrane and achromatic substances remain unat- tacked; and (4) in the fixed condition Feulgen's nucleal reaction (p. 897). Action of methyl green (Pollister and Leuchtenberger, 1949). There is no sharp demarcation between the vesicular and compact nuclei, since there are numerous nuclei the structures of which are Fig. 3. Parts of nacronuclei of Nyctotherus ovalis, showing chromatin spherules of different sizes, X650 (Kudo). 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- 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 44 PROTOZOOLOGY 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 a compact nucleus, the chromatin granules or spherules fill, as a rule, the intranuclear space compactly, in which one or more endo- somes (Fig. 2, i) may occur. In many nuclei these chromatin granules appear to be suspended freely, while in others a reticulum appears to make the background. The chromatin of compact nuclei gives a strong positive Feulgen's nucleal reaction. The macronuclear and micronuclear chromatin substances respond differently to Feulgen's nucleal reaction 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. The macro- nucleus and the micronucleus of vegetative Paramecium caudatum were found by Moses (1950) to possess a similar nucleic acid-protein composition; namely, similar concentrations of total protein, non- histone protein, desoxyribose nucleic acid and ribose nucleic acid. Of the two latter nucleic acids, ribose nucleic acid is said to be pres- ent in a larger amount than desoxyribose nucleic acid in both nuclei. It may be considered that the two nucleic acids occur in different proportions in the two nuclei. 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 cytoplasm of Actinosphaerium 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 cytoplasm. 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 Belaf that the lack of food caused the encystment rather than chromidia-formation in Actinosphaerium and, according MORPHOLOGY 45 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 Diffiugia, the chromidial-net is vacuolated or alveolated in the fall and in each alveolus appear glycogen granules which seem to serve as reserve 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, 1925). Apparently the widely distributed volutin (p. 114), and many inclusions or cytozoic parasites, such as Sphaerita (p. 893), which occur occasionally in different Sarcodina, have in some cases been called chromidia. By using Feulgen's nucleal reaction, 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 cytoplasm The extranuclear part of the protozoan body is the cytoplasm. It is composed of a colloidal system, which may be homogeneous, granu- lated, vacuolated, reticulated, or fibrillar in optical texture, and 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. Furthermore, pigment or crystals which are produced in the body may give protozoans vari- ous colorations. In several forms pigments are diffused throughout the cytoplasm. For example, many dinoflagellates are beautifully colored, which, according to Kofoid and Swezy, is due to a thorough diffusion of pigment in the cytoplasm. Stentor coeruleus is beautifully blue-colored. This coloration is due to the presence of pigment stentorin (Lankester, 1873) which occurs as granules in the ectoplasm (Fig. 14). The pigment is highly re- sistant to various solvents such as acids and alkalis, and the sun- light does not affect its nature. It is destroyed by bleaching with chlorine gas or with potassium permanganate, followed by immer- sion in 5 per cent oxalic acid (Weisz, 1948). Several species of Blepha- 46 PROTOZOOLOGY risma are rose- or purple-colored. The color is due to the presence of zoopurpurin (Arcichovskij, 1905) which is lodged in numerous gran- ules present in the ectoplasm. This pigment is soluble in alcohol, ether or acetone, and is destroyed by strong light (Giese, 1938). Weisz (1950) maintains that both pigment granules are chondrio- somes, and in Stentor, cytochrome oxidase appears to be localized in the pigment granules. The extent and nature of the cytoplasmic differentiation differ greatly among various groups. In the majority of Protozoa, the cytoplasm is differentiated into the ectoplasm and the endoplasm. The ectoplasm is the cortical zone which is hyaline and homogeneous in Sarcodina and Sporozoa. In the Ciliophora it is a permanent and distinct part of the body and contains several organelles. The endo- plasm 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 beginning of 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. 46). In numerous Sarcodina and certain Mastigophora, the body surface is naked and not protected by any form-giving organella. However, the surface layer is not only elastic, but solid, and there- fore 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, A. verrucosa (Howland, 1924), Pelomyxa carolinensis, P. illinoisensis (Kudo, 1946, 1951), etc., 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 organ- isms 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 body. In certain peritrichous ciliates, such as Vorticella monilata, Carchesium granulatum, etc., MORPHOLOGY 47 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 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 (1933) 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 accidental, since the specimens without the spicules are few and those with a short or broken spicules are not found. P. bowmanni, 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 constructed 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 48 PROTOZOOLOGY 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 Fig. 4. Diagram of the shell of Peneroplis pertusus, X about 35 (Carpenter), ep, external pore; s, septum; sc, stolon canal. of the spines results in production of highly complex and the most 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. 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. 806). 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. MORPHOLOGY 49 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 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. 46; 184). 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, Pelomyxa carolinensis (Fig. 186, b), etc., in which case it is a broadly tongue- like extension of the body in one direction and the progressive move- ment of the organisms is comparatively rapid. The lobopodia may occasionally be conical in general shape, as in Amoeba spumosa (Fig. 185, a). Although ordinarily the formation of lobopodia is by a gen- eral flow of the cytoplasm, in some it is sudden and "eruptive," as in Entamoeba blattae or Entamoeba histolytica in which the flow of the endoplasm presses against the inner zone of the ectoplasm and the accumulated pressure finally causes a break through the zone, result- ing 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 sometimes be 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 (Fig. 5), Peneroplis, etc., and in certain testaceans, such 50 PROTOZOOLOGY as Lieberkuhnia, Myxotheca, etc. The abundantly branching and anastomosing rhizopodia often produce a large network which serves almost exclusively for capturing prey. lift, \^i ;;:;;: ;l ; ;;\v„ I'll: Fig. 5. Pseudopodia of Elphidium strigilata, X about 50 (Schulze from Kiihn). 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- MORPHOLOGY 51 thocystis. The axial rod, which is composed of a number of fibrils (Doeflein; Roskin, 1925; Rumjantzew and Wermel, 1925), arises from the central body or the nucleus located in the approximate center of the body, from each of the nuclei in multinucleate forms, or from the zone between the ectoplasm and endoplasm (Fig. 6). Although semipermanent in structure, the axial rod is easily ab- sorbed and reformed. In the genera of Heliozoa not mentioned above and in numerous radiolarians, the radiating filamentous pseudopodia are so extremely delicate that it is difficult to determine en .-Ite c v kSX w 7\A\. -| '"•-/ ec Fig. 6. Portion of Actinosphaerium eichhorni, X800 (Kiihn). ar, axial rod; cv, contractile vacuole; ec, ectoplasm; en, endoplasm; n, nucleus. 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- 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. 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 52 PROTOZOOLOGY the often-quoted experiment of Verworn, a Umax amoeba changed into a radiosa amoeba upon addition of potassium hydroxide to the water (Fig. 7). Mast has recently shown that when Amoeba proteus or A . dubia 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 monopodal 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 Fig. 7. Form-change in a limax-amoeba (Verworn). a, b, contracted forms; c, individual showing typical form; d-f, radiosa-forms, after ad- dition of KOH solution to the water. 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- cised in using them for taxonomic consideration of the Protozoa. Flagella. The flagellum is a filamentous extension of the cytoplasm and is ordinarily extremely hue and highly vibratile, so that it is difficult to recognize it distinctly in life under the microscope. It is most clearly observed under a darkfield or phase microscope. Lugol's solution usually makes it more easily visible, though the organism is killed. In a small number of species, the flagellum can be seen in life under an ordinary microscope as a long filament, as for example in MORPHOLOGY 53 Peranema. As a rule, the number of flagella present in an individual is small, varying from one to eight and most commonly one or two; but in Hypermastigina there occur numerous flagella. A flagellum appears to be composed of two parts: an elastic axial filament or axoneme, made up of one to several fibrils and the con- tractile cytoplasmic sheath surrounding the axoneme (Fig. 8, a, b). In some flagella, both components extend the entire length and terminate in a bluntly rounded point, while in others the distal por- tion of the axoneme is apparently very thinly sheathed (Fig. 8, c). Fig. 8. Diagrams of flagella. a, flagellum of Euglena (Butschli); b, flagellum of Trachelomonas (Plenge); c, flagella of Polytoma uvella; d, flagella of Monas socialis (Vlk). In some flagellates, stained flagella show numerous lateral fibrils (Fig. 8, d) (Fischer, 1894; Dellinger, 1909; Mainx, 1929; Petersen, 1929; etc.). These flagella or ciliary flagella have also been noticed by several observers in unstained organisms under darkfield micro- scope (Vlk, 1938; Pitelka, 1949). In recent years, the electron micro- scope has been used by some to observe the flagellar structure (Schmitt, Hall and Jakus, 1943; Brown, 1945; Pitelka, 1949; Chen, 1950), but in all cases, the organisms were air-dried on collodion films for examination so that the flagella disintegrated more or less completely at the time of observation. Pitelka (1949) studied flagella of euglenoid organisms under light and electron microscopes. She found that the flagellum of Euglena 54 PROTOZOOLOGY gracilis, Astasia longa and Rhabdomonas incurva, consists of an axoneme, composed of about 9 fibrils, 350-600 A in diameter, ar- ranged in two compact, parallel bundles, and a sheath which is made up of fibrillar elements, a probably semi-fluid matrix and a limiting membrane. Under conditions always associated with death of the organism, the fibrils of the sheath fray out on one or more sides of the flagellum into fine lateral filaments or mastigonemes. The electron micrographs obtained by various investigators on supposedly one and the same flagellate present a varied appearance of the structure. Compare, for example, the micrographs of the frayed flagellum of Euglena gracilis by Brown (1945), Pitelka (1949) and Houwink (1951). The anterior flagellum of Peranema trichophorum frays out into three strands during the course of disintegration as first ob- served by Dellinger (1909) and by several recent observers. It can be easily demonstrated by treating the organism with reagents such as acidified methyl green. Under electron microscope, Petelka noted no frayed mastigonemes in the flagellum of Peranema, while Chen (1950) observed numerous mastigonemes extending out from all sides like a brush, except the basal portion of the flagellum. The electron micrographs of the flagellum of trypanosomes reveal that it also consists of an axoneme and a sheath of cytoplasm. The axoneme is composed of a number of long parallel fibrils, 8 in Tnjpanosoma lewisi, each with estimated diameters of 0. 055-0. 06m (Kleinschmidt and Kinder, 1950), and up to 9 in T. evansi, with estimated diameters of 0.04-0.05^ (Kraneveld, Houwink and Keidel, 1951). The cytoplasmic sheath of the latter species was said to be cross-striated at about 0.05m intervals. No mastigonemes occur in these flagella. The frayed condition of a flagellum which had become detached from the organism or which is still attached to a moribund indi- vidual, as revealed by the darkfield microscope, may also indicate a phase in disintegration of the flagellum. It is reasonable to assume that different flagella may have structural differences as revealed by the electron microscope, but evidence for the occurrence of mas- tigonemes on an active flagellum of a normally living organism ap- pears not to be on hand. A flagellum takes its origin in a blepharoplast of kinetosome im- bedded in the cytoplasm. The blepharoplast is a small compact granule, but in certain parasitic flagellates, it may be comparatively large and ovoid or short rod-shaped, surrounded often by a halo. Whether this is due to the presence of a delicate cortical structure enveloping the compact body or to desiccation or fixation is un- MORPHOLOGY 55 known. In such forms, the flagellum appears to arise from the outer edge of the halo. Certain observers such as Woodcock (1906), Min- chin (1912), etc., used the term kinetonucleus. It has since been found that the blepharoplast of certain trypanosomes often gives a positive Feulgen's reaction (Bresslau and Scremin, 1924). The blepharoplast and centriole are considered synonymous by some, since prior to the division of nucleus, it divides and initiates the division of the latter. A new flagellum arises from one of the daughter blepharoplasts. While the blepharoplast is inseparably connected with the flagellum and its activity, it is exceedingly small or absent in Trypanosoma equinum and in some strains of T. evansi. Furthermore, this condition may be produced by exposure of normal individuals to certain chemical substances (Jirovec, 1929; Piekarski, 1949) or spontaneously (p. 228) without decrease in flagellar activity. 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 posterior end of the body and would push the body forward by its vibration. Under favorable conditions, flagellates regenerate lost flagella. For example, Peranema trichophorum from which its an- terior flagellum w r as cut off, regenerated a new one in two hours (Chen, 1950). In certain parasitic Mastigophora, such as Trypanosoma (Fig. 9), 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. 127, 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. They may be uniformly long, as in Protociliata, or may be of differ- ent lengths, being longer 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. A cilium originates in a kinetosome embedded 56 PROTOZOOLOGY in the ectoplasm. In well-studied ciliates, there occurs a fine fibril, kinetodesma (Chatton and Lwoff, 1935), a short distance to the right of the kinetosome (Fig. 23). The ciliary row or kinety (Chatton and Lwoff) consists of the kinetosomes and kinetodesma (Fig. 23, a). In forms such as Suctoria in which cilia occur only in the swimming stage, the kinetosomes appear to be present as infraciliature (Chat- ton, Lwoff and Lwoff, 1929). Flagellum Undulating membrane Nucl( Blepharoplast Fig. 9. A diagram showing the structure of a trypanosome (Ktihn). As to its structure, a cilium appears to be made up of an axoneme and contractile sheath (Fig. 10, a). 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 (1929) 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, b, c). In electron micrographs of a dried cilium of Para- mecium, Jakus and Hall (1946) found that it consisted of a bundle of about 11 fibrils extending the full length (Fig. 10, d). These fibrils were about 300-500 A in diameter. As there was no visible sheath, the two observers remarked that if a sheath exists, it must be very fragile and easily ruptured. 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 MORPHOLOGY 57 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. Fig. 10. a, cilia of Coleps; b, cilium of Cyclidium glaucoma; c, basal por- tion of a cilium of Colpidium colpoda, all in silver preparations (Klein); d, electronmicrograph of a dried cilium of Paramecium, shadow-cast with chromium, XI 1,000 (Jakus and Hall). 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, a; 12, a), which was demonstrated by Taylor. Klein also showed by desiccation that each marginal cirrus of Stylonychia 58 PROTOZOOLOGY 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, Cirrus fiber Ectoplasmic granules Basal plate of the cirrus Kinetosomes of component cilia Adoral zone Frontal cirri Undulating membrane Marginal cirri Ventral cirri Anal cirri Caudal cirri Fig. 11. a, five anal cirri of Euplotes eurystomus (Taylo'r); b, schematic ventral view of Stylonychia to show the distribution of the cirri. caudal, and marginal cirri, according to their location (Fig. 11, b). Unlike cilia, the cirri may move in any direction so that the organ- isms bearing them show various types of locomotion. Oxytricha, MORPHOLOGY 59 Stylonychia, etc., "walk" on frontals, 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, b). A number of these membranellae occur on a margin of the peristome, forming the adoral zone of cpg Fig. 12. Diagrams of cirrus and membranella of Euplotes eurystomus, X1450 (Taylor), a, anal cirrus in side view; b, a membranella (cpg, co- agulated protoplasmic granules; cr, ciliary root; fp, fiber plate; k, kineto- some) . membranellae, which serves for bringing the food particles to the cytostome as well as for locomotion. The frontal portion of the zone, the so-called frontal membrane appears to serve for locomotion and Kahl considers that it is probably made up of three lamellae. The oral membranes which are often found in Holotricha and Heterotricha, are transparent thin membranous structures composed of one or two rows of cilia, which are 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 mem- branes. 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 60 PROTOZOOLOGY are either prehensile or usually suctorial. The prehensile tentacle appears to be essentially similar in structure to the axopodium (Roskin, 1925). The suctorial tentacles are tubular and this type is interpreted by Collin as possibly derived from cytostome and cyto- pharynx of the ciliate (Fig. 13). Although the vast majority of Protozoa possess only one of the three organelles of locomotion mentioned above, a few may possess jjjgjt Fig. 13. Diagrams showing the possible development of a suctorian tentacle from a cytostome and cytopharynx of a ciliate (Collin). pseudopodia in one stage and flagella in another during their de- velopment. Among several examples may be mentioned Naegleri- idae (Fig. 183), Tetramitus rostratus (Fig. 155), etc. Furthermore, there are some Protozoa which possess two types of organellae at the same time. Flagellum or flagella and pseudopodia occur in many Phytomastigina and Rhizomastigina, and a flagellum and cilia are present in Ileonema (Fig. 306, b, c). In the cytoplasm of Protozoa there occur various organellae, each of which will be considered here briefly. Fibrillar structures One of the fundamental characteristics of the protoplasm is its contractility. If a fully expanded Amoeba proteus is subjected to a 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 Protozoa appears to be Lieberkiihn (1857) who noted the "muscle MORPHOLOGY 61 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 3-7/x high by about lju wide and which appears cross-striated (Fig. 14). Roskin (1923) considers that mc gis Fig. 14. Myonemes in Stentor coeruleus (Schroder), a, cross-section of the ectoplasm; b, surface view of three myonemes; c, two isolated myonemes (cl, cilium; gis, granules between striae; k, kinetosome; m, myoneme; mc, myoneme canal). 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 (1911) studies show that the stalk is a pseudochitinous tube, enclosing an inner tube filled with granulated thecoplasm, which surrounds a cen- tral rod, composed of kinoplasm, on the surface of which are ar- 62 PROTOZOOLOGY ranged 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 Gregarinida aid apparently in locomotion, being arranged longitudinally, transversely and probably spirally (Roskin and Levinsohn, 1929) (Fig. 15, c). In certain Radiolaria, such as Acantho- Fig. 15. a, b, fibrillar structures of the stalk of Zoothamnium (Kolt- zoff); c, myonemes in Gregarina (Schneider), ef, elastic fiber; ie, inner envelope; k, kinoplasm; oe, outer envelope; t, thecoplasm. metron elasticum (Fig. 219, c), etc., each axial spine is connected with 10-30 myonemes (myophrisks) originating in the body surface. When these myonemes contract, the body volume is increased, thus in this case functioning as a hydrostatic organella. In Isotricha prostoma and /. 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 Nyctotherus ovalis (Zulueta; Kudo), ten Kate (1927, 1928) studied fibrillar systems in Opalina, Nycto- MORPHOLOCxY 63 therus, Ichthyophthirius, Didinium, and Balantidium, and found that there are numerous fibrils, each of which originates in the kine- tosome of a cilium and takes a transverse or oblique course through the endoplasm, ending in a kinetosome 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 ectoplasm of the operculum (opercular fibers). A similar apparatus has since been observed in many other ciliates: Euplotes (Yocom; 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 (Swezey), Oxytricha (Lund), Ancistruma and Conchophthirus (Kidder), etc. Ciliate fibrillar systems (Taylor, 1941). Euplotes, a common free-living hypotrichous ciliate, has been known for nearly 60 years to possess definite fibrils connecting the anal cirri with the anterior part of the body. Engelmann suggested that their function was more or less nervelike, while others main- tained that they were supporting or contracting in function. Yocom (1918) traced the fibrils to the motorium, a very small bilobed body (about 8/x by 2ju) located close to the right anterior corner of the triangular cytostome (Fig. 17, m). Joining with its left end are five 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; oc, oral cilia; od, oral disk; oef, oesophageal fibers; of, opercular fibers; p, pellicle; prs, pharyngeal retractor strands; si, skeletal laminae; vs, ven- tral skeletal area. MORPHOLOGY 65 long fibers (acf) 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 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 sm Fig. 17. Ventral view of Euplotes eurystomus (E. patella) showing neu- romotor system, X670 (Hammond), acf, fibril of anal cirrus; am, anterior adoral zone membranelle; m, motorium; mf, membranelle fibrils; oc, en- doral cilia; pf, post-pharyngeal fibril; pra, post-pharyngeal membrane; rf, radiating fibrils; sm, suboral membranelles; vm, ventral adoral zone membranelles. 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 (rf). By means of microdissection needles, Taylor demonstrated that these 66 PROTOZOOLOGY 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. Ham- mond (1937) and Hammond and Kofoid (1937) find the neuromotor system continuous throughout the stages during asexual reproduc- tion and conjugation so that functional activity is maintained at all times. 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 kinetosomes or basal 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 (1926-1942) subjected 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 kinetosomes 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 (Figs. 18- 20). Gelei, Chatton and Lwoff, Jlrovec, Lynch, Jacobson, Kidder. Lund, Burt, 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 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 is augmented by a distinct but connected external network of sensory fibrils. He however finds no motorium in this protozoan. MORPHOLOGY (17 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 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- Fig. 18. The silverline system of Ancistruma mytili, XlOOO (Kidder). a, ventral view; b, dorsal view. 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, 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 kine- tosomes in a longitudinal row of hexagons and of delicate transverse fibrils connecting granules of adjacent rows especially in the cyto- stomal region (Fig. 19). By using Sharp's technique, Lund found the neuromotor system 68 PROTOZOOLOGY of Paramecium multimicronucleatum constructed as follows: The subpellicular portion of the system is the longitudinal fibrils which connect the kinetosomes. In the cytostomal region, the fibrils of right and left sides curve inward forming complete circuits (the circular cytostomal fibrils) (Fig. 20). The postoral suture is separated at the point where the cytopyge is situated. Usually 40-50 fibrils Fig. 19. Diagram of the cortical region of Paramecium multimicronu- cleatum, showing various organellae (Lund), c, cilia; et, tip of trichocyst; k, kinetosome; If, longitudinal fibril; p, pellicle; t, trichocyst; tf, transverse fibril. 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 kinetosomes, 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 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. Silverline system (Klein, 1926-1942; Gelei, 1932); fibrils in ciliates Fig. 20. The neuromotor system of Paramecium multimicronucleahim (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; lbf, longitudinal body fibril; lof, longitudinal oesophageal fibrils; lpf, 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. 70 PROTOZOOLOGY (Jacobson, 1932; Taylor, 1941); argyrome in Astomata (Puytorac, 1951). Protective or supportive organ ellae The external structures as found among various Protozoa which serve for body protection, have already been considered (p. 47). 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 skeletal structures of Radiolaria vary in chemical com- position and forms, and are arranged with a remarkable regularity (p. 517). 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 furcula, observed by Lynch in Lechriopyla (p. 741) is said to be concerned with either the neuromotor system or protection. The members of the family Ophryoscolecidae (p. 816), 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 that are connected with the flagella (Lophomonas). The axostyle appears to be supportive in function, but in forms such as Saccino- baculus, it undulates and aids in locomotion (p. 379). In trichomonad flagellates there is often present along the line of MORPHOLOGY 71 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 flagella, blepharo- plast, parabasal body, or chromatin. 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. These rods are arranged to form the wall of the cyto- pharynx in a characteristic way. For example, the oral basket of Chilodonella cucullulus (Fig. 312, c, d) is made up of 12 long rods which are so completely fused in part that it appears to be a smooth tube; in other forms, the rods are evidently similar to the tubular trichocysts or trichites mentioned below. In numerous holotrichs, there occur unique organelles, trichocysts, imbedded in the ectoplasm, and usually arranged at right angles to the body surface, though in forms such as Cyclogramma, they are arranged obliquely. Under certain stimulations, the trichocysts "ex- plode" and form long filaments which extend out into the surround- ing medium. The shape of the trichocyst varies somewhat among different ciliates,, being pyriform, fusiform or cylindrical (Penard, 1922; Kriiger, 1936). They appear as homogeneous refractile bodies. The extrusion of the trichocyst is easily brought about by means of mechanical pressure or of chemical (acid or alkaline) stimulation. In forms such as Paramecium, Frontonia, etc., the trichocyst is elongate pyriform or fusiform. It is supposed that within an expansi- ble membrane, there is a layer of swelling body which is responsible for the remarkable longitudinal extension of the membrane (Kriiger) (Fig. 21, a). In other forms such as Prorodon, Didinium, etc., the tubular trichocyst or trichites are cylindrical in shape and the mem- brane is a thick capsule with a coiled thread, and when stimulated, the extrusion of the thread takes place. The trichites of Prorodon teres measure about 10—1 1 yu. long (Fig. 21, d) and when extruded, the whole measures about 20 /x; those of Didinium nasutum are 15- 20m long and after extrusion, measure about 40 m in length (Fig. 21, e,f). In Spathidium spathida (Fig. 21, c), 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 trichites which have been torn away during the process of prey ingestion, " 72 PROTOZOOLOGY Fig. 21. a, a schematic drawing of the trichocyst of Paramecium cau- datum (Kruger) (b, base of the tip; c, cap; m, membrane; mt, membrane of extruded trichocyst; s, swelling body; t, tip); b, an extruded trichocyst, viewed under phase dark contrast, X1800; c, trichites in Spathidium, spathula, X300 (Woodruff and Spencer); d, a diagram of the trichocyst of Prorodon teres (Kruger) (eg, capsule-granule; e, end-piece of filament; f, filament; w, capsule wall); e, f, normal and extruded trichocysts of Didin- ium nasutum (Kruger). MORPHOLOGY 73 Whether the numerous 12-20^ long needle-like structures which Kahl observed in Remanella (p. 727) are modified trichites or not, is not known. 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 toxicysts. But Kruger and Hayes (1938) found that the extruded trichocysts can be recog- nized. Perhaps the most frequently studied trichocysts are those of Paramecium. They are elongate pyriform, with a fine tip at the broad end facing the body surface. The tip is connected with the pellicle (Fig. 19, 0- Kruger found this tip is covered by a cap (Fig. 21, a) which can be seen under darkfield or phase microscope and which was demonstrated by Jakus (1945) in an electron micrograph (Fig. 22, a). When extruded violently, the entire structure is to be found outside the body of Paramecium. The extruded trichocyst is composed of two parts: the tip and the main body (Fig. 21, b). The tip is a small inverted tack, and may be straight, curved or bent. The main body or shaft is a straight rod, tapering gradually into a sharp point at the end opposite the tip. Extruded trichocysts meas- ure 20-40yu or more in length, and do not show any visible struc- tures, except a highly refractile granule present at the base of the tuck-shaped tip (Fig. 21, b). The electron microscope studies of the extruded trichocysts by Jakus (1945), Jakus and Hall (1946) and Wohlfarth-Bottermann (1950), show the shaft to be cross-striated (Fig. 22). Jakus considers that the main component of the tricho- cyst is a thin cylindrical membrane formed by close packing of longitudinal fibrils characterized by a periodic pattern (somewhat resembling that of collagen), and as the fibrils are in phase with re- spect to this pattern, the membrane appears cross-striated. As to the mechanism of the extrusion, no precise information is available, though all observers agree that the contents of the tricho- cyst suddenly increase in volume. Kruger maintains that the tricho- cyst cap is first lifted and the swelling body increases enormously in volume by absorbing water and lengthwise extension takes place, while Jakus is inclined to think that the membrane itself extends by the sudden uptake of water. 74 PROTOZOOLOGY How are these organelles formed? Tonniges (1914) believes that the trichocysts of Frontonia leucas originate in the endosomes of the macronucleus and development takes place during their migration to the ectoplasm. Brodsky (1924) holds that the trichocyst is com- posed of colloidal excretory substances and is first formed in the vicinity of the macronucleus. Chatton and Lwoff (1935) find how- Fig. 22. Electronmicrographs of extruded trichocysts of Paramecium, a, dried and stained with phosphotungstic acid, XI 1,000 (Jakus); b, a similarly treated one, X 15,000 (Jakus); c, shadow-cast with chromium, X 16,000 (Jakus and Hall). ever in Gymnodinioides the trichocysts are formed only in tomite stage and each trichocyst arises from a trichocystosome, a granule formed by division of a kinetosome (Fig. 23, a-c). In Polyspira, the trichocyst formation is not confined to one phase, each kinetosome is said to give rise to two granules, one of which may detach itself, migrate into other part of the body and develops into a trichocyst (d). In Foettingeria, the kinetosomes divide in young trophont stage into irichitosomes which develop into trichites (e). The two authors note that normally cilia-producing kinetosomes may give rise to MORPHOLOGY ::» trichocysts or trichites, depending upon their position (or environ- ment) and the phase of development of the organism. Although the trichocyst was first discovered by Ellis (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. On the other Fig. 23. Diagrams showing the formation of trichocysts in Gymnodini- oides (a-c) and in Polyspira (d) and of trichites in Foettingeria (e) (Chat- ton and Lwoff). a, a ciliary row, composed of kinetosomes, large satellite corpuscles and kinetodesma (a solid line); b, each kinetosome divides into two, producing trichocystosome; c, transformation of trichocystosomes into trichocysts; d, formation of trichocyst from one of the two division products of kinetosome; e, formation of trichites from the division prod- ucts of kinetosomes. hand, the trichocysts and trichites are clearly an offensive organelle in capturing food organisms in organisms such as Dileptus, Didinium, Spathidium, etc. Saunders (1925) considered that the extruded tri- chocysts of Paramecium serve for attachment of the body to other objects. But Wohlfarth-Bottermann (1950) saw Paramecium cauda- tum extruding up to 300 trichocysts without any apparent external stimulation and trichocyst-less individuals were able to adhere to foreign objects. This worker suggested that the trichocyst secretes calcium salt and probably also sodium and potassium, and thus may serve an osmoregulatory function. Some years ago Penard (1922) considered that some trichocysts may be secretory organellae to pro- duce material for loricae or envelope, with which view Kahl concurs, as granular to rod-shaped trichocysts occur in Metopus, Amphilep- 76 PROTOZOOLOGY tus, etc. Klein has called these ectoplasmic granules protrichocysts, and in Prorodon, Kruger observed, besides typical tubular tricho- cysts, torpedo-like forms to which he applied the same name. To this group may belong the trichocysts recognized by Kidder in Con- chophthirus mytili. The trichocysts present in certain Cryptomonad- ina (Chilomonas and Cyathomonas) are probably homologous with the protrichocysts (Kruger, 1934; Hollande, 1942; Dragesco, 1951). 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 Ahthophysis, 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. 235-237), 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 (Figs. 279, 286). The polar filament is considered as a temporary anchoring or- ganella of the spore at the time of its germination after it gained entrance into the alimentary canal of a suitable host. In the Micro- sporidia, the filament may or may not be enclosed within a capsule (Figs. 288; 289). The nematocysts (Fig. 132, b) of certain dino- flagellates belonging to Nematoidium and Polykrikos, are almost identical in structure with those found in the coelenterates. They are distributed through the cytoplasm, and various developmental stages were noticed by Chatton, and Kofoid and Swezy, which indi- cates 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 protozoans is not understood. MORPHOLOGY 77 Parabasal apparatus In the cytoplasm of many parasitic flagellates, there is frequently present a conspicuous structure known as the parabasal apparatus (Janicki, 1911), consisting of the parabasal body and often thread (Cleveland), which latter may be absent in some cases. This struc- ture varies greatly among different genera and species in appearance, structure and position within the body. It is usually connected with Fig. 24. Parabasal apparatus in: a, Lophomonas blattarujn (Kudo); b, Metadevescovina debilis; c, Devescovina sp. (Kirby). af, axostylar fila- ments; bl, blepharoplasts; f, food particles; fl, flagella; n, nucleus; pa, parabasal apparatus. 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. 24). 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" (1933) maintain 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 78 PROTOZOOLOGY 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 (1931), 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. Moreover the parabasal body is discarded or absorbed at the time of division of the body and two new ones are formed. The parabasal body of Lophomonas blattarum is discarded when the organism divides and two new ones are reformed from the cen- triole or blepharoplast (Fig. 65), and its function appears to be sup- portive. Possibly not all so-called parabasal bodies are homologous or analogous. A fuller comprehension of the structure and function of the organella rests on further investigations. Golgi apparatus With the discovery of a wide distribution of the so-called Golgi apparatus in metazoan cells, a number of protozoologists also re- 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 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- quently the cortical layer is of unequal thickness, and, therefore, crescentic forms appear. Ringform apparatus was noted in Chilo- donella and Dogielella by Nassonov (1925) and network-like forms were observed by Brown in Pyrsonympha and Dinenympha. The Golgi apparatus of Protozoa as well as of Metazoa appears to be composed of a lipoidal material in combination with protein sub- stance. 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 (1924) 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 MORPHOLOGY Table 2. — Golgi apparatus in Protozoa 79 Protozoa Golgi apparatus Observers Chromulina, Astasia Rings, spherules with a dark Hall Chilomonas nm Granules, vacuoles Hall Euglenoidina Stigma Grasse" Euglena gracilis Spherical, discoidal with dark rim; tend to group around or near nucleus Brown Peranema Rings, globules, granules Hall Pyrsonympha, Di- Rings, crescents, spherules; Brown nenympha granules break down to form network near pos- terior end Holomastigotes, Pyr- Parabasal bodies Dubocsq and sonympha, etc. Grass6 Amoeba proteus (Fig. Rings, crescents, globules, Brown 25) granules Endamoeba blattae Spheres, rings, crescents Hirschler Monocystis, Gregarina Spheres, rings, crescents Hirschler Aggregata, gregarines Crescents, rings Joyet-Lavergne Adelea Crescents, beaded grains King and Gatenby Blepharisma undidans Rings in the cytoplasm Moore Vorticella, Lionotus, The membrane of contrac- Nassonov Paramecium, Dogiel- tile vacuole and collecting ella, Nassula, Chilo- canals monas, Chilodonella 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, (Fig. 25), but fusion of minute vacuoles associated with crescentic Golgi bodies produces the vacuole and Park (1929) noted osmiophile knob-like elevations on the surface of the macronucleus of Stentor and Leucophrys, while the contractile vacuole system did not blacken. Duboscq and Grasse (1933) maintain that this body is a source of energy which is utilized by motor organelles. Joyet-Lavergne points out that in certain Sporozoa, the Golgi body is composed of granules and may be the center of enzyme production. Similar to Golgi ma- terial, the so-called vacuome, which consists of neutral red-staining and osmiophile globules, has been reported to occur in many Proto- 80 PROTOZOOLOGY zoa (Hall, 1931; Hall and Nigrelli, 1937). The exact morphological and physiological significance of these organellae and the relation between them must be looked for in future investigations. Golgi apparatus in Protozoa (Alexeieff, 1928; MacLennan, 1941; Grasse\ 1952). 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 IIS Fig. 25. The Golgi bodies in Amoeba proteus (Brown). 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 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- MORPHOLOGY 81 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 kinetosomes of cilia (Hor- ning). In Tillina canalifera, Turner (1940) noticed that the endo- plasmic chondriosomes are evenly distributed throughout the cyto- plasm (Fig. 26, b), while the ectoplasmic chondriosomes are ar- Sic. < x v } r a b m^' Fig. 26. Chondriosomes in Tillina canalifera (Turner), a, diagram show- ing the ectoplasmic chondriosomes (c, cilium; cf, coordinating fibril; ch, chondriosome; cr, ciliary rootlet; k, kinetosome I and II; p, pellicle); b, a section showing chondriosomes and food vacuoles. ranged in regular cross rows, one in the center of each square formed by four cilia (Fig. 2f6, a). In Peranema trichophorum, Hall (1929) ob- served peripheral chondriosomes located along the spiral striae, which Chadefaud (1938) considered as mucus bodies. Weisz (1949, 1950) finds that stentorin and zoopurpurin already mentioned (p. 45) are chondriosomes. 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 82 PROTOZOOLOGY 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 transversel}' 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 (1926) described "beta granules" in Amoeba proteus which are more abundantly found around the contractile vacuole. Mast and Doyle (1935, 1935a) noted that these spherical to rod-like beta granules are plastic and stain like chondriosomes and that there is a direct relation between the number of beta granules in the cyto- plasm and the frequency of contraction of the contractile vacuole. They maintained that these granules "probably function in trans- ferring substances from place to place in the cytoplasm." Similar granules are recognizable in the species of Pelomyxa (Andresen, 1942; Wilber, 1942; Kudo, 1951). 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 other hand, Calkins found that in Uroleptus, the chondriosomes became abundant in exconjugants, due to transformation of the macronu- clear material into the chondriosomes. 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' protoplasmic inclusions" (Piney, 1931). Condriosomes in Protozoa (MacLennan, 1941; Grasse, 1952). MORPHOLOGY 83 Numerous minute granules, less than l^u in diameter, occur usually abundantly suspended in the cytoplasm. They can most clearly be noted under phase microscope. Mast named those found in Amoeba "alpha granules." 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, Mastigophora and Ciliophora. Ma- rine or parasitic Sarcodina and Mastigophora do not ordinarily have a contractile vacuole. This organelle is present with a few exceptions in all marine and parasitic Ciliophora, while it is wholly absent in Sporozoa. In various species of free-living amoebae, the contractile vacuole is formed by accumulation of water 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- 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. 184, /). 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 Mastigophora, the contractile vacuole appears to be located in the anterior region. 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- 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- 84 PROTOZOOLOGY tile vacuoles are deeply seated, there is a delicate duct which con- nects the vacuole with the pore on the pellicle as in Paramecium 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 radiate, in which case the number of the canals varies among different species: one in Spirostomum, Stentor, etc., 2 in Clima- s£ i : a Fig. 27. Diagrams showing the contractile vacuole, the accessory vacu- oles and the aperture, during diastole and systole in Conchophthirus (Kidder). costomum, Eschaneustyla, etc., and several in Tillina. In Peritricha, the contractile vacuole occurs near the posterior region of the cyto- pharynx and its content is discharged through a canal into the vesti- bule and in Ophrydium ectatum, 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), in which Gelei (1939) demonstrated longitudinal fibrils, and each canal is made up of terminal portion, a proximal injection canal, and an ampulla between them. Surrounding the distal portion, there is osmi- ophilic cytoplasm which may be granulated or finely reticulated, and MORPHOLOGY 85 which Nassonov (1924) interpreted as homologous with 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 that which closed the end of the injection canal. These fluid 060 <3^=> _i^_ _ £5= Fig. 28. Diagrams showing the successive stages in the formation of the contractile vacuole in Paramecium multimicronucleatum (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. 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 (Fig. 30) . The canal wall expands and enlarg- ing accessory vacuoles fuse with one another, followed by a full ex- pansion of the canal. Through several excretory pores with short ducts the content of the contractile canal is excreted to the exterior. The function of the contractile vacuole is considered in the following 86 PROTOZOOLOGY Fig. 29. Contractile vacuoles of Paramecium multimicronucleatum, X1200 (King), a, early systole, side view; b, diastole, front view; c, com- plete systole, front view; d, systole, side view. MORPHOLOGY 87 chapter (p. 118). Comparative study of contractile vacuoles (Haye, 1930; Weatherby, 1941). Various other vacuoles or vesicles occur in different Protozoa. In the ciliates belonging to Loxodidae, there are variable numbers of Miiller's vesicles or bodies, arranged in 1-2 rows along the aboral sur- face. These vesicles (Fig. 31, a-c) vary in diameter from 5 to 8.5/* Fig. 30. Excretory canal of Haptophrya michiganensis (MacLennan). a, an individual in side view, showing a contraction wave passing down the canal; b, successive views of the same region of the contractile canal during a full pulsatory cycle (a-c, systole; d-g, diastole); c, diagram show- ing a contractile wave passing from left to right between two adjacent excretory pores. and contain a clear fluid in which one large spherule or several small highly refractile spherules are suspended. In some, there is a fila- 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 88 PROTOZOOLOGY complex. According to Dogiel (1929), the vacuole is composed of a pellicular cap, a permanent vacuolar wall, concrement grains and two fibrillar systems (Fig. 31, 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 Fig. 31. 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. 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 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 and the Mastigophora 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 MORPHOLOGY S9 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 protozoan. In the saprozoic Protozoa, which absorb fluid substances through the body surface, food vacuoles containing solid food, of course, do not occur. Chromatophores d Pvrenoids Fig. 32. 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, Chlamydomonas sp., showing the di- vision of pyrenoid (Geitler). Chromatophore and associated organellae In the Phytomastigina and certain other forms which are green- colored, one to many chromatophores (Fig. 32) containing chloro- phyll occur in the cytoplasm. The chromatophores vary in form among different species; namely, discoidal, ovoid, band-form, rod- like, cup-like, fusiform, network or irregularly diffused. The color of the chromatophore depends upon the amount and kinds of pig- ment which envelops the underlying chlorophyll substance. Thus the chromatophores of Chrysomonadina are brown or orange, as they contain one or more accessory pigments, including phycochrysin, and those of Cryptomonadina are of various types of brown with 90 PROTOZOOLOGY very diverse pigmentation. In Chloromonadina, the chromatophores are bright green, containing an excess of xanthophyll. In dinoflagel- lates, they are dark yellow or brown, because of the presence of pigments: carotin, phylloxanthin, and peridinin (Kylin, 1927), the last of which is said to give the brown coloration. A few species of Gymnodinium contain blue-green chromatophores for which phyco- cyanin 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, 1909; Pringsheim, 1914). Steinecke also noticed that the frequent yellow coloration of phytomonads in moorland pools is due to a development of carotin in the chromatophores as a result of deficiency in nitrogen. Johnson (1939) noted that the haematochrome granules of Euglena rubra be- come collected in the central portion instead of being scattered throughout the body when sunlight becomes weaker. Thus this Eu- glena appears green in a weak light and red in a strong light. The chromatophores undergo division at the time when the organism which contains them, divides, and therefore the number of chroma- tophores appears to remain about the same through different genera- tions (Fig. 32). In association with the chromatophores are found the pyrenoids (Fig. 32) 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. Pyrenoid (Geitler, 1926). Chromatophore-bearing Protozoa usually possess also a stigma (Fig. 32) 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 MORPHOLOGY 91 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 (1928), the pigment mass in Chlamydomonas, Pandorina, 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. 127, c, d, q, h) which is composed of amyloid lens and a dark pigment mass (melanosome) 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 (192G) considers that the stigma in the Vol- vocidae is an organella which determines the direction of the move- ment. References Alexeieff, A.: (1928) Sur la question des mitochondries et de l'ap- pareil de Golgi chez les protistes. Arch. Protist., 60:269. (1929) Nouvelles observations sur les chondriosomes chez les protozoaires. Ibid., 65:45. Arcichovskij, , V. : (1905) Ueber das Zoopurpurin, ein neues Pig- ment der Protozoa (Blcpharisma lateritium). Arch. Protist., 6: 227. Beers, C. D.: (1946) Tillina magna: micronuclear number, etc. Biol. Bull., 91:256. Belar, K.: (1926) Der Formwechsel der Protistenkerne. Ergebn. u. Fortschr. Zool., 6:235. 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Haeckel, E.: (1868) Monographic der Moneren. Jen. Zeit. Natur- wiss., 4. — (1870) Studien ueber Moneren und andere Protisten. Leip- zig. Hall, R. P.: (1929) Reaction of certain cytoplasmic inclusions to vital dyes and their relation to mictochondria, etc. J. Morphol. Physiol., 48:105. and Nigrelli, R. F. : (1937) A note on the vacuome of Para- mecium bursaria and the contractile vacuole of certain ciliates. Tr. Am. Micr. Soc, 56:185. Hammond, D. M. : (1937) The neuromotor system of Euplotes patella during binary fission and conjugation. Quart. J. Micr. Sc, 79: 507. MORPHOLOGY 93 and Kofoid, C. A.: (1937) The continuity of structure and function in the neuromotor system of Euplotes patella during its life cycle. Proc. Am. Phil. Soc, 77:207. Haye, A.: (1930) Ueber den Exkretionsapparat bei den Protisten, etc. Arch. Protist., 70:1. Hayes, M. L. : (1938) Cytological studies on Dileptus anser. Tr. Am. Micr. Soc, 57: 11. Herfs, A. : (1922) Die pulsierende Vakuole der Protozoen, etc. Arch. 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E.: (1946) Electron microscope observations of the trichocysts and cilia of Paramecium. Biol. Bull., 91:141. Janicki, C: (1911) Zur Kenntnis des Parabasalapparates bei para- sitischen Flagellaten. Biol. Zentralbl., 31:321. Jirovec, O. : (1929) Studien ueber blepharoplastlose Trypanosomen. Arch. Protist., 68:187. Kidder, G. W. : (1933) On the genus Ancistruma Strand (Ancistrum Maupas). Biol. Bull., 64:1. (1933a) Conchophthirus caryoclada sp. nov. Ibid., 65:175. (1934) Studies on the ciliates from freshwater mussels. I, II. Ibid., 66:69, 286. King, R. L. : (1935) The contractile vacuole of Paramecium multi- micronucleatum. J. Morphol., 58:555. Kirby, H. Jr.: (1931) The parabasal body in trichomonad flagel- lates. Tr. Am. Micr. Soc, 50:189. Klein, B. M.: (1926) Ueber eine neue Eigentumlichkeit per Pelli- cula von Chilodon uncinatus. Zool. Anz., 67:160. (1926a) Ergebnisse mit einer Silbermethode bei Ciliaten. Arch. Protist., 56:243. (1927) Die Silberliniensysteme der Ciliaten. 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Arch. Protist., 60:305. Mast, S. O.: (1926) Structure, movement, locomotion and stimula- tion in Amoeba. J. Morphol., 41:347. (1928) Structure and function of the eye-spot in unicellular and colonial organisms. Arch. Protist., 60:197. (1944) A new peritrich belonging to the genus Ophrydium. Tr. Am. Micr. Soc, 63:181. and Doyle, W. L. : (1935) Structure, origin and function of cytoplasmic constituents in Amoeba proteus. Arch. Protist., 86: 155. (1935a) II. Ibid., 86:278. Moses, M. J.: (1950) Nucleic acids and proteins of the nuclei of Paramecium. J. Morphol., 87:493. Nassonov, D.: (1924) Der Exkretionsapparat (kontractile Vacuole) MORPHOLOGY 95 der Protozoen als Homologen des Golgischen Apparatus der Metazoenzelle. Arch. mikr. Anat., 103:437. (1925) Zur Frage ueber den Bau und die Bedeutung des Lipoiden Exkretionsapparates bei Protozoen. Ztschr. Zell- forsch., 2:87. Owen, H. M.: (1947) Flagellar structure. I. Tr. Am. Micr. Soc, 66: 50. (1949) II. Ibid., 68:261. 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California Publ. Zool., 18:337. 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. In many Phytomastigina (Pringsheim, 1937a; Hall, 1939), afewciliates (Kidder and Dewey, 1951) and many blood-inhabiting flagellates (Lwoff, 1951) which have been cultivated in vitro free from other organisms, a much clearer information is becoming available. But for the majority of Protozoa a thorough comprehension of the nutri- tion is to be sought in future (Doyle, 1943; Lwoff, 1951; Most, 1951; Kidder, 1951). 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 (1910) oft-quoted observations, four methods of food-ingestion oc- cur in amoebae (Fig. 33) ; namely, (1) by "import," in which the food is taken into the body upon contact, with very little movement on 97 98 PROTOZOOLOGY 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 (6) ; (3) by "circumvalla- tion," in which the amoeba without contact with the food, forms pseudopodia which surround the food on all sides and ingest it (c) ; Fig. 33. Various ways by which amoebae capture food organisms, a, A moeba verrucosa feeding on Oscillatoria by 'import' (Rhumbler) ; b, A . 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). (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). In a species of Hartmannella, Ray (1951) reports an aggluti- nation of large numbers of motile bacteria over the body surface, which later form a large mass and are taken into a food cup. In certain testaceans, such as Gromia, several rhizopodia cooper- ate in engulfing the prey and, in Lieberkuhnia (Fig. 34), Verworn noted ciliates are captured by and digested in rhizopodia. Similar SIOLOGY 00 observation was made by Schaudinn in the heliozoan Camptonema in which several axopodia anastomose to capture a prey (Fig. 214, d). In the holozoic Mastigophora, such as Hypermastigina, which do not possess cytostome, the food-ingestion is by import or invagina- tion as noted in Trichonympha campanula (Cleveland, 1925a; Emik, 1941) (Fig. 35, a) and Lophomonas blattarum (Kudo, 1926). The food particles become attached to the pseudopodium and are held there on account of the viscid nature of the pseudopodium. The sudden immobility 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 Fig. 34. Rhizopodium of Lieberkiihnia, capturing and digesting Colpidium colpoda (Vervvorn). 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. 369, a). The prehensile tentacles of Ephelotidae are said to be similar in structure to the axopodia, in that each possesses a bundle of axial filaments around a cytoplasmic core (Roskin, 1925). These tentacles are capable of piercing through the body of a prey. In some suctorians, such as Choanophrya (Fig. 374, a), the tubular tentacles are clearly observable, 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 100 PROTOZOOLOGY it is taken into the endoplasm. Chen (1950) observed Peranema feed- ing on immobile organisms. When the tip of the anterior flagellum comes in contact with an immobile Euglena, the whole flagellum Fig. 35. a, eight outline sketches of a Trichonympha campanula, in- gesting a large particle of food, XI 50 (Emik); b, four outline sketches of a Peranema trichophorum feeding on an immobile Euglena (Chen). beats actively and the body contracts, followed by elongation. The process is repeated several times until the body touches Euglena. Then the cytostome stretches open, the oral rods move up, protrude from the body and become attached to Euglena. Peranema advances PHYSIOLOGY 101 toward the prey and the whole Euglena is engulfed in 2 to 15 min- utes (Fig. 35, b). In the ciliates, there are many types of cytostome and associated organelles, but the food-capturing seems to be in general of two kinds. When the cytostome is permanently open, the organism in- gests continuously food particles that are small enough to pass the cytostome and cytopharynx, as in the case of Paramecium. The other type is carried on by organisms bearing cytostome which is ordinarily closed such as seen in Coleps, Didinium, Perispira (Dewey and Kidder, 1940), but which expands to often an extraordinary size when the ingestion of prey takes place. Cannibalism in Protozoa (Dawson, 1919; Lapage, 1922; Gelei, 1925a; Tanabe and Komada, 1932; Giese and Alden, 1938; Chen, 1950). The ingested food particles are usually surrounded by a film of fluid which envelops the organism and the whole is known as the food vacuole (p. 88). 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) noticed 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- like elevation of the ectoplasm and Kirby (1932) figures ingestion of the brine containing no visible organisms by the cytostome of Rhopalophrya salina (Fig. 36). Mast and Doyle (1934) state that if Amoeba proteus, A. dubia, 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 a short cyclosis in the posterior half of the body. Some observers maintain that in ciliates there is a definite "diges- 102 PROTOZOOLOGY tive tubule" beginning with the cytostome and ending in the cyto- pyge, and the food vacuoles travel through it. Cosmovici (1931, 1932) saw such a canal in soluble starch-fed Colpidium colpoda upon staining with iodine, but Hall and Alvey (1933) could not detect such a structure in the same organism. Kitching (1938b) observed no such tubule in the peritrichous ciliates he studied, and concluded that the food vacuoles are propelled over the determined part of the course by the contraction of surrounding cytoplasm. In Vorticella sp., food vacuoles are formed one by one at the end of cytopharynx, migrate through different parts of the cytoplasm without order and food material is digested (Fig. 37, a). Old food vacuoles are defecated through a small papilla on the lower wall of the cytopharynx and thence to the outside (Hall and Dunihue, 1931) (Fig. 37, b-d). ^r\ n tr\ n n Fig. 36. Ingestion of brine by Rhopalophrya salina (Kirbj'). 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 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 (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 (1894) found in Carchesium that ingestion of PHYSIOLOGY 103 food particles stimulated the cytoplasm to secrete a mineral acid. 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 Fig. 37. Diagrams showing movements of food vacuoles in Vorticella sp. (Hall and Dunihue). a, diagram of the migration paths of six food vacuoles (vacuoles 1, 2, most recently formed; 3, 4, recently formed; 5, 6, formed some time before) ; b-d, stages in extrusion of a food vacuole (b, food vacuole entering gullet; c, a later stage; d, the food vacuole leaving cytostome, while another one is moving up toward the cytopyge). 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 in Paramecium caudatum 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 104 PROTOZOOLOGY 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 observations (1942) on the food vacuoles in Amoeba pro- teus and A. dubia 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 secretion, but is probably due to respiration in the in- gested 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. De La Arena (1941, 1942) found the maximum acidity of the fluid of food vacuoles in Pelomyxa carolinensis containing Colpidium striatum was pH 5.8 and was not fatal for the ciliate, but considered the possibility of the existence in the food vacuole of "some lethal agent" which kills the prey. Just exactly what processes take place in the food vacuole have been observed only in a few cases. Nirenstein (1925) noticed the ap- pearance 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 (1926) demonstrated the oxidase reaction in these granules. Hopkins and Warner (1946) believe that the digestion of food in Entamoeba histolytica is brought about by enzymes carried to the food vacuoles by "digestive spherules" which arise at the periphery of the nucleus, apparently due to the action of the substances diffusing from the nu- cleus into the cytoplasm. As to the localization or distribution of enzymes within protozoan body, definite information is not yet available. In centrifuged Amoeba proteus, Holter and Kopac (1937) found the peptidase ac- tivity independent of all cytoplasmic inclusions that were stratified by centrifugal forces. Holter and L0vtrup (1949) found peptidase in PHYSIOLOGY 105 centrifuged Pelomyxa carolinensis comparatively evenly distributed after centrifugation, possibly with a tendency to be concentrated in the lighter half, while proteinase was largely localized in the heavier half in which cytoplasmic granules were accumulated, and concluded that these two enzymes are bound, at least in part, to different cyto- plasmic components. A number of enzymes have been reported to occur in Protozoa, some of which are listed 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. The members of Vampyrella (p. 420) 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- 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 (Becker et al., 1930; Weineck, 1934). Dawson and Belkin (1928) injected oils into Amoeba dubia and found 1.4 to 8.3 per cent digested. Mast (1938) reported that the neutral fat globules of Colpidium are digested by Amoeba proteus and transformed into fatty acid and glycerine which unite and form neutral fat. Chen (1950) found that when Peranema trichophorum was fed on almond oil (stained dark blue with Sudan black), Sudan III-stainable droplets gradually increased in number in five to 10 hours, while ingested oil-droplets decreased in size, and considered that the droplets were "fat-substances" resynthesized from prod- ucts of digestion of almond oil by this flagellate. The digestion of rice starch is followed by the appearance of increasing number of ovoid paramylon granules, and the digestion of casein results in the formation of oil droplets and paramylon bodies. 106 PROTOZOOLOGY Table 3. — Enzymes in Protozoa Protozoa Enzymes Observers Amoeba proteus Peptidase Holter and Kopac (1 937) ; Holter and Doyle (1938); Andresen and Holter (1949); Holter and Ljtfvtrup (1949) Proteinase Andresen and Holter (1949); Holter and Ljrfvtrup (1949) Amylase Holter and Doyle (1938a) A. dubia Lipolytic substance Dawson and Belkin (1928) Pelornyxa palustris Diastatic enzyme Hartog and Dixon (1893) ; Stole (1900) Pepsin-like enzyme Hartog and Dixon (1893) Peptidase Andresen and Holter (1949) Proteinase u P. carolinensis Peptidase " Proteinase a Succinic dehydro- Andresen, Engel and Holter genase (1951) Lipase Wilber (1946) Soil amoeba " Amoebo-diastase, " a trypsin-like en- zyme Mouton (1902) Aethalium seftticum Pepsin-like enzyme Krukenberg (1886) Eitglena gracilis Proteolytic enzyme Jahn (1931) Xylophagous Poly- Cellulase Trager (1932) and Hyper-mas- Cellobiase Cleveland et ah (1934) tigina Didinium nasutum Dipeptidase Doyle and Patterson (1942) Tetrahymena pirifor- Proteolytic enzyme Lwoff (1932); Lawrie (1937) mis Peptidases Kidder and Dewey (1951) Acetylcholinesterase Seaman and Houlihan (1951) Colpidium striatum Proteolytic enzyme Elliott (1933) Paramecium cau- Peptidase Holter and Doyle (1938) datum Amylase a P. multimicronuclea- tum Frontonia sp. Dipeptidase Doyle and Patterson (1942) Peptidase Holter and Doyle (1938) Amylase a Balantidium coli Diastase Glaessner (1908) In certain Sarcodina such as Amoeba and Pelornyxa, refringent bodies occur conspicuously in the cytoplasm. They were first noticed in Pelornyxa palustris by Green" (1874) who called them "Glanz- korper." Stole (1900) and Leiner (1924) considered them as glycogen enclosed within a membrane and associated intimately with the PHYSIOLOGY 107 carbohydrate metabolism of the organism, since their number was proportionate to the amount of food obtained by the organism. Veley (1905) on the other hand found them albuminoid in nature. Studies of the refringent bodies in Amoeba proteus led Mast and Doyle (1935, 1935a) to conclude that the outer layer is composed of a protein stroma impregnated with lipid containing fatty acid, which gives positive reaction for Golgi substance; the envelope is made up of a carbohydrate which is neither starch nor glycogen; and the re- fringent bodies function as reserve food, since they disintegrate dur- ing starvation. The same function was assigned to those occurring in Pelomyxa carolinensis by Wilber (1945, 1945a), but Andresen and Holter (1945) do not agree with this view, as they observed the number of the refringent bodies ("heavy spherical bodies") remains the same in starvation. Thus a full comprehension of the nature and function of the refringent body must depend on future observations. The indigestible residue of the food is extruded from the body. The extrusion may take place at an}' 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 (Fig. 37, b-d). Permanent cytopyge is lack- ing in some forms. In Fabrea 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 outline is re- stored. The same seems to be the case with Spirostomum (Fig. 38), Blepharisma, etc. Cytopyge (Klein, 1939). Holophytic (autotrophic, prototrophic) 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. 89) in the presence of the sunlight, liberating the oxygen and combining the carbon with other elements derived from water and inorganic salts (photosynthesis). Aside from the Phytomastigina, chromato- phores were definitely observed in a ciliate Cyclotrichium meunieri (Figs. 300, o; 301) (Powers, 1932; Bary and Stuckey, 1950). In a number of other cases, the organism itself is without chromatophores, but is apparently not holozoic, because of the presence of chloro- phyll-bearing organisms within it. For example, in the testacean Paulinella (Fig. 206, c) in which occur no food vacuoles, chromato- phores 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 108 PROTOZOOLOGY of the Phytomastigina. A similar relationship seems to exist between Paramecium bursaria, Stentor pohjmorphus, etc. and zoochlorellae; Paraeuplotes tortugensis and a zooxanthella and others (p. 29). Pringsheim (1928) showed that organic matters from zoochlorellae are passed on to their host, Paramecium bursaria, to be used as food. Through studies of relationships between zooxanthellae and in- vertebrates, Yonge observed that the zooxanthellae utilize carbon dioxide, nitrogen and phosphorus which are the catabolic products of the host and supply in return oxygen, fats and carbohydrates to the host. Photosynthesis in Phytomastigina (Hutner and Provasoli, 1951). 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- Fig. 38. Outline sketches showing the defecation process in Spirostomum ambiguum (Blattner). stance in which the saprozoic nutrition is accomplished through a special organella is the pusules (Figs. 127, 129) in marine dinoflagel- lates which, according to Kofoid and Swezy (1921), appear to con- tain decomposed 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 flagellates nourish them- selves with this method. Recently a number of investigators found that saprozoic Protozoa could be cultivated in bacteria-free media of known compositions. For example, Pringsheim (1937) observed in Polytoma uvella (Fig. 113, h) that sodium acetate is needed from which the starch among others is produced and carbohydrates have no direct bearing upon the nutrition, but fatty acids derived from them participate in the metabolism. The Protozoa which live within the body of another organism are PHYSIOLOGY 109 able to nourish themselves by absorbing the digested or decomposed substances of the host and could be considered assaprozoic, though the term parasitic has sometimes been used. Coelozoic Protozoa be- long to this group, as for example, Protociliata, astomatous ciliates, Trypan osomatidae, 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 endo commensals, {Endamoeba blattae, Entamoeba coli, etc.) employ, of course, the holo- zoic nutrition. Many Protozoa nourish themselves by more than one method at the same or different times, subject to a change in external condi- tions. This is sometimes referred to as mixotrophic nutrition (Pfeif- fer). For example, Euglena gracilis, according to Zumstein (1900), Lwoff (1932) and Pringsheim and Hovasse (1948), loses its green coloration in the darkness 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 holo- phytic 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. 39), bacteria-feeders are tailed (50-75^ long), saprozoic forms are fusiform to ovoid (30-70/x long), forms feeding on sterile dead ciliates are fusiform (60-80^ 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. It is well known that Protozoa as any other organism, show atypical or abnormal morphological and physiological peculiarities. In the 110 PROTOZOOLOGY case of carnivorous forms, the condition of food organisms may pro- duce abnormalities in them, as was shown by Beers (1933) in Didi- nium fed on starved paramecia (Fig. 40). Some thirty years ago, Robertson (1921-1927) reported that when two ciliates, Enchelys and Colpoda, are placed in a small amount of fresh culture medium, the rate of reproduction following a "lag pe- Fig. 39. 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 campylum; e, starved individual from a killed-Colpidium 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. PHYSIOLOGY 111 riod" is more than twice (up to ten times) that of a single animal in the same amount of the medium. He assumed that this acceleration was due to a certain agent or substance produced within the animal, Fig. 40. Didinium nasutum, X265 (Beers), a, normal fully grown ani- mal; b-e, abnormal organisms which were fed on starved Paramecium. which diffused into the culture medium. When more than one animal is confined in a limited amount of culture fluid, this substance is present in a higher concentration than with one animal, and an in- creased rate of division is the result. Robertson called this "allelo- catalytic result," and the phenomenon, "allelocatalysis." 112 PROTOZOOLOGY Soon a large number of observers came forward with varying re- sults — some confirmatory, others contradictory. The vast majority of these observations including Robertson's own, were carried on ciliates which were grown in association with various bacteria, and naturally, the results lacked agreement. For a review of these ob- servations too numerous to mention here, the reader is referred to Allee (1931, 1934), Mast and Pace (1938) and Richards (1941). When bacteria-free cultivation became possible for some Protozoa, it was hoped that this problem might be solved under controlled conditions. How r ever, the results still lack agreement. For example, Phelps (1935) reported that in Tetrahymena (Glaucoma), the growth rate and the maximum yield were the same between tw r o cultures: one started with 0.014 organism and the other, with 1600 organisms per ml. Thus there was no allelocatalysis. On the other hand, Mast and Pace (1938) noted a significant acceleration of the growth rate in Chilomonas when up to 50 organisms were inoculated into 0.4 cc. of culture fluid as compared to the growth rate in cultures with one or more Chilomonas inocula, and furthermore, a single Chilomonas showed an increased rate of reproduction as the volume of the culture fluid was reduced. Various aspects of metabolic processes in Protozoa such as inor- ganic requirements, carbon and nitrogen metabolism, growth fac- tors, vitamins, etc., have recently been studied by a number of in- vestigators. For information, the reader is referred to Hall (1941) and Lwoff (1951). 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 glycogenous substances, 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. Gohre (1943) considers it a stabilized polymerization product of glycogen. Rumjantzew and Wermel (1925) demonstrated glycogen in Ac- tinosphaerium. In the cysts of Iodamoeba, glycogen body is con- PHYSIOLOGY 113 spicuously present and is looked upon as a characteristic feature of the organism. The iodinophile vacuole of the spores of Myxobolidae is a well-defined vacuole containing glycogenous substance and is also considered as possessing a taxonomic value. In many ciliates, both free-living (Paramecium, Glaucoma, Vorticella, Stentor, etc.) and parasitic (Ophryoscolecidae, Nyctotherus, Balantidium (Faure- Fremiet and Thaureaux, 1944)), glycogenous bodies are always present. According to MacLennan (1936), the development of the paraglycogen in Ichthyophthirius is associated with the chondrio- somes. In Eimeria tenella, glycogenous substance does apparently not occur in the schizonts, merozoites, or microgametocytes ; but becomes apparent first in the macrogametocyte, and increases in amount with its development, a small amount being demonstrable in the sporozoites (Edgar et al., 1944). c Fig. 41. a-d, two types of paramylon present in Euglena gracilis (Btitschli); e-h, paramylon of E '. sanguinea, X1100 (Heidt). (e, natural appearance; f, g, dried forms; h, strongly pressed body.) 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. 41) is spirally coiled which confirms Butschli's observation. The paramylon appears to be a polysac- 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 spheroid bodies which are known as leucosin, probably a carbohydrate which when boiled in water stains with iodine. Oil 114 PROTOZOOLOGY droplets occur in various Protozoa and when there is a large number of oil-producing forms in a body of water, the water may develop various odors as indicated in Table 4. 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, fishy (Whipple, 1927) Pelomyxa ripe cucumber (Schaeffer, 1937) Fats occur widely in Protozoa. They appear usually as small re- fractile globules. Zingher (1934) found that in the Sarcodina and Ciliata he studied, each species showed morphological characteristics of the fatty substance it contained. Fat globules occur abundantly in Amoeba and Pelomyxa which are easily seen by staining with Sudan III. In Tillina canalifera, fat droplets, 1-2/x in diameter, are present especially in the region to the right of the cytopharynx (Turner, 1940). According to Panzer (1913), the fat content of Eimeria gadi was 3.55 per cent and Pratje (1921) reports that 12 per cent of the dry matter of Noctiluca scintillans appeared to be the fatty substance present in the form of granules and is said to give luminescence upon mechanical or chemical stimulation. But the chemical nature of these "photogenic" granules is still unknown at present (Harvey, 1952). A number of other dinoflagellates, such as Peridinium, Ceratium, Gonyaulax, Gymnodinium, etc., also emit luminescence. 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. Luminescence in Protozoa (Harvey, 1952). Another reserve food-stuff which occurs widely in Protozoa, ex- 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 PHYSIOLOGY 115 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. 42) 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. 114, c, d), Doflein (1918) showed that an addition of sodium phos- phate resulted in an increase of volutin. Reichenow, Schumacher^ Fig. 42. 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. and others, hold that the volutin appears to be a free nucleic acid, and is a special reserve food material for the nuclear substance. Sas- 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. Volutin (Jirovec, 1926). Starvation. As in all living things, when deprived of food, Protozoa perish sooner or later. The changes noticeable under the microscope are: gradual loss of cytoplasmic movement, increasing number of vacuoles and their coalescence, and finally the disintegration of the body. In starved Pelomyxa carolinensis, Andresen and Holter (1945) noticed the following changes: the animals disintegrate in 10-25 days at 22°C. ; body volume decreases particularly during the early days of starvation and is about 20-30 per cent of the initial volume at the time of death; food vacuoles are extruded from the body in 24 to 48 hours; the cytoplasm becomes less viscous and many fluid vacuoles make their appearance; crystals and refringent bodies en- closed within vacuoles, form large groups as the vacuoles coalesce, some of which are extruded from the body; crystals and refringent bodies remain approximately constant during starvation and there 116 PROTOZOOLOGY is no indication that they are utilized as food reserves. The ratio of reduced weight and volume and the specific gravity remain reason- ably constant during starvation (Zeuthen, 1948). Andresen (1945) found starved Amoeba proteus to show a similar change on the whole, except that the number of chondriosomes decreased and in some cases dissolution of crystals occurred just before disintegration. 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 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 hydrogen 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 (1905) dem- onstrated 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 paraglycogen 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 PHYSIOLOGY 117 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 Paramedian caudatum is said to consume in one hour at 21°C. from 0.0052 c.c. (Kalmus) to 0.00049 c.c. (Howland and Bern- stein) of oxygen. The oxygen consumption of this ciliate in heavy suspensions (3X10 3 to 301 X10 3 in 3 c.c.) and associated bacteria, ranged, according to Gremsbergen and Reynaerts-De Pont (1952), from 1000 to 4000 nM 3 per hour per million individuals at 23.5°C. The two observers considered that P. caudatum possesses a typical cytochrome-oxidase system. Amoeba proteus, according to Hulpieu (1930), succumbs 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 proteus consumes 1.4 X10~ 3 mm 3 of oxygen per hour, while an enucleated amoeba only 0.2X10 -3 mm 3 . He suggests that "the oxygen-carriers concerned with 70 per cent of the normal respiration of an amoeba are related in some way to the presence of the nu- cleus." In Pelomyxa caroUnensis, the rate of oxygen consumption at 25°C. was found by Pace and Belda (1944) to be 0.244+0.028 mm 3 per hour per mm 3 cell substance and does not differ greatly from that of Amoeba proteus and Actinosphaervum eichhorni. The tem- perature coefficient for the rate of respiration is nearly the same as that in Paramecium, varying from 1.7 at 15-25°C. to 2.1 at 25-35°C. Pace and Kimura (1946) further note in Pelomyxa caroUnensis that carbohydrate metabolism is greater at higher than at lower tem- perature and that a cytochrome-cytochrome oxidase system is the mechanism chiefly involved in oxidation of carbohydrate. 118 PROTOZOOLOGY The Hypermastigina of termites are killed, according to Cleve- land (1925), when the host animals are kept in an excess of oxygen. Jahn found that Chilomonas paramedian in bacteria-free cultures in heavily buffered peptone-phosphate media at pH 6.0, required for rapid growth carbon dioxide which apparently brings about a favor- able intracellular hydrogen-ion concentration. Respiratory metabo- lism (Meldrum, 1934; Jahn, 1941). Excretion and secretion The catabolic waste material composed of water, carbon dioxide, and nitrogenous compounds, all of which are soluble, pass out of the body by diffusion through the surface or by means of the contractile vacuole (p. 83). The protoplasm of the Protozoa is generally con- sidered to possess a molecular make-up which appears to be similar among those living in various habitats. In the freshwater Protozoa the body of which is hypertonic to surrounding water, the water diffuses through the body surface and so increases the water content of the body protoplasm as to interfere with its normal function. The contractile vacuole, which is invariably present in all freshwater forms, is the means of getting rid of this excess water from the body. On the other hand, marine or parasitic Protozoa live in nearly iso- tonic media and there is no excess of water entering the body, hence the contractile vacuoles are not found in them. Just exactly why nearly all euciliates and suctorians possess the contractile vacuole regardless of habitat, has not fully been explained. It is assumed that the pellicle of the ciliate is impermeable to salts and slowly permeable to water (Kitching, 1936) or impermeable to water, salts and prob- ably gases (Frisch, 1937). If this is the case with 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 and of Yo- com (1934) on Paramecium caudatum and Euplotes patella, the con- tractile vacuoles of which nearly ceased functioning when the ani- mals were placed in 10 per cent sea water. Furthermore, marine amoebae develop contractile vacuoles de novo when they are trans- planted to fresh water as in the case of Vahlkampfia calkinsi (Hogue, 1923) and Amoeba biddulphiae (Zuelzer, 1927). Herfs (1922) studied PHYSIOLOGY 119 the pulsation of the contractile vacuoles of Paramecium caudatum in fresh water as well as in salt water and obtained the following meas- urements: 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 number of the contractile vacuoles present in a species is con- stant under normal conditions. The contraction period varies from a few seconds to several minutes in freshwater inhabitants, and is, as a rule, considerably longer in marine Protozoa. Kitching (1938a) estimated that a quantity of water equivalent to the body volume is eliminated by freshwater Protozoa in four to 45 minutes and by marine forms in about three to four hours. The size of contractile vacuole in diastole may vary. Botsford (1926) reported that the con- tractile vacuole in Amoeba proteus varied considerably within a short period of time in size and rate of contraction under seemingly identi- cal conditions. The rate of contraction is subject to change with the temperature, physiological state of the organism, amount of food substances, etc. For example, Rossbach noted in the three ciliates listed below, the contraction was accelerated first rapidly and then more slowly with rise of the temperature: 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 cucidlulus 9 7 5 4 4 — How much water enters through the body surface of Protozoa is not known, but it appears to be the major portion that is excreted through contractile vacuoles. Water also enters the protozoan body in food vacuoles. In Vampyrella lateritia which feeds on the cell con- tents of Spirogyra in a single feeding, many contractile vacuoles ap- pear within the cytoplasm and evacuate the Avater that has come in with the food (Lloyd, 1926) and the members of Ophryoscolecidae show an increased number and activity of contractile vacuoles while feeding (MacLennan, 1933). The amount of water contained in food vacuoles seems, however, to be far smaller than the amount evacu- ated by contractile vacuoles (Gelei, 1925; Eisenberg, 1925). Other evidences such as the contractile vacuole continues to pulsate when cytosome-bearing Protozoa are not feeding and its occurrence in automatons ciliates, would indicate also that the water entering 120 PROTOZOOLOGY through this avenue is not of a large quantity. How much water is produced during the metabolic activity of the organisms is un- known, but it is considered to be a very small amount (Kitching, 1938). The mechanism by which the difference in osmotic pressure can be maintained at the body surface is unknown. It may be, as suggested by Kitching (1934), that the contractile vacuole extrudes water but retains the solutes or some osmotically active substances must be continuously produced within the body. Attempts to detect catabolic products in the contractile vacuole, in the body protoplasm or in the culture fluid, were unsuccessful, be- cause of technical difficulties. Weatherby (1927) detected in the Fig. 43. Examples of crystals present in Protozoa, a-e, in Paramecium caudatum (Schewiakoff), (a-d, X1000, e, X2600); f, in Amoeba protetis; g, in A. discoides; h-1, in A. dubia (Schaeffer). spring water in which he kept a number of thoroughly washed Para- mecium, urea and ammonia after 30-36 hours and supposed that the urea excreted by the organisms gave rise to ammonia. He found also urea in similar experiments with Spirostomum and Didinium (Weatherby, 1929). Doyle and Harding (1937) found Glaucoma ex- creting ammonia, and not urea. Carbon dioxide is obviously ex- creted by the body surface as well as the contractile vacuole. At present the composition of the fluid in the contractile vacuole is not know 7 n. General reference (Weatherby, 1941); permeability of water in Protozoa (Belda, 1942; L0vtrup and Pigon, 1951); physiology of contractile vacuole (Stempell, 1924; Fortner, 1926; Gaw, 1936; Kitching, 1938a). Aside from the soluble forms, there often occur in the protozoan body insoluble substances in the forms of crystals and granules of various kinds. Schewiakoff (1894) first noticed that Paramecium often contained crystals (Fig. 43) composed of calcium phosphate, which disappeared completely in 1-2 days when the organisms were starved, and reappeared when food was given. Schewiakoff did not see the extrusion of these crystals, but considered that these crystals PHYSIOLOGY 121 were first dissolved and excreted by the contractile vacuoles, as they were seen collected around the vacuoles. When exposed to X-irradi- ation, the symbiotic Chlorella of Paramecium bursaria disappear gradually and crystals appear and persist in the cytoplasm of the ciliate (Wichterman, 1948a). These crystals varying in size from a few to 12m, are found mainly in the posterior region of the body. Wichterman notes that the appearance or disappearance of crystals seems to be correlated with the absence or presence of symbiotic Chlorella and with the holozoic or holophytic (by the alga) nutrition of the organism. In Amoeba proteus, Schubotz (1905) noted crystals of calcium phosphate which were bipyramidal or rhombic in form, were doubly refractile and measured about 2-5m in length. In three species of Amoeba, Schaeffer (1920) points out the different shape, number and dimensions of the crystals. Thus in Amoeba proteus, they are truncate bipyramids, rarely flat plates, up to 4.5m long; in A. discoides, abun- dant, truncate bipyramids, up to 2.5m long; and in .4. dubia, vari- ously shaped (4 kinds), few, but large, up to 10m, 12m, 30m long (Fig. 43). Bipyramidal or plate-like crystals are especially abundant in Pelom.yxa illinoisensis at all times (Kudo, 1951); the crystals of P. carolinensis remain the same during the starvation of the organism (Andresen and Holter, 1945; Holter, 1950). The crystals present in Protozoa appear to be of varied chemical nature. 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 which occur in many haemosporidians and which appear to be com- posed of a derivative of the haemoglobin of the infected erythrocyte (p. 605). In certain Radiolaria, there occurs a brownish amorphous mass which is considered as catabolic waste material and, in Foram- 122 PROTOZOOLOGY inifera, 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 Foraminifera, Heliozoa, Radiolaria, certain parasitic ciliates, etc., the extracellular secretions are easily recognizable as loricae, shells, envelopes, stalks, collars, mucous substance, etc. Furthermore, many Protozoa secrete, as was stated before, certain substances through the pseudopodia, tentacles or trichocysts 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 Butschli (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 due 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. 44, 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 in rotation as long as the amoeba continues to progress." Thus Amoeba verrucosa may be compared with an elastic sac filled with fluid. Dellinger (1906) studied the movement of Amoeba proteus, A. verrucosa and Difflugia spiralis. Studying in side view, he found that the amoeba (Fig. 45) extends a pseudopod, "swings it about, PHYSIOLOGY 123 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. Dellinger held thus that "the movements of amoebae are due to the presence Fig. 44. 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. of a contractile substance," which was said to be located in the endo- plasm as a coarse reticulum. Wilber (1946) pointed out that Pelo- myxa carolinensis carries on a similar movement at times. Fig. 45. Outline sketches of photomicrographs of Amoeba protexis during locomotion, as viewed from side (Dellinger). In the face of advancement of our knowledge on the nature of protoplasm, Rhumbler (1910) realized the difficulties of the surface tension theory and later suggested that the conversion of the ecto- plasm to endoplasm and vice versa were the cause of the cytoplasmic 124 PROTOZOOLOGY movements, which was much extended by Hyman (1917). Hyman considered 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. 44, 6) 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. 46). 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- lemma at this end (Fig. 47, a), but at other times it is closed by a thin sheet of gel which prevents the plasmasol from reaching the PHYSIOLOGY 12.5 Fig. 46. Diagram of Amoeba proteus, showing the solation and gelation ot the cytoplasm during amoeboid movement (Mast), c, crystal: cv con- tractile vacuole; f food vacuole; he, hyaline cap; n, nucleus; pg plasma- gel; pgs, plasmagel sheet; pi, plasmalemma; ps, plasmasol 126 PROTOZOOLOGY 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. 47. 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." The rate of amoeboid locomotion appears to be influenced by en- vironmental factors such as pH, osmotic pressure, salt concentration, substratum, temperature, etc. (Mast and Prosser, 1932). 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. 53) 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- PHYSIOLOGY 127 lum undergoes a series of lateral movements and, in so doing, a pres- 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 always 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 trichophorum 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. Krijgsman (1925) studied the movements of the long flagellum of Monas sp. (Fig. 48) which he found in soil cultures, under the dark- field microscope and stated: (1) when the organism moves forward with the maximum speed, the flagellum starting from c 1, 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 undergoes 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 passse 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 128 PROTOZOOLOGY permanently in the ciliates and vary in size and distribution among different species. Just as flagellates show various types of move- ments, so do the ciliates, though nearly all free-swimming forms swim in a spiral path (Bullington, 1925, 1930). Individual cilium on a Fig. 48. Diagrams illustrating flagellar 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 flagellum in backward movement; p, lateral movement; q, turning movement. PHYSIOLOGY 129 progressing ciliate bends throughout its length and strikes the water so that the organism tends to move in a direction opposite to that of the effective beat, while the water moves in the direction of the beat (Fig. 49, a-d). In the Protociliata and the majority of holotrichous and heterotrichous ciliates, the cilia 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 /" "^ 7 1 2 j '5 4 W/^mr^ll ^gc Fig. 49. 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. cilium (Fig. 49, e) in a single row is slightly in advance of the cilium behind it and slightly 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 Opalina among others, which is much like the waves passing over a wheat field on a windy day. The organized movements of cilia, cirri, membranellae and un- dulating membranes are probably controlled by the neuromotor system (p. 63) which appears to be conductile as judged by the results of micro-dissection experiments of Taylor (p. 65). Ciliary movement (Gray, 1928) ; spiral movement of ciliates (Bullington, 1925, 1930); movement of Paramecium (Dembowski, 1923, 1929a) and of Spirostomum (Blattner, 1926). The Protozoa which possess myonemes are able to move by con- 130 PROTOZOOLOGY 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 stimuli vary under dif- 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 only will 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 protozoans 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 ciliates 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 differently. Irri- tability (Jennings, 1906; Mast, 1941); in Spirostomum (Blattner, 1926). 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. 50, a), the amoe- boid movements proceeded on both sides of the filament (6), but soon motion ceased on one side, while it continued on the other, and PHYSIOLOGY 131 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 (rf), 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 Fig. 50. 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. (h). Positive reactions toward solid bodies account of course for the ingestion of food particles. 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 solid object, the response may be either negative or positive. In the former case, avoiding movement (Fig. 51, 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. Reaction to gravity. The reaction to gravity varies among dif- ferent Protozoa, according to body organization, locomotor organ- elle, etc. Amoebae, Testacea and others which are usually found attached to the bottom of the container, react as a rule positively 132 PROTOZOOLOGY 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- edly influenced by various factors (Koehler, 1922, 1930; Dembowski, 1923, 1929, 1929a; Merton, 1935). Reaction to current. Free-swimming Protozoa appear to move or orientate themselves against the current of water. In the case of Fig. 51. 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. 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. 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 within the PHYSIOLOGY 133 area of a drop of 0.02 per cent acetic acid introduced to the prepara- tion (Fig. 51, a); and if stronger acid is used, the organisms collect about its periphery where the acid is diluted by the surrounding water (b) . The reaction to chemical stimuli is probably of the great- est importance for the existence of Protozoa, since it leads them to proper food substances, the ingestion of which is the foundation of metabolic activities. In the case of parasitic Protozoa, possibly the reaction to chemical stimuli results in their finding specific host ani- mals 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. Chemotaxis in Peranema (Chen, 1950). 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- teus 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. Pelomyxa carolinensis reacts negatively to light (Kudo, 1946). 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. 229). Horvath (1950) exposed Kahlia sim- plex to ultraviolet rays and destroyed the micronucleus. The emi- cronucleate individuals lived and showed a greater vitality than nor- mal individuals, as judged by the division rate at 34°C. Mazia and 134 PROTOZOOLOGY Hirshfield (1951) subjected Amoeba proteus to ultraviolet radiation and noticed that irradiation of the whole and nucleated half amoebae delays division immediately following exposure; later progeny of the irradiated amoebae have a normal division rate; amputation of half of the cytoplasm greatly increases the radiation sensitivity as meas- ured by delayed division or by the dose required for permanent in- hibition of division (sterilization dose) ; individuals that have re- ceived this dose may survive for 20-30 days; and the survival time of an enucleate fragment is very much reduced by small (200-500 ergs/sq. mm) doses. The two workers consider that the overall radia- tion effect may have both nuclear and cytoplasmic components. By exposing Pelomyxa carolinensis to 2537 A ultraviolet irradiation, Wilber and Slane (1951) found the effects variable; however, all re- covered from a two minutes' exposure, none survived a 10-minute exposure, and 70 per cent of fat were released after two minutes' exposure. Zuelzer (1905) found the effect of radium rays upon various Pro- tozoa vary; in all cases, a long exposure was fatal to Protozoa, the first effect of exposure being shown by accelerated movement. Hal- berstaedter and Luntz (1929, 1930) studied injuries and death of Eudorina elegans by exposure to radium rays. Entamoeba histolytica in culture when subjected to radium rays, Nasset and Kofoid (1928) noticed the following changes: the division rate rose two to four times by the exposure, which effect continued for not more than 24 hours after the removal of the radium and was followed by a re- tardation of the rate; radium exposure produced changes in nuclear structure, increase in size, enucleation or autotomy, which were more striking when a larger amount of radium was used for a short time than a smaller amount acting on for a long time; and the effects persisted for four to six days after the removal of the radium and then the culture gradually returned to normalcy. Halberstaedter (1914) reported that when exposed to Beta rays, Trypanosoma brucei lost its infectivity, though remained alive. Halberstaedter (1938) exposed Trypanosoma gambiense to X-rays and found that 12,000r rendered the organisms not infectious for mice, while 600,000r was needed to kill the flagellates. Emmett (1950) exposed T. cruzi to X-rays and noticed that dosages between 51,000r and 100,000r were necessary to destroy the infectivity of this trypanosome; the cultures, after exposure to 100,000r, appeared to be thriving up to three months; and the effects of exposure were not passed on to new generations. When Paramecium bursaria were exposed to X-rays, Wichterman PHYSIOLOGY 135 (1948) noted: dosages higher than 100,0Q0r retard the locomotion of the ciliate; none survives 700,000r; the symbiotic Chlorella is de- stroyed by exposure to 300,000-000,000?' ; irradiation inhibits di- division temporarily, but the animals recover normal division rate after certain length of time; and mating types are not destroyed, though minor changes occur. In Pelomyxa carolinensis, Daniels (1951) observed: the median lethal dose of X-rays is 96,000r; with dosages 15,000-140,000r, the first plasmotomy is greatly delayed and the second plasmotomy is also somewhat delayed, but later plas- motomies show complete recovery; X-irradiation does not change the type of plasmotomy; and in individuals formed by plasmogamy of X-irradiated halves to non-irradiated halves, the nuclei divide simultaneously as in a normal individual. Reaction to temperature stimuli. As was stated before, there seems to be an optimum temperature range for each protozoan, 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. In the case of parasitic Protozoa which inhabit two hosts: warm-blooded and cold- blooded animals, such as Plasmodium and Leishmania, the difference in body temperature of host animals may bring about specific stages in their development. Reaction to electrical stimuli. Since Verworn's experiments, several investigators studied the effects of electric current which is passed through Protozoa in water. Amoeba shows negative re- action to the anode and moves toward the cathode either by revers- ing the cytoplasmic streaming (Verworn) or by turning around the body (Jennings). The free-swimming ciliates move mostly toward the cathode, but a few may take a transverse position (Spirostomum) or swim to the anode (Paramecium, Stentor, etc.). 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Protist., 86:404. Mast, S. O. : (1923) Mechanics of locomotion in amoeba. Proc. Nat. Acad. Sc, 9:258. PHYSIOLOGY 141 — (1926) Structure, movement, locomotion, and stimulation in amoeba. J. Morphol. Physiol., 41:347. — (1931) Locomotion in Amoeba proteus. Protoplasma 14:321. — (1938) Digestion of fat in Amoeba proteus. Biol. Bull., 75: 389. — ■ (1939) The relation between kind of food, growth and struc- ture in Amoeba. Ibid., 77:391. — (1941) Motor response in unicellular animals. In: Calkins and Summers (1941). — (1942) The hydrogen ion concentration of the content of the food vacuoles and the cytoplasm in Amoeba, etc. Biol. Bull., 83 : 173. — and Doyle, W. L.: (1934) Ingestion of fluid by amoeba. Pro- toplasma, 20:555. (1935) Structure, origin and function of cytoplasmic constituents in Amoeba proteus. I. Arch. Protist., 86:155. (1935a) II. Ibid., 86:278. — and Pace, D. M.: (1938) The effect of substances produced by Chilomonas Paramecium on the rate of reproduction. Physiol. 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Phelps, A.: (1935) Growth of Protozoa in pure culture. I. J. Exper. Zool., 70:109. Powers, P. B. A.: (1932) Cyclotrichium meunieri, etc. Biol. Bull., 63: 74. Pratje, A.: (1921) Makrochemische, quantitative Bestimmung des Fettes und Cholesterins, sowie ihrer Kennzahlen bei Noctiluca miliaris. Biol. Zentralbl., 41:433. Pringsheim, E. G. : (1923) Zur Physiologie der saprophytischer Flagellaten. Beitr. allg. Bot., 2:88. (1928) Physiologische Untersuchungen an Paramecium bur- saria, Arch. Protist., 64:289. (1937) Beitrage zur Physiologie der saprophytischer Algen und Flagellaten. I. Planta, 26:631. - (1937a) Algenreinkulturen. Arch. Protist., 88:143. and Hovasse, R. : (1948) The loss of chromatophores in Euglena gracilis. The New Phytologist., 47:52. Putter, A.: (1905) Die Atmung der Protozoen. Ztschr. allg. Phy- siol., 5:566. (1908) Methoden zur Forschung des Lebens der Protisten. Tigerstedt's Handb. physiol. Methodik., 1:1. Ray, D. 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(1924b) Allelocatalytic effect in cultures of Colpidium in hay infusion and in synthetic media. Biochem. J., 18:1240. — (1927) On some conditions affecting the viability of Infusoria PHYSIOLOGY 143 and the occurrence of allelocatalysis therein. Australian J. Ex- per. Biol.. 4:1. Roskin, G. : (1925) Ueber die Axopodien der Heliozoa und die Greiftentakel der Ephelotidae. Arch. Protist., 52:207. and Levinsohn, L. : (1926) Die Oxydasen und Peroxydasen bei Protozoen. Ibid., 56: 145. Rumjantzew, A. and Wermel, E.: (1925) Untersuchungen ueber den Protoplasmabau, etc. Arch. Protist., 52:217. Sassuchin, D. N.: (1935) Zum Studium der Protisten- und Bakte- rienkerne. I. Ibid., 84:186. Schaeffer, A. A.: (1920) Amoeboid movement, Princeton. Schewiakoff, W. : (1894) Ueber die Natur der sogennannten Ex- kretkorner der Infusorien. Ztschr. wiss. Zool., 57:32. Schulze, K. L. : (1951) Experimentelle Untersuchungen ueber die Chlorellen-symbiose bei Ciliaten. Biol. Gen., Vienna, 19:281. Seaman, G. R. and Houlihan, R. K. : (1951) Enzyme systems in Tetrahymena geleii S. II. J. Cell. Comp. Physiol., 37:309. Shapiro, N. N.: (1927) The cycle of hydrogen-ion concentration in the food vacuoles of Paramecium, Vorticella, and Stylonychia. Tr. Am. Micr. Soc, 46:45. Soule, M. H.: (1925) Respiration of Trypanosoma lewisi and Leish- mania tropica. J. Infect. Dis., 36:1245. Specht, H. : (1934) Aerobic respiration in Spirostomum ambiguum, etc. J. Cell Comp. Physiol., 5:319. Stempell, W. : (1924) Weitere Beitrage zur Physiologie der pul- sierenden Vakuole von Paramecium. I. Arch. Protist., 48:342. Stolc, A.: (1900) Beobachtungen und Versuche ueber die Ver- dauung und Bildung der Kohlenhydrate bei einen amoebenarti- gen Organismen, Pelomyxa palustris. Ztschr. wiss. Zool., 68: 625. Tanabe, M. and Komada, K. : (1932) On the cultivation of Balan- tidium colt. Keijo J. Med., 3:385. Taylor, C. V.: (1923) The contractile vacuole in Euplotes, etc. J. Exper. Zool., 37:259. Trager, W. : (1932) A cellulase from the symbiotic intestinal flagel- lates of termites, etc. Biochem. J., 26: 1762. Turner, J. P.: (1940) Cytoplasmic inclusions in the ciliate Tillina canalifera. Arch. Protist., 93:255. Veley, Lilian J.: (1905) A further contribution to the study of Pelomyxa palustris. J. Linn. Soc. Zool., 29:374. Verworn, M.: (1889) Psycho-physiologische Protisten-Studien. Jena. (1903) Allgemeine Physiologie. 4 ed. Jena. Weatherby, J. H. : (1927) The function of the contractile vacuole in Paramecium caudatum. Biol. Bull., 52:208. (1929) Excretion of nitrogenous substances in Protozoa. Physiol. Zool., 2:375. (1941) The contractile vacuole. In: Calkins and Summers (1941). 144 PROTOZOOLOGY Weineck, E. : (1934) Die Celluloseverdauung bei den Ciliaten des Wiederkauermagens. Arch. Protist., 82:169. Whipple, G. C: (1927) The microscopy of drinking water. 4th ed. New York. Wichterman, R. : (1948) The biological effects of X-rays on mating types and conjugation of Paramecium, bursaria. Biol. Bull., 94: 113. (1948a) The presence of optically active crystals in Para- mecium bursaria and their relationship to symbiosis. Anat. Rec, 101:97. Wilber, C. G.: (1945) Origin and function of the protoplasmic con- stituents in Pelomyxa carolinensis. Biol. Bull., 88:207. — — (1945a) The composition of the refractive bodies in rhizopod, etc. Tr. Am. Micr. Soc, 64:289. — (1946) Notes on locomotion in Pelomyxa carolinensis. Ibid., 65:318. and Slane, Gertrude M.: (1951) The effect of ultraviolet light on the protoplasm in Pelomyxa carolinensis. Ibid., 70:265. Yocom, H. B.: (1934) Observations on the experimental adaptation of certain freshwater ciliates to sea water. Biol. Bull., 67:273. Zeuthen, E. : (1948) Reduced weight and volume during starvation of the amoeba, etc. C. R. Lab. Carlesberg, Ser. Chim., 26:267. Zingher, J. A.: (1934) Beobachtungen an Fetteinschliissen bei ei- nigen Protozoen. Arch. Protist., 82:57. Zuelzer, M.: (1905) Ueber die Einwirkung der Radiumstrahlen auf Protozoen. Ibid., 5:358. (1907) Ueber den Einfiuss des Meerwassers auf die pul- sierende Vacuole. Berlin. Sitz.-Ber. Ges. naturf. Freunde, p. 90. (1927) Ueber Amoeba biddulphiae, etc. Arch. Protist., 57: 247. Zumstein, H.: (1900) Zur Morphologie und Physiologie der Euglena gracilis. Pringsheims Jahrb. wiss. Botanik., 34:149. 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 nearly 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. 165), 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. 52), 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. Furthermore, in some cases the nuclear components may undergo phase reversal, that is to say, the chromatin granules which are dis- persed phase in the non-staining fluid dispersion medium in the rest- ing nucleus, become dispersion medium in which the latter is sus- pended as dispersed phase. By using Feulgen's nucleal reaction, Reichenow (1928) demonstrated this reversal phenomenon in the di- vision of the macronucleus of Chilodonella cucullulus (Fig. 53). 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 occur, the type difference and certain other characters, according to 145 146 PROTOZOOLOGY 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. 52. Nuclear and cytoplasmic 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 REPRODUCTION 147 the macronucleus of Nyctotherus ovalis probably produce smaller spherules in their alveoli (Fig. 3). When the macronucleus is elongated as in Spirostomum, Stentor. Euplotes, etc., the nucleus becomes condensed into a rounded form prior to its division. During the "shortening period" of the elongated macronuclei prior to division, there appear 1-3 characteristic zones which have been called by various names, such as nuclear clefts, reconstruction bands, reorganization bands, etc. In Euplotes patella Fig. 53. The solation of chromatin during the macronuclear division of Chilodonella cucullulus, as demonstrated by Feulgen's nucleal reaction, Xl800(Reichenow). (E. eury stomas) , Turner (1930) observed prior to division of the macronucleus a reorganization band consisting of a faintly staining zone ("reconstruction plane") and a deeply staining zone ("solution plane"), appears at each end of the nucleus (Fig. 54, a) and as each moves toward the center, a more chromatinic area is left behind (b-d). The two bands finally meet in the center and the nucleus as- sumes 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 148 PROTOZOOLOGY moving along the left arm to its tip, and the other entering and pass- ing down the stem to join the posterior band. According to Summers (1935) a process similar to that of E. eurystomus occurs in Diophrys appendiculata and Stylonychia pustulata; but in Aspidisca lynceus (Fig. 55) a reorganization band appears first near the middle region of the macronucleus (6), divides into two and each moves toward an end, leaving between them a greater chromatinic content of the 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." Weisz (1950a) finds that the nodes of the moniliform macro- Fig. 54. Macronuclear reorganization before division in Euplotes eurystomus, X240 (Turner), a, reorganization band appearing at a tip of the macronucleus; b-d, later stages. nucleus of Stentor coeruleus contain different concentration of thymo- nucleic acid which is correlated with morphogenetic activity of indi- vidual nodes, and that fusion of ill-staining nodes results in a return of strong affinity to methyl green. It appears, therefore, concentra- tion of bandform or moniliform macronucleus prior to division may serve to recover morphogenetic potential prior to division. In a small number of ciliates, the macronucleus is distributed as small bodies throughout the cytoplasm. In Urostyla grandis, the macronuclear material is lodged in 100 or more small bodies scat- tered in the cytoplasm. Prior to fission, all macronuclear bodies fuse with one another and form one macronucleus which then divides three times into eight and the latter are evenly distributed between the two daughter individuals, followed by divisions until the number reaches 100 or more (Raabe, 1947). On the other hand, in Dileptus REPRODUCTION 149 anser (Fig. 310, 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. Fig. 55. 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. 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 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 150 PROTOZOOLOGY that during the nuclear division there appears and persists a small body within the nuclear figure, located at the division plane as in the case of Loxocephalus (Behrend), Eupoterion (MacLennan and Connell) and even in the widely different protozoan, Endamoeba blattae (Kudo, 1926). Kidder (1933) observed that during the division of the macronucleus of Conchophthirus my till (Fig. 56), the nucleus "casts out a part of its chromatin at every vegetative division," which "is broken down and disappears in the cytoplasm of either Fig. 56. Macronuclear division in Conchophthirus mytili, X440 (Kidder). 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), Allosphaerium convexa (Kidder and Summers), Colpoda inHata, C. maupasi, Tillina canalifera, Bresslaua vorax, etc. (Burt et al., 1941). Beers (1946) noted chromatin extrusion from the macro- nucleus during division and in permanent cysts in Tillina magna. What is the significance of this phenomenon? Kidder and his associ- ates believe that the process is probably elimination of waste sub- stances of the prolonged cell-division, since chromatin extrusion does not take place during a few divisions subsequent to reorganization REPRODUCTION 151 after conjugation in Conchophthirus mytili and since in Colpidium and Glaucoma, the chromatin elimination appears to be followed by a high division rate and infrequency of conjugation. Dass (1950) noticed a dark body between two daughter macronuclei of a ciliate designated by him as Glaucoma piriformis and considered it as sur- plus desoxyribonucleic acid about to be converted by the cytoplasm to ribonucleic acid necessary for active growth. In Paramecium aurelia, Woodruff and Erdmann (1914) reported the occurrence of "endomixis." At regular intervals of about 30 days, the old macronucleus breaks down and disappears, while each of the two micronuclei divides twice, forming eight nuclei. Of these, six disintegrate. The animal then divides into two, each daughter indi- vidual receiving one micronucleus. This nucleus soon divides twice into four, two of which develop into two macronuclei, while the other two divide once more. Here the organism divides again into two individuals, each bearing one macronucleus and two micronuclei. This process, they maintained, is "a complete periodic nuclear re- organization without cell fusion in a pedigreed race of Paramecium." The so-called endomixis has since been reported to occur in many ciliates. However, as pointed out by Wilson (1928), Diller (1936), Sonneborn (1947) and others, there are several difficulties in holding that endomixis is a valid process. Diller considers that endomixis may have been based upon partial observations on hemixis (p. 206) and autogamy (p. 203). Sonneborn could not find any indication that this process occurs in numerous stocks and varieties of Para- mecium aurelia, including the progeny of the strains studied by Woodruff, and maintained that endomixis does not occur in this spe- cies of Paramecium. As has been stated already, 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. 188) and autogamy (p. 203), the macronucleus becomes trans- formed, in many species, into irregularly coiled thread-like structure (Fig. 85) which undergoes segmentation into pieces and finally is absorbed by the cytoplasm. New macronuclei are produced from 152 PROTOZOOLOGY some of the division-products of micronuclei by probably incor- porating the old macronuclear material. In most cases this sup- position is not demonstrable. However, Kidder (1938) has shown in S'd © © d e f g Fig. 57. Diagram showing the macronuclear regeneration in Parame- cium aurelia (Sonneborn). a, an individual before the first division after conjugation or autogamy, containing two macronuclear (stippled) an- lagen, two micronuclei (rings) and about 30 disintegrating (solid black) masses of the old macronucleus; b, two individuals formed by the first division, each containing one macronuclear anlage, two micronuclei and macronuclear masses; c, two individuals produced by the second division: one (above) with the new macronucleus, two micronuclei and macro- nuclear masses, and the other without new macronucleus; d-f, binary fissions in which the two micronuclei divide, but old macronuclear masses are distributed equally between the two daughters until there is one large regenerated macronucleus and two micronuclei; g, division following f, goes on in an ordinary manner. 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 to form a macronuclear anlage which then develops into a macronucleus after passing through "ball-of-yarn" stage simi- lar to that which appears in an exconjugant of Nyctotherus (Fig. 85). REPRODUCTION 153 Since the macro-nucleus 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, 1947) (Fig. 57) ob- served that when the animal in conjugation is exposed to 38°C. from the time of the synkaryon-formation until before the second postzygotic nuclear division (a-c), the development of the two newly formed macronuclei is retarded and do not divide as usual with the result that one of the individuals formed by the second postzygotic division receives the newly formed macronucleus, while the other lacks this (c). In the latter, however, division continues, during which some of the original 20-40 pieces of the old macronucleus that have been present in the cytoplasm segregate in approximately equal number at each division (d, e) until there is only one in the animal (/). Thereafter the macronucleus divides at each division (g). Sonne- born found this "macronuclear regeneration" in the varieties 1 and 4, but considered that it occurs in all stocks. Thus the macronucleus in this ciliate appears to be a compound structure with its 20-40 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 nucleus composed of many "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. While the macronuclear division usually follows the micronuclear division, it takes place in the absence of the latter as seen in amicro- nucleate individuals of ciliates which possess normally a micronu- cleus. Amicronucleate ciliates have been found to occur naturally or produced experimentally in the following species: Didinium nasutum (Thon, 1905; Patten, 1921), Oxytricha hymenostoma (Dawson, 1919), O.fallax, Urostyla grandis (Woodruff , 1921), Paramecium caudatum (Landis, 1920; Woodruff, 1921), etc. Amicronucleate Oxytricha f alia x which were kept under observation by Reynolds (1932) for 29 months, showed the same course of regeneration as the normal indi- viduals. Beers (1946b) saw no difference in vegetative activity be- tween amicronucleate and normal individuals of Tillina magna. In Euplotes patella, amicronucleates arise from "double" form (p. 229) with a single micronucleus, and Kimball (1941a) found that the mioronucleus is not essential for continued life in at least some 154 PROTOZOOLOGY clones, though its absence results in a marked decrease in vigor. The bi-micronucleate Paramecium bursaria which Woodruff (1931) iso- lated, developed in the course of 7 years of cultivation, unimicronu- cleate and finally amicronucleate forms, in which no marked varia- tion in the vitality of the race was observed. These data indicate that amicronucleates are capable of carrying on vegetative activity and multiplication, but are unable to conjugate or if cell-pairing occurs, the result is abortive, though Chen (1940c) reported conjugation be- tween normal and amicronucleate individuals of P. bursaria (p. 189). Horvath (1950) succeeded in destroying the micronucleus in Kahlia simplex (p. 133) and found the emicronucleates as vigorous as the normal forms, judged by the division rate, but were killed within 15 days by proactinomycin, while normal individuals resisted by en- cystment. This worker reasons that the emicronucleates are easily destroyed by unfavorable conditions and, therefore, ciliates without a micronucleus occur rarely in nature. Fig. 58. Amitosis of the vegetative nucleus in the trophozoite of Myxosoma catostomi, X2250 (Kudo). Other examples of amitosis are found in the vegetative nuclei in the trophozoite of Myxosporidia, as for example, Myxosoma catos- tomi (Fig. 58), Thelohanellus notatus (Debaisieux), etc., in which the endosome divides first, followed by the nuclear constriction. In Streblomastix 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 a few examples will suffice. A veritable mitosis was noted by Dobell in the heliozoan Oxnerella maritima (Fig. 59), 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 REPRODUCTION 155 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 \\«m\ ymmmn ••fin Hi' '».'. ' / f'n Fig. 59. Nuclear and cytoplasmic division in Oxnerella maritime/,, X about 1000 (Dobell). a, a living individual; b, stained specimen; c-g, prophase; h, metaphase; i, anaphase; j, k, telophase; 1, division completed. passing through an equatorial-plate stage, the two groups of 24 chromosomes move toward the opposite poles (g-i). As the spindle fibers become indistinct, radiation around the centrioles becomes 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 156 PROTOZOOLOGY Stern, very similar to the above. Aside from these two species, the centriole has been reported in many others, such as Hartmannella (Arndt), Euglypha, Monocystis (Bglaf), Aggregata (Dobell; Bglaf; Fig. 60. Mitosis in Trichonympha campanula, X800 (Kofoid and Swezy). a, resting nucleus; b-g, prophase; h, metaphase; i, j, anaphase; k, telophase; 1, a daughter nucleus being reconstructed. REPRODUCTION 157 Naville), various Hypermastigina (Kofoid; Duboscq and 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- cording to Kofoid and Swezy (1919), in Trichonympha campanula (Fig. 60), 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 desmose. From the posterior end of each centriole, astral rays extend out and the split chromosomes form loops and pass through "tangled skein" stage. In the metaphase, the equatorial plate is made up of V-shaped chromosomes as each of the split chromosomes is still connected at one end, which finally becomes separate in anaphase, followed by reformation of two daughter nuclei. As to the origin and development of the achromatic figure, vari- ous observations and interpretations have been advanced. Certain Hypermastigina possess very large filiform centrioles and a large rounded nucleus. In Barbulanympha (Fig. 61), Cleveland (1938a) found that the centrioles vary from 15 to 30^ 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 30/x 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. 158 PROTOZOOLOGY Fig. 61. 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. In Holomastigotoides tusitala (Fig. 172, a, b), Cleveland (1949) brought to light the formation of the achromatic figure, and the minute structure and change in chromosomes (Fig. 62). In the late telophase, after cytoplasmic division, the centrioles follow the flagel- lar bands 4 and 5 for 1.5 turns (a). The two chromosomes are an- chored to the old centriole. When the new centriole has become as REPRODUCTION 159 a M>, Fig. 62. Mitosis in Holomastigotoides tusitala (Cleveland), a, anterior region showing flagellar bands, centrioles, centromeres and chromosomes, b-h, telophase; i, j, prophase; k, metaphase; 1, anaphase; m, telophase, b, c, new and old centrioles forming achromatic figure; d, one chromosome has shifted its connection from old to new centriole; e, f, flattening out of centrioles and achromatic figure; g, h, beginning of chromosomal twist- ing; i, chromosomes duplicated, producing many gyres of close-together relational coiling of chromatics, and centromeres duplicated; j, chroma- tids losing their relational coiling by unwinding; k, relational coiling dis- appeared, achromatic figure elongating and separating sister chromatids; 1, central spindle bent, chromatids in two groups; m, central spindle pulled apart. 160 PROTOZOOLOGY long as the old one, the centrioles begin to produce astral rays (b) which soon meet and form the central spindle (c). An astral ray from the new centriole becomes connected with the centromere of one of the chromosomes (d). The spindle grows in length and enters resting stage (e-j), later the spindle fibers lengthen (k, /) and pull apart (m). The chromosome is composed of the matrix and chromonema (Fig. 63), of which the former disintegrates in the telophase and re- appears in the early prophase of each chromosome generation, while the latter remains throughout. From late prophase to mid-telophase, minor coils are incorporated in major coils (a-c) ; from mid-telophase to late telophase, they are in very loose majors (d); and after the majors have disappeared completely, they become free (e). Soon after cytoplasmic division, the majors become looser and irregular and finally disappear, while minors and twisting remain. Each chro- mosome presently divides into 2 chromatids (f) and a new matrix is formed for each. As the matrix contracts the chromatids lose their relational coiling and the minors become bent and thus the new generation of major coils makes its appearance (g). With the further concentration of the matrix, the majors become more conspicuous (h), the minors being incorporated into them. When most of the re- lational coiling has been lost and majors are close together, the chromosomal changes cease for days or weeks. This is the late pro- phase. After the resting stage, the achromatic figure commences to grow again (i, j) and the two groups of chromatids are carried to the poles, followed by transverse cytoplasmic division (Fig. 64). The coils remain nearly the same during metaphase to early telophase. Thus Cleveland showed the continuity of chromosomes from genera- tion to generation. He finds that the resting stage of chromosomes varies in different types of cells: some chromosomes rest in inter- phase, some in early prophase and others in telophase, and that the centromere is an important structure associated with the movement of chromatids and in the reduction of chromosomes in meiosis. For fuller information the reader is referred to the profusely illustrated original paper (Cleveland, 1949). In Lophomonas blattarum, the nuclear division (Fig. 65) 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 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 REPRODUCTION 161 ?, aft Fig. 63. Chromosomal changes in Holomastigotoides tusitala, X1050 (Cleveland), a, telophase shortly after cytoplasmic division, new fifth band and new centriole are growing out and chromosomes are twisted; b, c, the same chromosome showing major and minor coils respectively; d, later telophase, showing minor coils; e, matrix completely disinte- grated, showing minor coils; f, a prophase nucleus, showing division of chromosomes into two chromatids; g, later prophase, in which majors are developing with minors; h, later prophase; i, metaphase in which distal halves of the chromatids have not yet separated, showing minor coils; j, anaphase, showing major and minor coils of chromonemata. 162 PROTOZOOLOGY Fig. 64. Cytoplasmic division in Holomastigotoides tusitala, X about 430 (Cleveland), a, fifth flagellar band has separated from others; b, one nucleus and fifth band moving toward posterior end; c, the movement of the band and nucleus has been completed; d, e, anterior and posterior daughter individuals, produced by transverse division. REPRODUCTION 163 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. In some forms, such as Noctiluca (Calkins), Actinophrys (Belaf), etc., there may appear at each pole, a structureless mass of cyto- plasm (centrosphere), but in a very large number of species there Fig. 65. Nuclear division in Lophomonas blattarum, X1530 (Kudo), a, resting nucleus; b, c, prophase; d, metaphase; e-h, anaphase; i-k, telo- phase. appear no special structures at poles and the spindle fibers become stretched seemingly between the two extremities of the elongating nuclear membrane. Such is the condition found in Pelomyxa (Kudo) (Fig. 66), Cryptomonas (Belaf), Rhizochrysis (Doflein), Aulacantha (Borgert), and in micronuclear 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 schubergi (Schaudinn), Euglena viridis (Tschenzoff), Oxyrrhis marina (Hall), 164 PROTOZOOLOGY Colacium vesiculosum (Johnson), Haplosporidium limnodrili (Gran- 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 (Belar), Acantho- cystis (Stern), Chilomonas (Doflein), Dinenympha (Kirby), etc. Fig. 66. Mitosis in Pelomyxa carolinensis, X1150 (Kudo), a, c, 1, in life; b, d-k, in acidified methyl green, a, b, resting nuclei; c-g, prophase; h, metaphase; i-k, anaphase; 1, front and side view of a young daughter nucleus. In the vegetative division of the micronucleus of Conchophthirus anodontae, Kidder (1934) found that prior to division the micronu- cleus moves out of the pocket in the macronucleus and the 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 transform them- REPRODUCTION 105 selves into two compact daughter nuclei. A detailed study of micro- nuclear division (Fig. 67) of Urostyla grandis was made by Raabe (1946). The micronucleus is a compact body in the interphase (a), Fig. 67. Micronuclear division of Urostyla grandis, X2100 (H. Raabe). a, resting stage; b-j, prophase (b-e, stages in the formation of spireme; f, g, spireme ribbon; h, i, twelve segments of ribbon arranged in the direc- tion of the elongating nuclear axis; j, a polar view of the same); k, 1, metaphase, condensation of the segments; m-o, anaphase; p, late ana- phase; q, a daughter nucleus in telophase; r-t, reconstruction stages; u, a resting daughter nucleus. 166 PROTOZOOLOGY but increases in size and the chromatin becomes grouped into small masses (6, c), which become associated into a spiral ribbon (d-g). The latter then breaks up into 12 segments that are arranged paral- lel to the axis of the elongating nucleus (h-i). Each segment con- denses into a chromosome which splits longitudinally into two (k) and the two groups of chromosomes move to opposite poles (l-P). In Zelleriella elliptica (Fig. 295) and four other species of the genus in- habiting the colon of Bufo valliceps, Chen (1936, 1948) observed the formation of 24 chromosomes, each of which is connected with a fiber of the intranuclear spindle and splits lengthwise in the meta- phase. While in the majority of protozoan mitosis, the chromosomes split longitudinally, there are observations which suggest a transverse di- vision. As examples 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 by assuming, as Hall (1937) showed in Euglena gracilis, that the splitting is not completed at once and the pulling force act- ing upon them soon after division, brings forth the long chromo- somes still connected at one end. Thus the chromosomes remain to- gether 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. Cytoplasmic division The division of the nucleus is accompanied by division of extranu- clear organelles such as chromatophores, pyrenoids, etc. The blepha- roplast of the flagellates and kinetosomes of the ciliates undergo di- REPRODUCTION Table 5. — Chromosomes in Protozoa 167 Protozoa Number of chromosomes Observers Rhizochrysis scherffeli 22 Doflein H aematococcus pluvialis 20-30 Elliott Polytomella agilis 5 Doflein Chla7?iydomonas spp. 10 (haploid) Pascher Polytoma uvella 16 (diploid) Moewus Euglena pisciformis 12-15(?) Dangeard E. viridis 30 or more Dangeard Phacus pyrum 30-40 Dangeard Rhabdomonas incurva About 12 Hall Vacuolaria virescens About 30 Fott Syndinium turbo 5 Chatton Anthophysis vegetans 8-10 Dangeard Cercomonas longicauda 4-5 Dangeard Collodictyon triciliatum About 20 Belaf Chilomastix gallinarum About 12 Boeck and Tanabe Eutrichomastix serpentis 5 Kofoid and Swezy Dinenympha fimbricata 25-30 Kirby Metadevescovina debilis About 4 Light Trichomonas tenax 3 Hinshaw T. gallinae 6 Stabler T. hominis 5 or 6 Bishop T. vaginalis 5 Hawes Tritrichomonas atigusta 5 Kofoid and Swezy 4 or 8 Kuczynski 6 Samuels T, batrachorum 4 or 8 Kuczynski 6 Bishop T. muris 6 Wenrich Hexamita salmonis 5 or 6 Davis Giardia intestinalis 4 Kofoid and Swezy G. muris 4 Kofoid and Christiansen Calonympha grassii 4 or 5 Janicki Spirotrichonympha polygyra 2 doubles Cup 2 Cleveland S. bispira 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 Barbidanympha laurabuda 40 Cleveland B. uf alula 50 Cleveland Rhynchonympha tarda 19 Cleveland Urinympha talea 14 Cleveland Staurojoenia assimilis 24 Kirby Trichony mpha campanula 52 or 26 doubles Kofoid and Swezy 168 PROTOZOOLOGY Table 5. — Continued Protozoa Number of chromosomes Observers T. grandis 22 Cleveland Plasmodiophora brassicae 8 (diploid) Terby Naegleria gruberi 14-16 Rafalko N. bistadialis 16-18 Kiihn Amoeba protevs 500-600 Liesche Endamoeba disparata About 12 Kirby Entamoeba histolytica 6 Kofoid and Swezy; Uribe E. coli 6 Swezy; Stabler 4 Liebmann E. gingivalis 5 Stabler; Noble Dientamoeba fragilis 4 Wenrich 6 Dobell Uydr amoeba hydroxena 8 Reynolds and Threlkeld Spirillina vivipara 12 (diploid) Myers Patellina corrugata 24 (diploid) Myers Pontigulasia vas 8-12 Stump Actinophrys sol 44 (diploid) Belaf Oxnerella maritima About 24 Dobell Thalassicolla nucleata 4 Belaf Aulacantha scolymantha More than 1600 Borgert 4 in gamogony Belaf Zygosoma globosum 12 (diploid) Noble Diplocystis schneideri 6 (diploid) Jameson Gregarina blattarum 6 (diploid) Sprague Nina gracilis 5 (haploid) L6ger and Duboscq Actinocephalus parvus 8 (diploid) Weschenfelder Aggregata eberthi 12 (diploid) Dobell; Belaf; Naville Merocystis kathae 6 (haploid) Patten Adelea ovata 8-10 (diploid) Greiner Adelina deronis 20 (diploid) Hauschka Orcheobius herpobdellae 10-12 Kunze Chloromyxum leydigi 4 (diploid) Naville Sphaerospora polymorpha 4 (diploid) Kudo Myxidium lieberkuhni 4 Bremer M. serotinum 4 (diploid) Kudo Sphaeromyxa sabrazesi 6 Debaisieux; Belaf 4 Naville S. balbianii 4 Naville Myxobolus pfeifferi 4 Keysselitz; Mercier; Georgevitch Protoopalina intestinalis 8 (diploid) Metcalf Zelleriella antilliensis 2(?) Metcalf Z. intermedia 24 Chen Didinium nasutum 16 (diploid) Prandtl Cyclotrichium meunieri 6 Powers REPRODUCTION Table 5. — Continued Protozoa Number of chromosomes Observers Chilodonella uncinata 4 (diploid) Enrique; MacDougall C. uncinata (tetraploid) 8; 4 MacDougall Conchophthirus anodontae 12 (diploid) Kidder C. mytili 16 (diploid) Kidder Ancistruma isseli About 5 (haploid) Kidder Paramecium aurelia 30-40 Diller About 35 Sonneborn P. caudatum About 36 Perm Stentor coeruleus 28 (diploid) Mulsow Tetrato.vum unifasciculatum About 14 Davis Oxytricha bifaria 24 (diploid) Kay 0. fallax 24 (diploid) Gregory Uroleptus halseyi 24 (diploid) Calkins Pleurotricha lanceolata About 40 (dipl.) Manwell Stylonychia pustulata 6 Prowazek Eaplotes patella 6 (diploid) Yocom; Ivanic E. eurystomus 8 (diploid) Turner Vorticella microstoma 4 Finley Carchesium polypinum 16 (diploid) Popoff Trichodina sp. 4-6 Diller vision, giving rise to daughter blepharoplasts and kinetosomes that become organized into characteristic locomotor organelles. Morpho- genesis in the apostomes (Chatton and Lwoff, 1935; Lwoff, 1950); mechanism of morphogenesis in ciliates (Faure-Fremiet, 1948; Guil- cher, 1950; Weisz, 1951, 1951a). 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. In Amoeba proteus, Chalkley and Daniel found that there is a definite correlation between the stages of nuclear divi- sion and external morphological changes (Fig. 68). During the pro- 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 (b, 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, 170 PROTOZOOLOGY as in Arcella, Pyxidicula, 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 into two parts. In the majority of the Mastigophora, the division is longitudinal, as is shown by that of Rhabdomonas incurva (Fig. 69). In certain dinoflagellates, such as Ceratium, Cochliodinium, etc., the division plane is oblique, while in forms such as Oxyrrhis (Dunk- b ^ ssfisSk Fig. 68. 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. erly; Hall), the fission is transverse. In Streblomastix strix (Kofoid and Swezy, 1919), Lophomonas striata (Kudo, 1926b), Spirotricho- nympha bispira (Cleveland, 1938), Holomastigotoides tusitala (Fig. 64) and others (Cleveland, 1947), and Strombidium clavellinae (Bud- denbrock, 1922), 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. REPRODUCTION 171 In the Ciliophora the division is as a rule transverse (Fig. 52), in which the body 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- Fig. 69. Nuclear and cytoplasmic division in Rhabdomonas incurva, X about 1400 (Hall), a, resting stage; b, c, prophase; d, equatorial plate; e, f, anaphase; g, telophase. 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. 256) 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. 208); the Radiolaria (Fig. 218), 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 172 PROTOZOOLOGY 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. 127), Myxosporidia (Fig. 70, b), astomatous ciliates (Fig. 298), Chono- tricha, Suctoria (Fig. 371, k), etc. Endogenous budding has been lit /im Fig. 70. a, b, budding in Myxidium lieberkiihni; c, d, plasmotomy in Chloromyxum leydigi; e, plasmotomy in Sphaeromyxa balbianii. found in Testacea, Gregarinida, Myxosporidia (Figs. 279, e; 281, j), and other Sporozoa as well as Suctoria (Fig. 371, h). 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 and leaves sooner or later the parent pellicle. Plasmotomy. Occasionally the multinucleate body of a protozoan divides into two or more small, mutinucleate individuals, the cyto- plasmic division taking place independently of nuclear division. This has been called plasmotomy by Doflein. It has been observed in the REPRODUCTION 173 trophozoites of several coelozoic myxosporidians, such as Chloro- ■myxumleydigi, Sphaeromyxa balbianii (Fig. 70), etc. It occurs further in certain Sarcodina such as Mycetozoa (Fig. 179) and Pelomyxa (Fig. 71), and Protociliata. Fig. 71. Eight individuals of Pelomyxa carolinensis, seen undisturbed in culture dishes, in which mitotic stages occurred as follows, X40 (Kudo) : a, early prophase; b, c, later prophase; d, metaphase; e, f, early and late anaphase; g, h, late telophase to resting nuclei (g, plasmotomy into two individuals; h, plasmotomy into three daughters). Colony formation When the division is repeated without a complete separation of the daughter individuals, a colonial form is produced. The compon- 174 PROTOZOOLOGY 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: Catenoid or linear colony. The daughter individuals are attached endwise, forming a chain of several individuals. It is of compara- tively uncommon occurrence. Examples: Astomatous ciliates such as Radiophrya (Fig. 298), Protoradiophrya (Fig. 298) and dinoflagel- lates such as Ceratium, Haplozoon (Fig. 130) and Polykrikos (Fig. 132). Arboroid or dendritic colony. The individuals remain connected with one another in a tree-form. The attachment may be by means of the lorica, stalk, or gelatinous secretions. It is a very common colony found in different groups. Examples: Dinobryon (Fig. 108), Hyalobryon (Fig. 108), etc. (connection by lorica); Colacium (Fig. 121), many Peritricha (Figs. 362; 364), etc. (by stalk); Poterioden- dron (Fig. 139), Stylobryon (Fig. 151), etc. (by lorica and stalk); Hydrurus (Fig. 109), Spongomonas (Fig. 150), Cladomonas(Fig. 150) and Anthophysis (Fig. 151) (by gelatinous secretions). Discoid colony. A small number of individuals are arranged in a single plane and grouped together by a gelatinous substance. Exam- ples: Cyclonexis (Fig. 108), Gonium (Fig. 116), Platydorina (Fig. 117), Protospongia (Fig. 138), Bicosoeca (Fig. 139), 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. 108, c), Uroglenopsis (Fig. 108, d), Volvox (Fig. 115), Pandorina (Fig. 117,/), Eudorina (Fig. 117, h), etc. Such forms as Stephanoon (Fig. 117, 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. REPRODUCTION 175 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 ncrease to a much greater number by multiple division or schizog- ony and the trophozoites are called schizonts. Under certain conditions, the trophozoite undergoes encystment (Fig. 72). Prior to encystment, the trophozoites cease to ingest, and extrude remains of, food particles, resulting in somewhat smaller forms which are usually rounded and less active. This phase is some- Fig. 72. Encystment of Lophomonas blattarum, X1150 (Kudo). times called the precystic stage. The whole organism becomes de- differentiated; namely, various cell organs such as cilia, cirri, flagella, axostyle, peristome, etc., become usually absorbed. Finally the organism secretes substances which become solidified into a re- sistant 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. Protozoa appear to encyst under various conditions. Low tem- perature (Schmahl, 1926), evaporation (Belaf, 1921; Bodine, 1923; Garnjobst, 1928), change in pH (Koffman, 1924; Darby, 1929), low or high oxygen content (Brand, 1923; Rosenberg, 1938), accumula- tion of metabolic products (Belaf, 1921; Mast and Ibara, 1923; Beers, 1926) or of associated bacteria (Mouton, 1902; Belaf, 1921) and over-population (Barker and Taylor, 1931) in the water in which Protozoa live, have been reported to bring about encystment. While 17(3 PROTOZOOLOGY lack of food in the culture has been noted by many observers (Oehler, 1916; Claff, Dewey and Kidder, 1941; Singh, 1941; Beers, 1948; etc.) as a cause of encystment in a number of Protozoa such as Blepharisma (Stolte, 1922), Polytomella (Kater and Burroughs, 1926), Didinium (Mast and Ibara, 1931), Uroleptus (Calkins, 1933), etc., an abundance of food and adequate nourishment seem to be prerequisite for encystment. Particular food was found in some in- stances to induce encystment. For example, Singh (1948) employed for culture of Leptomyxa reticulata, 40 strains of bacteria, of which 15 led to the production of a large number of cysts in this sarcodinan. Encystment of Entamoeba histolytica is easily obtained by adding starch to the culture (Dobell and Laidlow, 1926; Balamuth, 1951). The age of culture, if kept under favorable conditions, does not influence encystment. Didinium after 750 generations, according to Beers (1927), showed practically the same encystment rate as those which had passed through 10 or 20 generations since the last encyst- ment. When Leptomyxa mentioned above is cultured for more than a year, no encystment occurred, but young cultures when supplied with certain bacteria encysted (Singh, 1948). In some cases, the organisms encyst temporarily in order to un- dergo nuclear reorganization and multiplication as in Colpoda (Fig. 73) (Kidder and Claff, 1938; Stuart, Kidder and Griffin, 1939), Til- lina (Beers, 1946), etc. In Ichthyophthirius, the organism encysts after leaving the host fish and upon coming in contact with a solid object, and multiplies into numerous "ciliospores" (MacLennan, 1937). Pelomyxa carolinensis (Illinois stock) has not encysted since its discovery in 1944, although the cultures were subjected to vari- ous environmental changes, but P. illinoisensis has been found to encyst and excyst frequently in flourishing cultures (Kudo, 1951). Thus it may be assumed that some unknown internal factors play as great a part as do the external factors in the phenomenon of en- cystment (Ivanic, 1934; Cutler and Crump, 1935). The cyst is covered by one to three membranes. Though generally homogeneous, the wall of cyst may contain siliceous scales as in Euglypha (Fig. 74). While chitinous substance is the common ma- terial of which the cyst wall is composed, cellulose makes up the cyst membrane of many Phytomastigina. Entz (1925) found the cysts of various species of Ceratium less variable in size as com- pared with the vegetative form, and found in all, glycogen, oil and volutin. The capacity of Protozoa to produce cyst is probably one of the REPRODUCTION 177 reasons why they are so widely distributed over the surface of the globe. The minute protozoan cysts are easily carried from place to 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 Fig. 73. Diagram showing the life cycle of Colpoda cucullus (Kidder and Claff). a-j, normal reproductive activity repeated (j-b) under favorable cultural conditions; k-o, resistant cyst (k-n, nuclear reorganization and chromatin elimination). animals to which they become readily attached. The cyst is capable of remaining viable for a long period of time : eight years in Haema- tococcus pluvialis (Reichenow, 1929), four yaers in Spathidium spath- ula and Oxytricha sp. (Dawson and Mitchell, 1929), five years in Colpoda cucullus (Dawson and Hewitt, 1931), 10 years in Didinium nasutum (Beers, 1937), etc. 178 PROTOZOOLOGY When a cyst encounters a proper environment, redifferentiation takes place within the cyst. Various organellae which characterize the organism, are regenerated and reformed, and the young tropho- zoite excysts. The emerged organism returns once more to its trophic phase of existence. Experimental data indicate that excystment takes place under conditions such as addition of fresh culture me- dium (Kiihn, 1915; Rosenberg, 1938), hypertonic solution (Ilowai- sky, 1926), distilled water (Johnson and Evans, 1941), organic in- fusion (Mast, 1917; Beers, 1926; Barker and Taylor, 1933), and bac- terial infusion (Singh, 1941; Beers, 1946a) to the culture medium. Change in pH (Koffman, 1924), lowering the temperature (John- son and Evans, 1941) and increase in oxygen content (Brand, 1923; Finley, 1936) of the medium have also been reported as bringing about excystment. Excystment in Colpoda cucullus is said to be due Fig. 74. Encystment of Euglypha acanthophora, X320 (Kiihn). to specific inducing substances present in plant infusion (Thimann and Barker, 1934; Haagen-Smit and Thimann, 1938). Experiment- ing with two soil amoebae, "species 4 and Z," Crump (1950) found that the excystment in species Z took place without the presence of bacteria and regardless of the age of the cysts, but species 4 excysted only in the presence of certain bacteria (Aerobacter sp. or "4036") and the excystment diminished with the age of cysts. Crump sug- gested that the two strains of bacteria appeared to produce some material which induced excystment in Amoeba species 4. In Tillina magna, Beers (1945) found, however, the primary excystment-in- ducing factor to be of an osmotic nature and inducing substances, a secondary one. As to how an aperture or apertures are formed in the cyst wall prior to the emergence of the content, precise information is not yet on hand, though there are many observations. In the excyst- ment in Didinium and Tillina, Beers (1935, 1945, 1945a) notes that REPRODUCTION 179 an increased internal pressure due to the imbibition of water, re- sults in the rupture of the cyst wall which had lost its rigidity and resistance (Fig. 75). Apertures in the cyst wall of Pelomyxa illi- noisensis are apparently produced by pseudopodial pressure (Kudo, 1951). Seeing a similar aperture formation in the cyst of Entamoeba histolytica, Dobell (1928) "imagined that the amoeba secretes a fer- ment which dissolves the cyst wall." Fig. 75. Excystment in Didinium nasutum, as seen in a single indi- vidual, X250 (Beers), a, resting cyst; b, appearance of "excystment" vacuole; c, rupture of the cyst membrane, the vacuole is becoming en- larged; d, e, emergence of the cyst content, the vacuole increasing in size; f, the empty outer cyst membrane; g, the free organism with the inner membrane; h, organism after discharge of vacuole; i, j, later stages of emergence of the ciliate. Although encystment seems to be an essential phase in the life cycle of Protozoa in general, there are certain Protozoa including such common and widely distributed forms as the species of Para- mecium in which this phenomenon has not been definitely observed (p. 744). In some Sporozoa, encystment is followed by production of large numbers of spores, while in others there is no encystment. Here at the end of active multiplication of trophozoite, sexual re- 180 PROTOZOOLOGY production usually initiates the production of the spores (Fig. 76). The spores which are protected by a resistant membrane are capa- ble of remaining viable for a long period of time outside the host body. Fig. 76. 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. Sexual reproduction and life-cycles Besides reproducing by the asexual method, numerous Protozoa reproduce themselves in a manner comparable with the sexual re- 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) , REPRODUCTION 181 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. 77, 256) with that which exists between the spermatozoa and the 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. 77). ^^ Fig. 77. a, macrogamete, and b, microgamete of Volvox aureus, X1000 (Klein). 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. 276) 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 secreted by the other. Kiihn, 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- 182 PROTOZOOLOGY Fig. 78. Sexual fusion in Copromonas subtilis, X1300 (Dobell). 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. 79. Sexual fusion in Trinema linearis, X960 (Dunkerly). a, an organism in life, with the resting nucleus and two contractile vacuoles; b, union of two individuals; c, fusion of the organisms in one test, sur- rounded by cyst membrane; d, older cyst; e, still older cyst with a single nucleus. REPRODUCTION 183 The isogamy is typically represented by the flagellate Copro- monas subtilis (Fig. 78), in which there occurs, according to Dobell, Fig. 80. The life-cycle of Stephanosphaera pluvialis (Hieronymus). a-e, asexual reproduction; f-m, sexual reproduction. 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 Trinerna lineare (Fig. 79), Dunkerly (1923) saw isogamy in which Fig. 81. Sexual reproduction in Trichonympha of Cryptocercus (Cleveland), a, vegetative individual; b, gametocyte in early stage of encystment; c, anterior end of the same organism (chromosomes have been duplicated, nuclear sleeve is opening at seams and granules are flowing into the cytoplasm); d, further separation of the male and fe- male chromosomes; e, the nuclear division has been completed, few old flagella remain and new post rostral flagella are growing; f, the cytoplas- mic division has begun at the anterior end; g, the gametes just before ex- cystment, the female showing the developing ring of fertilization granules; h, a female gamete; i, a female gamete with a fertilization ring, a, X350; b, X320; c, X600; d-i, X280. REPRODUCTION 185 two individuals undergo a complete fusion within one test and en- cyst. In Stephanosphaera pluvialis (Fig. 80), both asexual and sexual reproductions 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 morum, Pringsheim observed that each cell develops asex- ually into a young colony or into anisogametes which undergo sexual fusion and encyst. The organism emerges from the cyst and develops into a young trophozoite. A similar life-cycle was found by Goebel in Eudorina elegc.ns The wood-roach inhabiting flagellates belonging to Trichonympha, Oxymonas, Saccinobaculus, Notila and Eucomonympha, were found by Cleveland (1949a-1951a) to undergo sexual reproduction when the host insect molts. It has been observed that the gamete-forma- tion is induced by the molting hormone produced by the prothoracic glands of the host insect. The sexual reproduction of Trichonympha, possessing 24 chromosomes, as observed and described by Cleve- land, is briefly as follows (Figs. 81, 82): About three days before its host molts, the haploid nucleus in the flagellate divides, in which two types of daughter chromosomes (or chromatids) become sepa- rated from each other: the dark-staining male gamete nucleus and light-staining female gamete nucleus (Fig. 81, b-d); in the mean- time, a membrane is formed to envelop the organism (b, d). When the cytoplasmic division is completed (e-g), the two gametes "ex- cyst" and become free in the host gut (h; Fig. 82, b). In the female gamete, there appear "fertilization granules" (Fig. 81, h), which gather at the posterior extremity (i), through which a fluid-filled vesicle ("fertilization cone") protrudes (Fig. 82, a). A male gamete (6) comes in touch with a female gamete only at this point (c), and enters the latter (d-f). The two gamete nuclei fuse into a diploid synkaryon (g, h). The zygote and its nucleus begin immediately to increase in size, and undergo two meiotic divisions (i-k), finally giv- ing rise to vegetative individuals (Fig. 81, a). Among the Sporozoa, anisogamy is of common occurrence. In Coccidia, the process was well studied in Eimeria schubergi (Fig. 243), Aggregata eberthi (Fig. 246), Adelea ovata (Fig. 253), etc., and the resulting products are the oocysts (zygotes) in which the spores or sporozoites develop. Similarly in Haemosporidia such as Plasmo- 186 PROTOZOOLOGY Fig. 82. Sexual reproduction in Trichonympha of Cryptocercus (Cleveland), a, a female gamete with a fetilization ring and cone; b, a male gamete; c-g, stages in fusion and fertilization; h, a zygote; i, telo- phase of the first meiotic division of the zygote nucleus; j, k, prophase and anaphase of the second meiotic division, a-g, X280;h, X215;i-k, X600. REPRODUCTION 187 dium vivax (Fig. 256), 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- tion, is still lacking. Naville, however, states that in the trophozoite of Sphaeromyxa sabrazesi (Fig. 277), 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 Endamoeba blattae (Mercier, 1909). Cultural studies of various para- sitic amoebae in recent years show, however, no evidence of sexual reproduction. Among the Ciliophora, the sexual fusion occurs only in Protociliata (Fig. 294). 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. 83), the process of conjugation has been studied by many workers, including Biitschli (1876), Maupas (1889), Calkins and Cull (1907), and others. Briefly the process is as follows: Two similar individuals come in contact on their oral surface (a). The micronucleus in each conjugant divides twice (b-e), forming four micronuclei, 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 pro- nucleus (/, g). The wandering pronucleus in each of the conjugants enters the other individual and fuses with its stationary pronucleus (h, r). The two conjugants now separate from each other and be- come 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). Cytoplasmic fision follows then, producing first, two individuals with four nuclei (p) and then, four small in- dividuals, each containing a micronucleus and a macronucleus (a). Jennings maintained that of the four smaller nuclei formed in the exconjugant (o), only one remains active and the other three de- generate. This active nucleus divides prior to the cytoplasmic divi- 188 PROTOZOOLOGY Fig. 83. Diagram illustrating the conjugation of Paramecium caudatum. a-q, X about 130 (Calkins); r, a synkaryon formation as in h, X1200 (Dehorne). REPRODUCTION 1S9 sion so that in the next stage (p), there are two developing macro- nuclei and one micronucleus which divides once more before the second and last cytoplasmic division (q). During these changes, the original macronucleus disintegrates, degenerates, and finally be- comes absorbed in the cytoplasm. Although this is the general course of events in the conjugation of this ciliate, recent observations revealed a number of different nuclear behavior. For example, there may not be pronuclear ex- change between the conjugants (cytogamy, p. 204), thus resulting in self fertilization (Diller, 1950a). In a number of races, Diller (1950) found that one of the two nuclei produced by the first divi- sion of the synkaryon degenerates, while the other nucleus divides three times, forming 8 nuclei, and furthermore, an exconjugant may conjugate occasionally with another individual before the reorgani- zation has been completed. The conjugaton of P. bursaria has also received attention of many workers. According to Chen (1946a), the first micronuclear division is a long process. One daughter nucleus degenerates and the other undergoes a second division. Here again one nucleus de- generates, while the other divides once more, giving rise to a wan- dering and a stationary pronucleus. Exchange of the wandering pronuclei is followed by the fusion of the two pronuclei in each conjugant. The synkaryon then divides. One of the two nuclei formed by this division degenerates, while the other gives rise to four nuclei by two divisions. The latter presently become dif- ferentiated into two micronuclei and two macronuclei, followed by a cytoplasmic division. The time two conjugants remain paired is said to be 20-38 or more hours (Chen, 1946c). In this Paramecium also, various nuclear activities have been reported. Chen (1940a, c) found that conjugation between a micronucleate and an amicronu- cleate can sometimes occur. In such a case, the micronucleus in the normal individual divides three times, and one of the pronuclei mi- grates into the amicronucleate in which there is naturally no nu- clear division. The single haploid nucleus ("hemicaryon") in each individual divides three times as mentioned above and four nuclei are produced. Thus amicronucleate becomes micronucleated. Con- jugating pairs sometimes separate from each other in a few hours. Chen (1946c) found that when such pairs are kept in a depression slide, temporary pairing recurs daily for many days, though there is seemingly no nuclear change. Chen (1940) further observed that the micronucleus in this species is subject to variation in size and 190 PROTOZOOLOGY in the quantity of chromatin it contains, which gives rise to dif- ferent (about 80 to several hundred) chromosome numbers during conjugation in different races, and that polyploidy is not uncom- mon in this ciliate. This investigator considers that polyploidy is a result of fusion of more than two pronuclei which he observed on several occasions. The increased number of pronuclei in a conju- gant may be due to: (1) the failure of one of the two nuclei produced by the first or second division to degenerate; (2) the conjugation between a unimicronucleate and a bimicronucleate, or (3) the fail- ure of the wandering pronucleus to enter the other conjugant; with this latter view Wichterman (1946) agrees. Apparently polyploidy occurs in other species also; for example, in P. caudatum (Calkins and Cull, 1907; Penn, 1937). In P. trichium, Diller (1948) reported that the usual process of conjugation is the sequence of three micronuclear divisions, pro- ducing the pronuclei (during which degeneration of nuclei may oc- cur at the end of both the first and second divisions), cross- or self-fertilization and three divisions of the synkarya. Ordinarily four of the eight nuclei become macronuclei, one remains as the micro- nucleus and the other three degenerate. The micronucleus divides at each of the two cytoplasmic divisions. Exchange of strands of the macronuclear skein may take place between the conjugants. Diller found a number of variations such as omission of the third prefer- tilization division, autogamous development, etc., and remarked that heteroploidy is pronounced and common. In P. aurelia possessing typically two micronuclei, the process of conjugation was studied by Maupas (1889), Hertwig (1889), Dil- ler (1936), Sonneborn (1947), etc., and is as follows: Soon after bi- association begins, the two micronuclei in each conjugant divide twice and produce eight nuclei, seven of which degenerate, while the remaining one divides into two gametic nuclei (Maupas, Woodruff, Sonneborn) Diller notes that two or more of the eight nuclei divide for the third time, but all but two degenerate; the two gametic nu- clei may or may not be sister nuclei. All agree that there are two functional pronuclei in each conjugant. As in other species of Para- mecium already noted, there is a nuclear exchange which results in the formation of a synkaryon in each conjugant. The synkaryon di- vides twice and the conjugants separate from each other at about this time. Two nuclei develop into macronuclei and the other two into micronuclei. Prior to the first cytoplasmic division of the excon- jugant, the micronuclei divide once, but the macronucleus does not divide, so that each of the two daughters receives one macronucleus REPRODUCTION 191 and two micronuclei. The original macronucleus in the conjugant becomes transformed into a skein which breaks up into 20 to 40 small masses. These are resorbed in the cytoplasm as in other species. As to when these nuclear fragments are absorbed, depends upon the nutritive condition of the organism (Sonneborn); namely, under a poor nutritional condition the resorption begins and is completed early, but under a better condition this resorption takes place after several divisions. During conjugation reciprocal migration of a pronucleus thus oc- curs in all cases. During biassociation and even in autogamy (p. 203), there develops a conical elevation ("paroral cone") and the nuclear migration takes place through this region. Although there is ordi- narily no cytoplasmic exchange between the conjugants, this may occur in some cases as observed by Sonneborn (1943a, 1944). P. aurelia of variety 4, according to Sonneborn, do occasionally not separate after fertilization, but remain united by a thin strand in the region of the paroral cones. In some pairs, the strand enlarges into a broad band through which cytoplasm flows from one individual to the other. The first division gives off a normal single animal from each of the "parabiotic twins" and the two clones derived from the two individuals belong to the same mating type (p. 192). Conjugation between different species of Paramecium has been attempted by several workers. Muller (1932) succeeded in producing a few pairings between normal P. caudatum and exconjugant P. multimicronucleatum. The nuclear process ran normally in cauda- tum, which led Muller to believe that crossing might be possible, but without success. De Garis (1935) mixed "double animals" (p. 228) of P. caudatum and conjugating population of P. aurelia. Pairing be- tween them occurred readily, in which the aurelia mates remained attached to caudatum for five to 12 hours. Four pairs remained to- gether, but aurelia underwent cytolysis on the second day. The separated aurelia from other pairs died after showing "cloudy swell- ing" on the second or third day after biassociation. The caudatum double-animals on the other hand lived for two to 12 (average six) days during which there was neither growth nor division and finally perished after "hyaline degeneration." No information on nuclear behavior in these animals is available. Apparently, the different spe- cies of Paramecium are incompatible with one another. In 1937, Sonneborn discovered that in certain races of P. aurelia, 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. The members of 192 PROTOZOOLOGY a class or caryonide (Sonneborn, 1939) are progeny of one of the two individuals formed by the first division of an exconjugant and thus possess the same macronuclear constitution. These classes were des- ignated by Sonneborn (1938) as mating types. Soon a similar phe- nomenon was found by several workers in other species of Para- A ' ,r ,- , .- • *~K * * #* ^ ^*% % •» 4. it _• ' . im. o _ • _i .*_, J Fig. 84. Mating behavior of Paramecium bursaria (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. mecium; namely, P. bursaria (Jennings, 1938), P. caudatum (Gil- man, 1939; Hiwatashi, 1949-1951), P. trichium, P. calkinsi (Sonne- born, 1938) and P. multimicronucleatum (Giese, 1939). When organ- isms which belong to different mating types are brought together, they adhere to one another in large clumps ("agglutination") of numerous individuals (Fig. 84, b). After a few to several hours, the REPRODUCTION 193 large masses break down into small masses (c) and still later, con- jugants appear in pairs (d). The only other ciliate in which mating types are definitely known to occur is Euplotes patella in which, ac- cording to Kimball (1939), there occurs no agglutination mating re- action. How widely mating types occur is not known at present. But as 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, Stylonychia pustulata, and Onychodromus grandis, 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 of mating types among different ciliates depends on future research. In Paramecium aurelia, Sonneborn distinguishes seven varieties which possess the same morphological characteristics of the species, but which differ in addition to mating types, also in size, division rate, conditions of temperature and light under which mating reac- tion may occur, etc. (Sonneborn, 1947). There occurs ordinarily no conjugation between the clones of different varieties. Within each of 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 G). Under optimum breeding conditions two mating types of the same variety give 95 per cent immediate agglutination and conjugation. But exceptions occur. Sonneborn and Dipell (1946) place the 7 va- rieties of aurelia under two groups: A (varieties 1, 3, 5 and 7) and B (varieties 2, 4 and 6) on the basis of their conjugational reactions. Mating types in group A do not conjugate with those of group B; no mating type of group B is known to conjugate with any type of other varieties in this group; but a number of combinations of mating types belonging to different varieties of group A conjugate with each other. For example, varieties 1 and 5 conjugate (namely, type I with type X and type II with type IX); however these interparietal mat- ing reactions are (1) always less intense than intra varietal reaction, (2) dependent upon the degree of reactivity of the culture, and (3) different from the intravarietal reaction with respect to the condi- tions for optimum reaction. Furthermore in most cases, the progeny of intervarietal matings are not viable. In the varieties of group A, the mating types appear to be of a more general sort. Therefore, Sonneborn (1947) designated even- and odd-numbered types as + and — respectively. 194 PROTOZOOLOGY Table 6. — Groups, varieties and mating types in Paramecium aurelia (Sonneborn) indicates that conjugation does not occur; numbers show the maximum percentage of conjugant-pairs formed; Inc. indicates incomplete mating reaction Group A B Variety 1 3 5 7 2 4 6 Mating type I II V VI IX X XIII III IV VII VIII XI XII General Type 1 I II 95 1 40 40 10 + A 3 V VI 95 3 Inc. + 5 IX X 95 1 Inc. + 7 XIII - 2 III IV 95 B 4 VII VIII 95 6 XI XII 95 In P. bursaria, Jennings (1938, 1939) found three varieties. Va- rieties 1 and 3 contain 4 mating types each, while variety 2, eight mating types. Jennings and Opitz (1944) further found variety 4 (Russian), composed of tw r o mating types and variety 5 under which several Russian clones were placed. Chen (1946a) added variety 6 (originating in Europe) containing four mating types. Thus in this species of Paramecium, there are now six varieties, containing 23 mating types (Table 7), and mating reaction occurs even among enucleate fragments of animals of different mating types of the same variety (Tartar and Chen, 1941). In Euplotes patella, Kimball (1939) observed six mating types which he designated as type I to type VI (Table 8). Though the members of a clone are of the same mating type and therefore do not conjugate, a clone may undergo at very long inter- vals (some 2000 culture days), "self -differentiation" into tw r o mating types which then conjugate (Jennings, 1941). Furthermore, Jennings REPRODUCTION 195 Table 7. — Varieties and mating types in Paramecium bursaria (Jennings; Jennings and Opitz; Chen) + indicates that conjugation occurs; — indicates that it does not Variety 1 2 3 4 5 6 Mating type A B C D EFGHJKLM N O P Q R S T U V W X A B C D - + + + - + + - + 1 2 E F G H J K L M - + + + + + + + - + + + + + + - + + + + + - + + + + - + + + - + + - + + - + - + - + - N O P Q + + + - + + - + 3 4 R S - + 5 T 6 U V w X - + + + - + + - + and Opitz (1944) found that mating type R (variety 4) conjugated with E, K, L or M (variety 2), but all conjugants or exconjugants perished without multiplication. Chen (1946a) made a cytological study of them and observed that the nuclear changes which are Table 8. — Mating types in Euplotes patella (E .imball) Mating type I II III IV V VI I _ + + + + + II — + + + + III — + + + IV — + + V — + VI — 19G PROTOZOOLOGY seemingly normal during the first 16 hours, become abnormal sud- denly after that time, and the micronuclei divide only once and there is no nuclear exchange. The death of conjugants or exconjugants is possibly due to physiological incompatibility between the varieties upon coming in contact or probably due to "something that diffuses from one conjugant to the other." Studies of mating types have revealed much information re- garding 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. bursaria, 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 (p. 235). Pre- sumably certain substances are secreted by the organisms and be- come diffused in the culture fluid. In Paramecium aurelia, Sonne- born (1943) found that of the four races of variety 4, race 51 was a "killer," while the other three races, "sensitive." Fluid in which the killer race grew, kills the individuals of the sensitive races. As has been mentioned already, P. bursaria designated as type T (variety 5) (Table 7) conjugates with none. But Chen (1945) found that its culture fluid induces conjugation among a small number of the indi- viduals of one mating type of varieties 2, 3, 4 and 6, in which nuclear changes proceed as in normal conjugation. Furthermore, this fluid is capable of inducing autogamy in single animals. Other visible in- fluences of the fluid on organisms are sluggishness of movement and darker coloration and distortion of the body. REPRODUCTION 197 Boell and Woodruff (1941) noticed that in P. calkinsi, living indi- viduals of one mating type will agglutinate with dead ones of the complementary mating type. A similar phenomenon was also ob- served by Metz (194(5, 1947, 1948) who employed various methods of killing the animals. The pairs composed of living and formaldehyde- killed animals, behave much like normal conjugating pairs; there is of course no cross-fertilization, but the living member of the pair undergoes autogamy. While the "mating type substances" can be destroyed by exposure to 52°C. for five minutes; by X-irradiation; by exposure of formaldehyde-killed reactive animals to specific anti- sera or to 100°C, etc., Metz demonstrated that animals may be killed by many reagents which do not destroy these substances. Furthermore, all mating activities disappear when the animals are thoroughly broken up, which suggests that Paramecium might re- lease some mating substance inhibitory agent. This agent was later found in this Paramecium (Metz and Butterfield, 1950). Metz (4948) points out that the mating reaction involves substances present on the surfaces of the cilia, and supposes that the interaction between two mating-type substances initiates a chain of reactions leading up to the process of conjugation and autogamy. Hiwatashi (1949a, 1950) using four groups (each composed of two mating types) of P. caudatum, confirmed Metz's observation. Metz and Butterfield (1951) more recently report that non-proteolytic enzymes (lecithin- ase, hyaluronidase, lysozyme, ptyalin, ribonuclease) have no de- tectable effect on the mating reactivity of P. calkinsi; but proteo- lytic enzymes such as trypsin and chymotrypsin destroy the mating reactivity, and mating substance activity was not found in the digest of enzyme-treated organisms. The two observers believe that the mating reactivity is dependent upon protein integrity. 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. This seems to be the case with the majority, al- though more than one micronucleus may divide for the third time to produce several pronuclei, for example, two in Euplotes patella, Sty- lonychia pustulata; two to three in Oxytricha fallax and two to four in Uroleptus mobilis. This third division is often 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 198 PROTOZOOLOGY Fig. 85. 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 macronucleus; k-n, exconjugants found nearly exclu- sively in recently transformed host; o, mature trophozoite; p-s, binary fission stages; t, precystic stage. REPRODUCTION 199 conjugation. According to Wichterman (1936), conjugation in Nyctotherus cordiformis (Fig. 85) 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- zoites, because of the preconjugation 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 exconjug- ant, the synkaryon divides once to form the micronucleus and the macronuclear anlage (k-l) which 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' (1888) classical work on Vorticella nebulifera, the ordinary vegetative form divides twice, forming four small indi- viduals, which become detached from one another and swim about independently. 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 di- vides twice, forming four nuclei, of which three degenerate, and the other dividing into two. During these changes 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. In Vorti- cella microstoma (Fig. 86), Finley (1943) notes that a vegetative indi- vidual undergoes unequal division except the micronucleus which divides equally (a), and forms a large stalked macroconjugant and a small free microconjugant (b). The conjugation which requires 18- 24 hours for completion, begins when a microconjugant attaches it- self to the lower third of a macroconjugant. The protoplasm of the microconjugant enters the macroconjugant (c). The micronucleus of the microconjugant divides three times, the last one of which being reductional (d, e), while that of the macroconjugant divides twice (one mitotic and one meiotic). Fusion of one of each produces a synkaryon (/) which divides three times. One of the division products becomes a micronucleus and the other seven macronuclear anlagen (g, h) which are distributed among the progeny (i,j). Another example of this type has been observed in Metopus es 200 PROTOZOOLOGY (Fig. 87). 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 ib-e). But the cytoplasm and both pronuclei of one conjugant pass into the other (J), leaving the degenerating macronucleus and a small Fig. 86. Sexual reproduction in Vorticella microstoma, X800 (Fin- ley), a, preconj ligation division which forms a macroconjugant ami a microconjugant; b, a macroconjugant with three microconjugants; c, a microconjugant fusing with a macroconjugant; d, the micronucleus of the microconjugant divided into four nuclei; e, with 12 nuclei formed by di- visions of the two micronuclei of conjugants; f, synkaryon; g, eight nu- clei after three divisions of synkaryon; h, seven enlarging macronuclear anlagen and a micronucleus in division; i, first division; j, a daughter in- dividual with a micronucleus, four macronuclear anlagen. and old macro- nuclear fragments. REPRODUCTION 201 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. While ordinarily two individuals participate in conjugation, three Fig. 87. 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 newlj r formed micronucleus and macronucleus j, small exconjugant with degenerating macronucleus; k-m, development of two nuclei, a, X290; b-j, X250, k-m, X590. 202 PROTOZOOLOGY or four individuals are occasionally involved. For example, conjuga- tion of three animals was observed in P. caudatum by Stein (1867), Jickeli (1884), Maupas (1889) and in Blepharisma vndulans by Giese (1938) and Weisz (1950). Chen (1940b, 1948) made a careful study of such a conjugaion which he found in Paramecium bur- Fig. 88. Conjugation of three individuals in Paramecium bursaria, X365 (Chen), a, late prophase of the first nuclear division (the individual on right is a member of a race with "several hundred chromosomes," while the other two belong to another race with "about 80 chromosomes") ; b, anaphase of the third division (each individual contains 2 degenerating nuclei); c, beginning of pronuclear exchange between two anterior ani- mals; d, e, synkaryon formation; f, after the first division of synkaryon, one daughter nucleus undergoing degeneration in all animals. REPRODUCTION 203 saria (Fig. 88). He found that the usual manner of association is conjugation between a pair with the third conjugant attached to the posterior part of one of them (a). Nuclear changes occur in all three individuals, and in each, two pronuclei are formed by three divisions (c) . But the exchange of the pronuclei takes place only between two anterior conjugants (c-e) and autogamy (see below) occurs in the third individual. Fig. 89. 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. 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 which undergo a complete fusion are present in a single cell, the process is called autogamy, but, if they are in two 204 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. 276; 277) and Myxosoma cato- stomi (Fig. 275). In the Microsporidia, autogamy appears to initiate the spore-formation at the end of schizogonic activity of individuals as in Thelohania legeri (Fig. 76). Diller (1936) observed in solitary Paramecium aurelia (Fig. 89), 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 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 (Z). 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 (1940) observed a similar process in P. caudatum and named it cytog- amy. Two small (about 200/x long) individuals of P. caudatum 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. Sonne- born (1941) finds the frequency of cytogamy in P. aurelia to be cor- related with temperature. At 17°C, conjugation occurs in about 95 per cent of the pairs and cytogamy in about 5 per cent; but at 10° and 27°C, cytogamy takes place in 47 and 60 per cent respectively. In addition, there is some indication that sodium decreases and calcium increases the frequency of occurrence of cytogamy. The paedogamy occurs in at least two species of Myxosporidia, namely, Leptotheca ohlmacheri (Fig. 279) and Unicapsula muscularis (Fig. 280). 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- morphose into a uninucleate amoebula. Perhaps the classical exam- ple of the paedogamy is that which was found by Hertwig (1898) in Actinosphaerium eichhorni. The organism encysts and the body di- REPRODUCTION 205 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 (1923) observed a similar process in Actinophrys sol (Fig. 90). This heliozoan withdraws its axopodia and divides into two uninucleate bodies which become surrounded by a common Fig. 90. Paedogamy in Actinophrys 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. 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, 206 PROTOZOOLOGY without involving micronuclei. He coined the term hemixis for this reorganization. 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 of 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. Fig. 91. 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. 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. 91), he observed 16 chromosomes in each of the 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. 168). In various species of Paramecium and many other forms, the number of chromosomes appears to be too great to allow a precise counting, but the observations of Sonneborn, as quoted elsewhere (p. 234) and of Jennings (1942) on P. aurelia and P. bursaria respec- REPRODUCTION 207 tively, indicate clearly the occurrence of meiosis prior to nuclear ex- change during conjugation. Information on the meiosis involved in the complete fusion of gam- etes is even more scanty and fragmentary. In Monocystis rostrata (Fig. 92), a parasite of the earthworm, Mulsow (1911) noticed that f ' ^^V m -m ^tzm&j^ w Fig. 92. Mitosis and meiosis in Monocystis rostrata (Mulsow). a-g, mitosis; h-j, meiosis. a, a resting nucleus in the gametocyte; b, develop- ment of chromosomes; c, polar view of equatorial plate; d, longitudinal splitting of eight chromosomes; e, separation of chromosomes in two groups; f, late anaphase; g, two daughter nuclei; h, i, polar view of the equatorial plate in the last division; j, anaphase, the gamete nucleus is now haploid (4). a-c, X1840; d-g, X1400; h-j, X3000. the nuclei of two gametocytes which encyst together, multiply by mitosis in which eight chromosomes are constantly present (a-g), but in the last division in gamete formation, each daughter nucleus receives only 4 chromosomes (h-j). In another species of Monocystis, Calkins and Bowling (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 (Fig. 90), Belaf (1923) finds 44 chromosomes in the first nuclear division, but after two meiotic divisions, the remaining functional nucleus contains only 22 chromo- somes 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 syn- karyon divides twice, and during the first division reduction division takes place. 208 PROTOZOOLOGY In the coccidian, Aggregata eberthi (Fig. 246), according to Dobell (1925), Naville (1925) and Belaf (1926) and in the gregarine, Diplo- cystis schneideri, according to Jameson (1920), 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 reduction takes place also in Actinocephalus parvus (8 to 4, after Weschenf elder, 1938), Greg arina blattarum (6 to 3, after Sprague, 1941), Adelina deronis (20 to 10, after Hauschka, 1943), etc. Tri- chonympha and other flagellates (p. 185) of woodroach, Polytoma Fig. 93. 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. and Chlamydomonas (p. 276) also undergo postzygotic meiosis. Thus in these organisms, the zygote is the only stage in which the nucleus is diploid. Some seventy 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 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 REPRODUCTION 209 which they can be cultivated in the laboratory, the majorhVy 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 February 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 structure, until the animals could no longer divide and died (Fig. 93). 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 often becomes restored. Perhaps the most thorough experiment was carried on by Calkins (1919, 1933) with Uroleptus mobilis. Starting with an exconjugant on 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 spathula (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 continued asexual reproduc- tion. Other examples of longevity of ciliates without conjugation are: Glaucoma for 2701 generations (Enriques, 1916), Paramecium caudatum for 3967 generations (Metalnikov, 1922), Spathidium spa- thula for 1080 generations (Woodruff and Moore, 1924), Didinium nasutum for 1384 generations (Beers, 1929), etc. With Actinophrys sol, Belaf (1924) carried on isolation cultures for 1244 generations for a period of 32 months and noticed no decline in the division rate. 210 PROTOZOOLOGY Hartmann (1921) made a similar observation on Eudorina 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. 150) and that this elimination, though not complete, enables the protoplasm of the products of divi- sion to carry on their metabolic functions more actively. 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 (1944) distinguished four successive periods in various clone cultures of Paramecium bursaria; namely, (1) a period of sexual immaturity during which neither sexual reaction nor conjugation occurs; (2) a period of transition during which weak sexual reactions appear in a few individuals; (3) a period of maturity in which conjugation takes place readily when proper mating types are brought together; and (4) a period of decline, ending in death. The length of the first two periods depends on the cultural conditions. Exconjugant clones that are kept in condition under which the animals multiply rapidly, reach maturity in three to five months, while those subjected to de- pressing condition require 10 to 14 months to reach maturity. The third period lasts for several years and is followed by the fourth period during which fission becomes slower, abnormalities appear, many individuals die and the clones die out completely. Does conjugation affect the longevity of clones in Paramecium busaria? A comparative study of the fate of exconjugants and non- conjugants led Jennings (1944a) to conclude that (1) conjugation results in production of one of the following four types: (a) excon- jugants perish without division, (b) exconjugants divide one to four times and then die, (c) exconjugants produce weak abnormal clones which may become numerous, and (d) exconjugants multiply vigor- ously and later undergo conjugation again; at times the latter are REPRODUCTION 211 more vigorous than the parent clones, thus showing rejuvenescence through conjugation; (2) conjugation of young clones results in little or no mortality, while that of old clones results in high (often 100 per cent) mortality; (3) conjugation between a young and an old clone, results in the death of most or all of the exconjugants; (4) the two members of a conjugating pair have the same fate; and (5) what other causes besides age bring about the death, weakness or ab- normality of the exconjugants, are not known. 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 a long chain of generations indefinitely. Jennings (1929, 1942) who reviewed the whole problem states: "Some Protozoa are so con- stituted 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." 212 PROTOZOOLOGY 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. It is now a well established 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 undertake ana- bolic activities, and consequently degenerates sooner or later. Brandt (1877) studied regeneration in Actinosphaerium eichhorni and found that only nucleate portions containing at least one nucleus regener- ated and enucleate portions or isolated nuclei degenerated. Similarly Gruber (1886) found in Amoeba proteus the nucleate portion regener- ated 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-Allermann (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 zymo- gens to be activated in the dedifferentiating cytoplasm. According to Brachet (1950), the enucleated half of an amoeba shows a steady decrease in ribonucleic acid content, while the nucleated half retains a much larger amount of this substance. Thus it appears that the synthesis of the cytoplasmic particles containing ribonucleic acid is under the control of the nucleus. In Arcella (Martini; Hegner) and Difflugia (Verworn; Penarcl), when the tests are partially destroyed, the broken tests remain un- 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 all times. 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. REPRODUCTION 213 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 ambiguum and 1/70-1/75 of Dileptus anser regenerated and Phelps showed portions down to 1/80 of an amoeba were able to regenerate. In Stentor coeruleus, pieces as small as 1/27 (Lilly) or 1/64 (Morgan) of the original speci- mens or about 70/jl in diameter (Weisz) regenerate. Burnside cut 27 specimens 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. 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. Information is now available on the process of morphogenesis in regeneration and reorganization in certain ciliates (Chatton and Lwoff, 1935; Bala- muth, 1940; Summers, 1941; Faure-Fremiet, 1948; Weisz, 1948, 1951). References Balamuth, W. : (1940) Regeneration in Protozoa: a problem of morphogenesis. Quart. Rev. Biol., 15:290. (1951) Biological studies on Entamoeba histolytica. III. J. Infect. Dis., 88:230. Barker, H. A. and Taylor, C. V.: (1931) A study of the conditions of encystment of Colpoda cucullus. Physiol. Zool., 4:620. — (1933) Studies on the excystment of Colpoda cucul- lus. Ibid., 6:127. 214 PROTOZOOLOGY Beers, C. D.: (1926) The life-cycle in the ciliate Didinium nasutum with reference to encystment. J. Morphol., 42:1. (1927) Factors involved in encystment in the ciliate Didin- ium nasvtum. J. Morphol. Physiol., 43:499. (1928) Rhythms in Infusoria with special reference to Didin- ium nasutum. J. Exper. Zool., 51:485. 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Belar, K. : (1921) Untersuchungen ueber Thecamoeben der Chlamydophrys-Gruppe. Arch. Protist., 43:287. (1923) Untersuchungen an Actinophrys sol. I. Ibid., 46: 1. (1924) II. Ibid., 48:371. (1926) Der Formwechsel der Protistenkerne. Ergebn. u. Fortsch. Zool., 6:235. Bodine, J. H.: (1923) Excystation of Colpoda cucullus. J. Exper. Zool., 37:115. Boell, E. J. and Woodruff, L. L.: (1941) Respiratory metabolism of mating types in Paramecium calkinsi. J. Exper. Zool., 87:385. Brachet, J.: (1950) Un etude cytochimique des fragments nuclees et enuclees d'amibes. Experientia, 6:294. Buddenbrock. W. v. : (1922) Ueber eine neue Strombidium-Art aus Heligoland. Arch. Protist., 45:129. Butschli, O.: (1876) Studien liber die ersten Entwicklungsvorgange der Eizelle, die Zelltheilung und die Conjugation der Infusorien. Abh. Senk. Nat. Ges. Frankf., 10:1. Btjrnside, L. H.: (1929) Relation of body size to nuclear size in Stentor coeruleus. J. Exper. Zool., 54:473. Burt, R. L., Kidder, G. W. and Claff, C. L.: (1941) Nuclear re- organization in the family Colpodidae. J. Morphol., 69:537. Calkins, G. N.: (1919) Uroleptus mobilis. II. J. Exper. Zool., 29: 121. - (1933) The biology of the Protozoa. 2nd ed. Philadelphia. REPRODUCTION 215 — and Bowling, R. C: (1926) Gametic meiosis in Monocystis. Biol. Bull., 51:385. — and Cull, S. W. : (1907) The conjugation of Paramecium aurelia (caudatum). Arch. Protist., 10:375. and Summers, F. M.: (editors) (1941) Protozoa in biological research. New York. Chalkley, H. W. : (1936) The behavior of the karyosome and the "peripheral chromatin" during mitosis and interkinesis in Amoeba proteus, etc. J. Morphol., 60:13. and Daniel, G. E.: (1933) The relation between the form of the living cell and the nuclear phases of division in Amoeba proteus. Physiol. Zool., 6:592. Chatton, E. and Lwoff, A.: (1935) Les Cilies Apostomes. I. Arch. zool. exper. gen., 77:1. Chen, T. T.: (1936) Observations on mitosis in opalinids. I. Proc. Nat. Acad. Sc, 22:594. (1940) Polyploidy and its origin in Paramecium. J. Hered., 31:175. (1940a) Conjugation in Paramecium bursaria between ani- mals with diverse nuclear constitutions. Ibid., 31:185. ■ (1940b) Conjugation of three animals in Paramecium bur- saria. Proc. Nat. Acad. Sc, 26:231. (1940c) Conjugation in Paramecium bursaria between ani- mals with very different chromosome numbers, etc. Ibid., 26: 243. (1945) Induction of conjugation in Paramecium bursaria, etc. Ibid., 31:404. (1946) Conjugation in Paramecium bursaria. I. J. Morphol., 78:353. (1946a) II. Ibid., 79:125. (1946b) Varieties and mating types in Paramecium bursaria. I. Proc. Nat. Acad. Sc, 32:173. (1946c) Temporary pair formation in Paramecium bursaria. Biol. Bull., 91:112. (1948) Chromosomes in Opalinidae, etc J. Morphol., 83: 281. Clark, A. M.: (1942) Some effects of removing the nucleus from Amoeba. Australian J. Exper. Biol., 20:241. (1943) Some physiological functions of the nucleus in Amoeba, etc. Ibid., 21:215. Cleveland, L. R. : (1938) Longitudinal and transverse division in two closely related flagellates. Biol. Bull., 74:1. (1938a) Origin and development of the achromatic figure. Ibid., 74:41. ■ (1949) The whole life cycle of chromosomes and their coiling systems. Tr. Am. Philos. Soc, 39:1. — (1949a) Hormone-induced sexual cycles of flagellates. J. Morphol., 85:197. — (1950) II. Ibid., 86:185. - (1950a) III. Ibid., 86:215. 216 PROTOZOOLOGY (1950b) IV. Ibid., 87:317. (1950c) V. Ibid., 87:349. (1951) VI. Ibid., 88:199. (1951a) VII. Ibid., 88:385. Hall, S. R., Sanders, E. P. and Collier, J.: (1934) The wood-feeding roach Cryptocercus, etc. Mem. Am. Acad. Arts & Sc., 17:185. Crump, Lettice M.: (1950) The influence of bacterial environment on the excystment of amoebae from soil. J. Gen. Microbiol., 4: 16. Cutler, D. W. and Crump, L. M.: (1935) The effect of bacterial products on amoebic growth. Brit. J. Exper. Biol., 12:52. Daniel, G. E. and Chalkley, H. W. : (1932) The influence of tem- perature upon the process of division in Amoeba proteus. J. Cell. Comp. Physiol., 2:311. Darby, H. PL: (1929) The effect of the hydrogen-ion concentration on the sequence of protozoan forms. Arch. Protist., 65: 1. Dass, C. M. S.: (1950) Chromatin elimination in Glaucoma pyrifor- mis. Nature, 165:693. Davis, T. G.: (1941) Morphology and division in Tetratoxum uni- fasciculatum. Tr. Am. Micr. Soc, 60:441. Dawson, J. A.: (1919) An experimental study of an amicronucleate Oxytricha. I. J. Exper. Zool., 29:473. and Hewitt, D. C: (1931) The longevity of encysted Col- poda. Am. Nat., 65:181. and Mitchell, W. H.: (1929) The vitality of certain in- fusorian cysts. Ibid., 63:476. De Garis, C. F.: (1935) Lethal effects of conjugation between Paramecium aurelia and double-monsters of P. caudatum. Am. Nat., 69:87. Diller, W. F.: (1936) Nuclear reorganization processes in Para- mecium aurelia, etc. J. Morphol., 59:11. (1948) Nuclear behavior of Paramecium trichium during con- jugation. Ibid., 82:1. (1950) An extra postzygotic nuclear division in Paramecium caudatum. Tr. Am. Micr. Soc, 69:309. (1950a) Cytological eivdence for pronuclear interchange in Paramecium caudatum. Ibid., 69:317. Dobell, C. : (1908) The structure and life history of Copromonas subtilis, etc. Quart. J. Micr. Sc, 52:75. (1917) On Oxnerella maritima, etc. Ibid., 62:515. (1925) The life history and chromosome cycle of Aggregata eberthi. Parasitology, 17:1. (1928) Researches on the intestinal Protozoa of monkeys and man. I, II. Ibid., 20:357. and Laidlaw, P. P.: (1926) On the cultivation of Entamoeba histolytica, etc. Ibid., 18:283. Enriques, P.: (1916) Duemila cinquecento generazioni in un in- fusorio, senza conjugazione ne partenogenesi, ne depressioni. Rev. Acad. Sc. Bologna, 20:67. REPRODUCTION 217 Entz, G.: (1925) Ueber Cysten und Encystierung der Siisswasser- Ceratien. Arch. Protist., 51:131. Everritt, Martha G.: (1950) The relationship of population growth, etc. J. Parasit., 36:586. Faure-Fremiet, E.: (1948) Les mecanismes de la morphogenese chez les cilies. Folia Bioth., 3:25. Finley, H. E.: (1936) A method for inducing conjugation within Vorticella cultures. Tr. Am. Micr. Soc, 55:323. (1943) The conjugation of Vorticella microstoma. Ibid., 62: 97. Frosch, P.: (1897) Zur Frage der Reinzuchtung der Amoeben. Zen- tralbl. Bakt. I. Abt., 21:926. Garnjobst, L.: (1928) Induced encystment and excystment in Ewplotes taylori, etc. Physiol. Zool., 1:561. Giese, A. C.: (1938) Size and conjugation in Blepharisma. Arch. Protist., 91:125. (1939) Studies on conjugation in Paramecium multimicro- nucleatum. Am. Nat., 73:432. (1939a) Mating types in Paramecium caudatum. Am. Nat., 73:445. Gilman, L. C.: (1941) Mating types in diverse races of Paramecium caudatum. Biol. Bull., 80:384. Grasse, P.-P.: (1952) Traite de Zoologie. I. Fasc. 1. Paris. Guilcher, Yvette: (1950) Contribution a l'etude des cilies gemmi- pares, etc. Univ. de Paris thesis, Ser. A. no. 2369. Haagen-Smit, A. J. and Thimann, K. V.: (1938) The excystment of Colpoda cucullus. I. J. Cell. Comp. Physiol., 11:389. Hall, R. P.: (1923) Morphology and binary fission of Menoidium incurvum. Univ. California Publ. Zool., 20:447. (1937) A note on behavior of the chromosomes in Euglena. Tr. Am. Micr. Soc, 56:288. Hartmann, M.: (1917) Ueber die dauernde rein agame Zuchtung von Eudorina elegans, etc. Ber. preuss. Akad. Wiss., Phys.- Math. Kl., p. 760. Hauschka, T. S.: (1943) Life history and chromosome cycle of the coccidian, Adelina deronis. J. Morphol., 73:529. Hertwig, R.: (1889) Ueber die Conjugation der Infusorien. Abh. bayerl. Akad. Wiss., 17:151. Hinshaw, H. C: (1926) On the morphology and mitosis of Tri- chomonas buccalis. Univ. California Publ. Zool., 29:159. Hiwatashi, K.: (1949) Studies on the conjugation of Paramecium, caudatum. I. Sc. Rep. Tohoku Univ. Ser. IV, 18:137. (1949a) II. Ibid., 18:141. (1950) III. Ibid., 18:270. (1951) IV. Ibid., 19:95. Horvath, J.: (1950) Vitalitatsausserung einer mikronucleuslose Bodenziliate in der vegetativen Fortpflanzung. Oesterr. zool. Ztschr., 2:336. Ilowaisky, S. A.: (1926) Material zum Studium der Cysten der Hypotrichen. Arch. Protist., 54:92. 218 PROTOZOOLOGY Ivanic, M. : (1934) Ueber die Ruhestadienbildung und die damit am Kernapparate verbundenen Veranderungen bei Lionotus cygnus. Zool.Anz., 108:17. (1938) Ueber die mit der Chromosomenbildung verbundene promitotische Grosskernteilung bei den Vermehrungsruhe Sta- tien von Chilodon uncinatus. Arch. Protist., 91:61. Jameson, A. P.: (1920) The chromosome cycle of gregarines with special reference to Diplocystis schneideri. Quart. J. Micr. Sc, 64:207. Jennings, H. S.: (1929) Genetics of the Protozoa. Bibliogr. Gen., 5: 105. — ■ (1938) Sex relation types and their inheritance in Parame- cium bursaria. I. Proc. Nat. Acad. Sc, 24:112. (1939) Genetics of Paramecium bursaria. I. Genetics, 24:202. (1941) II. Proc. Am. Philos. Soc, 85:25. (1942) III. Genetics, 27:193. (1942a) Senescence and death in Protozoa and invertebrates. E. V. Cowdry's Problems of ageing. 2 ed. Baltimore. (1944) Paramecium bursaria: Life history. I. Biol. Bull., 86: 131. ■ (1944a) II. J. Exper. Zool., 96:17. and Opitz, Pauline: (1944) Genetics of Paramecium bur- saria. IV. Genetics, 29:576. Raffel, D., Lynch, R. S. and Sonneborn, T. M.: (1932) The diverse biotypes produced by conjugation within a clone of Paramecium aurelia. J. Exper. Zool., 62:363. Jickeli, C. F.: (1884) Ueber die Kernverhaltnisse der Infusorien. Zool. Anz., 7:491. Johnson, W. H. and Evans, F. R.: (1940) Environmental factors affecting encystment in Woodruffia metabolica. Physiol. Zool., 13:102. — — — (1941) A further study of environmental factors af- fecting cystment in Woodruffia metabolica. Ibid., 14:227. Kater, J. M. and Burroughs, R. D.: (1926) The cause and nature of encystment in Polyiomella citri. Biol. Bull., 50:38. Kay, M. M.: (1946) Studies on Oxytricha bifaria. III. Tr. Am. Micr. Soc, 65:132. Kidder, G. W. : (1933) Studies on Conchophthirus mytili de Morgan. I. Arch. Protist., 79:1. (1938) Nuclear reorganization without cell division in Para- clevelandia simplex, etc. Ibid., 91:69. and Claff, C. L.: (1938) Cytological investigations of Gol- poda cucullus. Biol. Bull., 74:178. and Diller, W. F. : (1934) Observations on the binary fission of four species of common free-living ciliates, etc Ibid., 67:201. and Stuart, C. A.: (1939) Growth studies on ciliates. II. Physiol. Zool., 12:341. and Summers, F. M.: (1935) Taxonomic and cytological studies on the ciliates associated with the amphipod family, etc. Biol. Bull, 68:51. REPRODUCTION 219 Kimball, R. F.: (1939) Change of mating type during vegetative reproduction in Paramecium aurelia. J. Exper. Zool., 81:165. (1939a) Mating types in Euplotes. Amer. Nat., 73:451. (1941) The inheritance of mating type in the ciliate protozoan Euplotes patella. Genetics, 26:158. (1941a) Double animals and amicronucleate animals, etc. J. Exper. Zool., 86:1. (1942) The nature and inheritance of mating types in Eu- plotes patella. Genetics, 27:269. — — — • (1943) Mating types in the ciliate Protozoa. Quart. Rev. Biol., 18:30. Koffman, M.: (1924) Ueber die Bedeutung der Wasserstoffionenkon- zentration fur die Encystierung bei einigen Ciliatenarten. Arch. mikr. Anat., 103:168. Kofoid, C. A. and Swezy, Olive: (1919) Studies on the parasites of the termites. I. Univ. California Publ. Zool., 20:1. (1919a) III. Ibid., 20:41. Korschelt, E.: (1927) Regeneration und Transplantation. Vol. 1. Berlin. Kudo, R. R. : (1926) Observation on Endamoeba blattae. Am. J. Hyg., 6:139. (1926a) Observations on Lophomonas blattarum, etc. Arch. Protist., 53:191. — — — (1926b) A cytological study of Lophomonas striata. Ibid., 55: 504. — (1936) Studies on Nyctotherus ovalis, etc. Ibid., 87:10. (1947) Pelomyxa carolinensis Wilson. II. J. Morphol., 80: 93. ■ (1951) Observations on Pelomyxa illinoisensis. Ibid., 88: 145. Kuhn, A.: (1915) Ueber Bau, Teilung und Encystierung von Bodo edax. Arch. Protist., 36:212. Landis, E. M.: (1920) An amicronucleate race of Paramecium cau- datum. Anat. Rec, 54:453. Liebmann, H.: (1944) Beitrag zur Kenntnis der Kernteilung bei vegetativen Stadien von Entamoeba coli. Arch. Protist., 97: 1. Liesche, W. : (1938) Die Kern- und Fortpflanzungsverhaltnisse von Amoeba proteus. Ibid., 91:135. Lund, E. J.: (1917) Reversibility of morphogenetic processes in Bursaria. J. Exper. Zool., 24:1. Lwoff, A. : (1950) Problems of morphogenesis in ciliates. New York. MacLennan, R. F.: (1937) Growth in the ciliate Ichthyophthirius. I. J. Exper. Zool, 76:243. Manwell, R. D.: (1928) Conjugation, division and encystment in Pleurotricha lanceolata. Biol. Bull., 54:417. Mast, S. O. and Ibara, Y.: (1923) The effect of temperature, food and the age of the culture on the encystment of Didinium nasu- tum,. Ibid., 45:105. Maupas, E.: (1888) Recherches experimentales sur la multiplica- tion des infusoires cilies. Arch. zool. exper. (2), 6:165. 220 PROTOZOOLOGY (1889) Le rejeunissement karyogamique chez les cilies. Ibid., 7:149. Metalnikov, S.: (1922) Dix aus de culture des infusoires sans con- jugasion. C. R. Acad. Sc, 175:776. Metz, C. B.: (1946) Effects of various agents on the mating type substance of Paramecium aurelia variety 4. Anat. Rec, 93:347. (1947) Induction of "pseudo selfing" and meiosis in Para- mecium aurelia by formalin killed animals of opposite mating type. J. Exp. Zool., 105:115. (1948) The nature and mode of action of the mating type substances. Am. Nat., 82:85. and Butterfield, Winifred: (1950) Extraction of a mating reaction inhibiting sgent from Paramecium calkinsi. Proc. Nat. Acad. Sc, 36:268. Mouton, H.: (1902) Recherches sur la digestion chez les amibes, etc. Ann. Inst. Pasteur, 16:457. Muller, W. : (1932) Cytologische und vergleichend-physiologische Untersuchungen ueber Paramecium, etc. Arch. Protist., 78:361. Mulsow, K. : (1911) Ueber Fortpflanzungserscheinungen bei Mono- cystis rostrata, n. sp. Ibid., 22:20. Naville, A. : (1925) Recherches sur le cycle sporogonique des Aggre- gata. Rev. Suiss. Zool., 32:125. Noble, E. R. : (1947) Cell division in Entamoeba gingivalis. Univ. California Publ. Zool., 53:263. Noland, L. E.: (1927) Conjugation in the ciliate Metopus sygmoides. J. Morphol. Physiol., 44:341. Oehler, R. : (1916) Amoebenzucht auf reinem Boden. Arch. Pro- tist., 37:175. Patten, M. W. : (1921) The life history of an amicronucleate race of Didinium nasutum. Proc. Soc. Exper. Biol., 18:188. Penn, A. B. K.: (1927) Reinvestigation into the cytology of conju- gation in Paramecium caudatum. Arch. Protist., 89:46. Powers, E. L.: (1943) The mating types of double animals in Eu- plotes patella. Am. Midland Nat., 30:175. Prandtl, H.: (1906) Die Konjugation von Didinium nasutum. Arch. Protist., 7:251. Raabe, H.: (1946) L'appareil nucleaire d'Urostyla grandis. I. Ann. Uni. Mar. Curie-Ski., Lublin, Sec. C, 1:18. (1947) II. Ibid., 1:151. Rafalko, J. S.: (1947) Cytological observations on the amoebo- flagellate, Naegleria gruberi. J. Morphol., 81:1. Reichenow, E.: (1928) Ergebnisse mit der Nuclealfarbung bei Pro- tozoen. Arch. Protist., 61:144. (1929) In: Doflein-Reichenow's Lehrbuch der Protozoen- kunde. Jena. Reynolds, Mary E. C: (1932) Regeneration in an amicronucleate infusorian. J. Exper. Zool, 62:327. Rhumbler, L.: (1888) Die verschiedenen Cystenbildungen und die Entwicklungsgeschichte der holotrichen Infusoriengattung Col- poda. Zeitschr. wiss. Zool., 46:449. Rosenberg, L. E.: (1938) Cyst stages of Opisthonecta henneguyi. Tr. Am. Micr. Soc, 57:147. REPRODUCTION 221 Schmahl, O.: (1926) Die Neubildung des Peristoms bei der Teilung von Bursaria truncatella. Arch. Protist., 54:359. Singh, B. N.: (1941) The influence of different bacterial food sup- plies on the rate of reproduction in Colpoda steini, etc. Ann. Appl. Biol, 27:65. (1948) Studies on giant amoeboid organisms. I. J. Gen. Mi- crob., 2:8. Sokoloff, B.: (1924) Das Regenerationsproblem bei Protozoen. Arch. Protist., 47:143. Sonneborn, T. M. : (1937) Sex, sex inheritance and sex determina- tion in Paramecium aurelia. Proc. Nat. Acad. Sc., 23:378. (1938) Mating types in Paramecium aurelia, etc. Proc. Am. Phil. Soc, 79:411. (1939) Paramecium aurelia: mating types and groups, etc. Am. Nat., 73:390. (1940) The relation of macronuclear regeneration in Para- mecium aurelia to macronuclear structure, etc. Anat. Rec 78: 53. (1941) The occurrence, frequency and causes of failure to undergo reciprocal cross-fertilization, etc. Ibid., 81, Suppl.:66. — — — (1942) Sex hormones in unicellular organisms. Cold Spr. Harb. Symp. Quant. Biol., 10:111. (1942a) Inheritance in ciliate Protozoa. Am. Nat., 76:46. (1943) Gene and cytoplasm. I. Proc. Nat. Acad. Sc, 29: 329. (1943a) II. Ibid., 29:338. (1944) Exchange of cytoplasm at conjugation in Paramecium aurelia, variety 4. Anat. Rec, 89:49. (1947) Recent advances in the genetics of Paramecium and Euplotes. Adv. Genetics, 1:263. (1950) The cytoplasm in heredity. Heredity, 4:11. and Dippell, Ruth V.: (1943) Sexual isolation, mating types, and sexual responses to diverse conditions in variety 4, Paramecium aurelia. Biol. Bull., 85:36. (1946) Mating reactions and conjugation between varieties of Paramecium aurelia, etc. Physiol. Zool., 19:1. Sprague, V.: (1941) Studies on Gregarina blatlarum, etc., 111. Biol. Monogr., 18, no. 2. Stein, F.: (1867) Der Organismus der Infusionsthiere. Pt. 2:1. Stolte, H. A.: (1922) Verlauf, Ursachen und Bedeutung der En- cystierung bei Blepharisma. Verh. deutsch. zool. Gesell., 27:79. Stuart, C. A., Kidder, G. W. and Griffin, A. M.: (1939) Growth studies on ciliates. III. Physiol. Zool., 12:348. Summers, F. M.: (1935) The division and reorganization of the macronuclei of Aspidisca lynceus, etc Arch. Protist., 85: 173. (1941) The Protozoa in connection with morphogenetic problems. In: Calkins and Summers' Protozoa in biological re- search. Swezy, Olive: (1922) Mitosis in the encysted stages of Entamoeba coli. Univ. Calif orina Publ. Zool., 20:313. Tartar, V. and Chen, T. T.: (1941) Mating reactions of enucleate fragments in Paramecium bursaria. Biol. Bull., 80:130. 222 PROTOZOOLOGY Taylor, C. V. and Strickland, A. G. R.: (1938) Reactions of Col- poda duodenaria to environmental factors. I. Arch. Protist., 90: 398. Thimann, K. V. and Barker, H. A.: (1934) Studies on the excyst- ment of Colpoda cucullus. II. J. Exper. Zool., 69:37. and Haagen-Smit, A. J.: (1937) Effects of salts on emer- gence from the cyst in Protozoa. Nature, 140:645. Thon, K.: (1905) Ueber den feineren Bau von Didinium nasutum. Arch. Protist., 5:282. Turner, J. P.: (1930) Division and conjugation in Euplotes patella, etc. Univ. California Publ. Zool., 33:193. von Brand, T. : (1923) Die Encystierung bei Vorticella microstoma und hypotrichen Infusorien. Arch. Protist., 47:59. Weisz, P. B.: (1948) Time, polarity, size and nuclear content in the regneration of Stentor fragments. J. Exper. Zool., 107:269. (1950) Multiconjugation in Blepharisma. Biol. Bull., 98: 242. (1950a) A correlation between macronuclear thymonucleic acid concentration and the capacity of morphogenesis in Sten- tor. J. Morphol., 87:275. (1951) An experimental analysis of morphogenesis in Stentor coeruleus. J. Exper. Zool., 116:231. (1951a) A general mechanism of differentiation based on morphogenetic studies in ciliates. Am. Nat., 85:293. Wenrich, D. H.: (1939) Studies on Dientamoeba fragilis. III. J. Parasitol., 25:43. Weschenfelder, R.: (1938) Die Entwicklung von Actinocephalus parvus. Arch. Protist., 91:1. Wichterman, R. : (1936) Division and conjugation in Nyctotherus cordiformis, etc. J. Morphol., 60:563. (1940) Cytogamy: a sexual process occurring in living joined pairs of Paramecium caudatum, etc. Ibid., 66:423. (1946) Further evidence of polyploidy in the conjugation of green and colorless Paramecium bursaria. Biol. Bull., 91:234. Wilson, E. B.: (1928) The cell in development and heredity. New York. Wolff, E.: (1927) Un facteur de l'enkystment des amibes d'eau douce. C. R. Soc. Biol., 96:636. Woodruff, L. L.: (1921) Micronucleate and amicronucleate races of Infusoria. J. Exper. Zool., 34:329. (1931) Micronuclear variation in Paramecium bursaria. Quart. J. Micr. Sc, 74:537. (1932) Paramecium aurelia in pedigree culture for 25 years. Tr. Am. Micr. Soc, 51:196. and Erdmann, Rhoda: (1914) A normal periodic reorganiza- tion process without cell fusion in Paramecium. J. Exper. Zool., 17:425. and Spencer, H.: (1921) The survival value of conjugation in the life history of Spathidium spathula. Proc. Soc. Exper. Biol., 18:303. Chapter 6 Variation and heredity IT IS generally recognized that individuals of all species of organ- ism vary in morphological and physiological characteristics. Pro- tozoa are no exception, 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. Wesen- berg-Lund (1908) noticed a definite seasonal morphological variation in Cerctium hirundinella in Danish lakes, while Schroder (1914) found at least nine varieties of this organism (Fig. 94) occurring in various bodies of water in Europe, and List (1913) reported 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 inhabiting the same deep lakes; namely, individuals from the deep water may reach 200m in length, while those from the surface layer measure only about 100^ 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, Orbulina universa inhabiting surface water has a com- paratively thin shell, 1.28-18^ thick, while individuals living on the bottom have a thick shell, up to 24/x in thickness. According to Uyemura, a species of Amoeba living in thermal waters, showed a distinct dimensional difference in different springs. It measured 10— 40/x in diameter in sulphurous water and 45-80^ 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 usually dis- appear 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 al- ready been mentioned (p. 109). Chlamydomonas debaryana are repre- sented by many races differing in form, size, and structure, in various localities as well as under different laboratory conditions. Moewus 223 224 PROTOZOOLOGY (1934) distinguished 12 such varieties and showed that any variety could be changed into another by using different culture media. This transformation, however, did not occur at the same rate among dif- ferent races. It was found that the longer a strain has remained under Fig. 94. Varieties of Ceratium hirundinella from various European waters (Schroder), a, furcoides-type (130-300> by 30-45/x); b, brachy- ceroides-type (130-145/z by 30-45^); c, silesiacum-type (148-280/x by 28— 34ju) ; d, carinthiacum-type (120-145/z by 45-60/x); e, gracile-type (140-200/* by 60-75/x); f, austriacum-type (120-160/x by 45-60/x); g, robustum-type (270-310/x by 45-55/x); h, scotticum-type (160-210/z by 50-60m); i, piburgense-type (180-260/* by 50-60/x). conditions producing a given type, the greater the time and the num- ber of generations needed to change it to a new type under a new condition, as is shown in Table 9. 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- VARIATION AND HEREDITY 225 Table 9. — 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 1 to type 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 acters. To the last-mentioned category belongs perhaps a strain of Tetrahymena pyriformis in which, according to Furgason (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 became more marked in older cultures. The first comprehensive study dealing with the variation in size and its inheritance in asexual reproduction of Protozoa was conducted by Jennings (1909). From a "wild" lot of Paramec- ium caudatum, eight races or biotypes with the relative mean lengths of 206, 200, 194, 176, 142, 125, 100, and 45/x 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 progeny 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 corona, 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 operation, and found that operated mouth or teeth were restored to 226 PROTOZOOLOGY 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." Races or strains have been recognized in almost all intensively studied Protozoa. For example, Ujihara (1914) and Dobell and Jepps (1918) noticed five races in Entamoeba histolytica on the basis of dif- ferences 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. Sapiro, Hakansson and Louttit (1942) similarly notice two races which can be distinguished by the diameters of cysts, the division line being 10/x and 9m in living and balsam-mounted specimens respectively. The race with large cysts gives rise to trophozoites which are more actively motile, ingest erythrocytes, and culture easily, is patho- genic to man and kitten, while the race with small cysts develops into less actively motile amoebae which do not ingest erythrocytes and are difficult to culture, is not pathogenic to hosts, thus not being histozoic. It is interesting to note, however, that Cleveland and Sanders (1930) found the diameter of the cysts produced in a pure- line culture of this sarcodinan, which had originated in a single cyst, varied from 7 to 23m- Furthermore, the small race of Frye and Meleney mentioned above was later found by Meleney and Zucker- man (1948) to give rise to larger forms in culture, which led the last two observers to consider that the size range of the strains of this amoeba is a characteristic which may change from small to large or vice versa under different environmental conditions. Investigations by Boyd and his co-workers and others show that the species of Plasmodium appear to be composed of many strains which vary in diverse physiological characters. In an extended study on Trypanosoma 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- VARIATION AND HEREDITY 227 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. The two strains of T. cruzi isolated from human hosts and maintained for 28 and 41 months by Hauschka (1949), showed well defined and constant strain-specific levels of virulence, different degrees of affinity for certain host tissues, unequal suscepti- bility to the quinoline-derivative Bayer 7602, and a difference in re- sponse to environmental temperature. The five strains of Tricho- monas gallinae studied by Stabler (1948) were found to possess a marked variation in virulence to its hosts. According to Kidder and his associates, the six strains (H, E, T, T-P, W, GHH) of Tetrahymena pyriformis and the two strains (V, PP) of T. vorax differ in biochemical reactions. They found the ap- pearance of a biochemical variation between a parent strain (T) and a daughter strain (T-P) during a few years of separation and a greater difference in the reactions between the two species than that between the strains of each species. These strains show further dif- ferences in antigenic relationships. Five strains of pyriformis con- tain qualitatively identical antigens, but differ quantitatively with respect to amount, concentration or distribution of antigenic ma- terials. The sixth strain (T) contains all the antigens of the other five strains and additional antigens. The two strains of vorax are said to be nearly identical antigenically. The antigenic differences between the two species were marked, since there is no cross-reaction within the standard testing time. In these cases, thus, some aspects of the physiological difference among different strains are understood. Jollos (1921) subjected Paramecium caudatum to various environ- 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 228 PROTOZOOLOGY 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 brucei loses its blepharoplast when inoculated into mice which have been treated with pyronin, acridin, oxazin and allied dyes, and Piekarski (1949) showed that trypaflavin and organic metal com- pounds which act as nuclear poisons and interfere with nuclear di- vision, also bring about the loss of blepharoplast in this trypano- some. Laveran and Roudsky (1911) found that the dyes mentioned above have a special affinity for, and bring about the destruction by auto-oxidation of, the blepharoplast. Such trypanosomes lacking a blepharoplast behave normally and remain in that condition during many passages through mice. When subjected to small doses of cer- tain 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 modifications 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 mobilis (Fig. 95, b) which was formed by a complete fusion of two conjugants. This abnormal animal underwent fission 367 times for 405 days, but finally reverted back to normal forms, without reversion to double form. The double animal of Euplotes patella (d) is, according to Kim- ball (1941) and Powers (1943), said to be formed by incomplete di- vision and rarely through conjugation. De Garis (1930) produced double animals in Paramecium caudatum through inhibition of di- vision by exposing the animals to cyanide vapor or to low tempera- tures VARIATION AND HEREDITY 229 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- Fig. 95. a-c, Uroleptus mobilis (Calkins) (a, a pair in conjugation; b, an individual from the third generation by division of a double organism which had been formed by the coalescence of a conjugating pair; c, a product of reversion); d, a double animal of Euplotes patella (Kimball). 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, 1934; Moewus, 1934; Sonneborn, 1947). 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 230 PROTOZOOLOGY were inherited through successive generations and hence considered as mutations. The mutants were triploid, tetraploid, and tailed diploid forms (Fig. 96), 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. Fig. 96. Chilodonella uncinata (MacDougall). a, b, ventral and side view of normal individual; c, d, ventral and side view of the tailed mutant. Kimball (1950) exposed Paramecium aurelia to beta particles from plaques containing P 32 and obtained many clones which multiplied more slowly than normal animals or died, which conditions were interpreted by him to be due to mutational changes induced in the micronuclei by the radiation. Kimball found that the radiation was less effective if given just before the cytoplasmic division than if given at other times during the division interval and that exposure of the organisms to ultraviolet ray of wave length 2537 A inactivates the Kappa (p. 239). The loss of the blepharoplast in trypanosomes mentioned above occurs also spontaneously in nature. A strain of Trypanosoma evansi which had been maintained in laboratory animals for five years, sud- denly lost the blepharoplast (Wenyon, 1928) which condition re- mained for 12| years (Hoare, 1940). Hoare and Bennett (1937) found five camels out of 100 they examined infected by the same species of trypanosome that was without a blepharoplast. One strain inocu- lated into laboratory animals has retained this peculiarity for nearly VARIATION AND HEREDITY 231 three years. Nothing is known as to how such strains arise, though some workers suggest mutational change. In sexual reproduction, the nuclei of two individuals participate in producing new combinations which would naturally bring about diverse genetic constitutions. The new combination is accomplished 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 the most complete information was obtained by Moewus through his extended studies of certain Phytomonadina. In Polytoma (p. 281), Chlamydomonas (p. 276), and allied forms, the motile indi- viduals are usually haploid. Two such individuals (gametes) fuse with each other and produce a diploid zygote which encysts. 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 multiply asexually by division for many generations, the descendants 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. 97, a). Form B: Oval (F), without papilla (p), without stigma (s), large (D) (Fig. 97, b). P. pascheri Form C: Pyriform (f), with papilla (P), without stigma (s), large (D) (Fig. 97, c). Form D: Pyriform (f), with papilla (P), without stigma (s), small (d) (Fig. 97, d). 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 232 PROTOZOOLOGY of course found. It was found that about half the zygotes gives rise to the two parental combinations (Fig. 97, b, c), while the other half gives rise to FPsD (e) and fpsD (/). 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. a b c d e ( Fig. 97. a, b. Polytoma uvella. a, Form A; b, P^orm B. c, d. P. pascheri. c, Form C; d, Form D. e, f. Crosses between Forms B and C. (Moewus) 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. 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 VARIATION AND HEREDITY 233 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 Chlamydo?no?ias 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, etc. The discovery of mating types in Paramecium and in Euplotes, and in- tensive studies of conjugation and related phenomena, are bringing to light hitherto unknown information on some of the fundamental problems in genetics. Sonneborn (1939) has made extended studies of variety 1 of Paramecium aurelia (p. 194) and found that genetically diverse ma- terials show different types of inheritance, as follows: (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 therefore con- siders that the mating types are determined by macronuclei, as 234 PROTOZOOLOGY judged by segregation at first or sometimes second division in excon- jugants 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-type condition, thus approximating an expected Men- delian ratio of 3 dominants to 1 recessive. That the F 2 dominants are composed of two-thirds heterozygotes (Aa) and one-third homo- zygotes (AA) was confirmed by the results obtained by allowing F 2 dominants to conjugate with the recessive parent stock (aa). Of 19 pairs of conjugants, 6 pairs gave rise to only dominant progenj^, which shows that they were homozygous (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 (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 considered only on cytological ground. In Euplotes patella, Kimball (1942) made various matings with respect to the inheritance of the mating type. The results obtained can be explained if it is assumed that mating types I, II, and V, are determined by different heterozygous combinations of three allelic genes which if homozygous determine mating types III, IV, and VI. VARIATION AND HEREDITY 235 Upon this supposition, type I has one allele in common with type II, and this allele is homozygous in type IV. It has one allele in common with type V, and this allele is homozygous in type VI. Type II has one allele in common with type V and this is homozygous in type III. These alleles were designated by Kimball, mt 1 , mt 2 , and mt 3 . The genotypes of the six mating types may be indicated as follows: imVmtMI), rn^mt 3 (II), mt 3 mt 3 (III), mtfmt 1 (IV), mt 2 mt 3 (V), and mt 2 mt 2 (VI). 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 combi- nation. Kimball (1939, 1941) had shown that the fluid obtained free of Euplotes from a culture of one mating type will induce conjuga- tion 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 induces 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 may be 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 double animals of Euplotes patella (p. 228) conjugate with double animals or with single animals in appropriate mixtures and at times a double animal gives rise by binary fission to a double and two single animals instead of two animals (Fig. 98). Powers (1943) obtained doubles of various genotypes for mating types which were determined by observing the mating type of each of the two singles that arose from the doubles. Doubles of type IV (m^mt 1 ) with a single micronucleus (Fig. 98, a) were mated with singles of type VI (mt 2 mt 2 ) (6). The double exconjugants (d) were "split" into their component singles belonging to mating types IV and VI (g), while the doubles were type I (/) . Thus it was found that the phenotype of a double animal with separate nuclei was the same as though the alleles present in the nuclei were located within one nucleus. The fact that loss of one micronucleus had no effect on the type of doubles, tends to show that the micronucleus has no direct effect on 236 PROTOZOOLOGY mating types. Sonneborn's view (p. 233) that the macro-nucleus is the determiner of the mating types in Paramecium aurelia appears to hold true in Euplotes also. The relation between the cytoplasm and nucleus in respect to in- heritance has become better known in recent years in some ciliates. Sonneborn (1934) crossed two clones of Paramecium aurelia differing markedly in size and division rate, and found the difference persisted Type VI Type J Type I Type I Type VI Fig. 98. Diagram showing conjugation between a double (type IV) and a single (type VI) of Euplotes patella (Powers), a, a double organism with one micronucleus (genotype mt'mt 1 ); b, a normal single with a mi- cronucleus (genotype mt 2 mt 2 ); c, conjugation of the single with the ami- cronucleate half of the double (one of the pronuclei produced in the sin- gle migrates into the double, while the two pronuclei of the double un- dergo autogamy); d, the exconjugant double is shown to be type I (mtmit 2 ); e, exconjugant single remains type VI; f, the double divides into two type I doubles; g, occasionally the anterior half of the double is widely "split," and division produces a double and two singles, the latter testing as type IV and type VI; h, line of exconjugant single. Newly formed macronuclei are stippled. VARIATION AND HEREDITY 237 for a time between the two Fi clones produced from the two mem- bers of each hybrid pair of exconjugants, but later both clones be- came practically identical in size and division rate (Sonneborn, 1947). De Garis (1935) succeeded in bringing about conjugation in Paramecium caudatum, between the members of a large clone (198m long) (Fig. 99, a) and of a small clone (73ju long) (b). The excon- jugants of a pair are different 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. However, as division is repeated, the descend- 0." oooooOOOOO Fig. 99. Diagram showing the size changes in two clones derived from a pair of conjugants of Paramecium caudatum, differing in size (a, b). Gradual change in dimensions in each clone during 22 days resulted in intermediate size (Jennings) . ants of the large clone become gradually smaller after successive fissions, while the descendants of the small clone become gradually larger, until at the end of 22 days (in one experiment) both clones produced individuals of intermediate size (about 135ju long) which remained in the generations that followed. Since the exconjugants differed in the cytoplasm only, it must be considered probable that at first the cytoplasmic character was inherited through several vegetative divisions, but ultimately the influence of the new nucleus gradually changed the cytoplasmic character. The ultimate size be- tween the two clones is however not always midway between the mean sizes of the two parent clones, and is apparently dependent upon the nuclear combinations brought about by conjugation. It has also become known that different pairs of conjugants between the same two clones give rise to diverse progeny, similar to those of sexual reproduction in Metazoa, which indicates that clones of Para- 238 PROTOZOOLOGY mecium caudatum are in many cases heterozygous for size factors and recombination of factors occurs 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 excellent review writes: "The primary source of diversities in inherited characters lies in the nucleus. But the nucleus by known material interchanges im- presses 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." Since the first demonstration some forty years ago of "cytoplas- mic inheritance" in higher plants, many cytoplasmic factors have been observed in various plants (Michaelis and Michaelis, 1948). Information on similar phenomena in Metazoa and Protozoa is of recent origin. As was already mentioned (p. 196), Sonneborn found in four races of variety 4 of Paramecium aurelia a pair of characters which he designated as "killer" and "sensitive." The killers liberate para- mecin, a desoxyribonucleoprotein (Wagtendonk and Zill, 1947), into the culture fluid, to which they are resistant. When the sensitive races are exposed to paramecin in the fluid in which the killer race 51 lived, they show after hours a hump on the oral surface toward the posterior end which becomes enlarged, while the anterior part of the body gradually wastes away. The body becomes smaller and rounded; finally the organisms perish (Fig. 100). Sensitives can be mated to the killers, however, without injury if proper precaution is taken, since paramecin does not affect them during conjugation. The two exconjugants obtain identical genotypes, but their progeny VARIATION AND HEREDITY 239 are different; that is, one is a killer and the other is a sensitive culture. F 2 progeny obtained by selfing show no segregation. There- fore, the difference between the killer and the sensitive is due to a cytoplasmic difference and not to a genie difference. The same observer noted that the thin cytoplasmic paroral strand which appears between conjugating pair that ordinarily breaks off within a minute, occasionally may remain for a long time, and if the strand persists as long as 30 minutes, there occurs an interchange of cytoplasm between the pair (Fig. 101). When this happens, both exconjugants produce killer clones. In F 2 no segregation takes place. Thus killers can introduce the killer trait to sensitives through a cytoplasmic connection between them. Sonneborn supposed that the killers contain a cytoplasmic genie factor or a plasmagene which de- Fig. 100. Paramecium aurelia. The changes leading up to death when the sensitives are exposed to the killer stock 51 (variety 4) (Sonneborn). termines the killer trait and called it kappa. Preer (1948) demon- strated that this kappa is a particle which can be recognized in Giemsa-stained specimens (Fig. 102). It was further found that kill- ers can be irreversibly transformed into hereditary sensitives by eliminating kappa particles by exposure to high temperature (Sonne- born, 1946), x-irradiation (Preer, 1948b) or nitrogen mustard (Geck- ler, 1949) and that sensitives can be transformed to hereditary killers by placing them in concentrated suspensions of broken bodies of killers (Sonneborn, 1948a). Therefore, it became clear that kappa is a self-multiplying cytoplasmic body which is produced when some are already present. Killer races of variety 2 differ from each other and from that of variety 4 mentioned above, in the effects produced on sensitives be- fore the latter are killed. These sensitives possess a gene different from that of the killers and cannot be changed into killers by im- mersing it to kappa suspensions of broken bodies of killers. When this sensitive is mated with a killer, F 2 generation produced by self- 240 PROTOZOOLOGY ing among the killer Fi clones, shows segregation of sensitives and killers in the ratio of a single gene difference. In the presence of dominant gene K, kappa is maintained, but in recessive k homozy- gotes, kappa cannot be maintained and any kappa carried over from killers is rapidly lost. Thus it is evident, Sonneborn points out, that the plasmagene kappa is dependent on gene K. Dipell (1948, 1950) found a number of killer mutants in variety 4. End oi- conjuqa+iorv.: Separated except at paroral cone. Time until separation is completed More +h