u n 6^-^ l^ iO = ^ s ^ r^ = = CO ■■"i — r^ o^s ^s i-n r S^ r-q = i n- r D fY\ n- PROTOZOOLOGY PROTOZOOLOGY <^1 ^^' By RICHARD ROKSABRO KUDO, D.Sc. Associate Professor of Zoology The University of Illinois Enlarged and completely rewritten edition of HANDBOOK OF PROTOZOOLOGY With two hundred and ninety-one illustrations n 3^ CHARLES C THOMAS • PUBLISHER SPRINGFIELD- ILLINOIS • BALTIMORE • MARYLAND COPYRIGHT, 1931, BY CHARLES C THOMAS COPYRIGHT, 1939, BY CHARLES C THOMAS Printed in the United States of A merica All rights reserved. This book may not be re- produced, in whole or in part, in any form (ex- cept by reviewers for the public press), with- out written permission from the publisher. " 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 Preface THE present work is similar in its primary aim to that of its predecessor, Handbook of Protozoology (1931), in presenting "introductory information on the common and representative genera of all groups of both free-living and parasitic Protozoa," to advanced undergraduate and graduate students in zoology in colleges and universities. With the expansion of courses in proto- zoology at the University of Illinois and elsewhere, it seemed ad- visable to incorporate more material for lecture and discussion, in addition to the enlargement of the taxonomic section. The change of the text-contents has, therefore, been so extensive that a new title. Protozoology , is now given. Chapters 1 to 6 deal with introduction, ecology, morphology, physiology, reproduction, and variation and heredity, of Proto- zoa, Each subject-matter has been considered in the light of more recent investigations as fully as the space permitted. Selection of material from so great a number of references has been a very difficult task. If any important papers have been omitted, it was entirely through over-sight on the part of the author. The taxonomic portion (Chapters 7 to 43) has also been com- pletely rewritten and enlarged. Numerous genera and species, both old and new, have been added; synonymy of genera and species has as far as possible been brought down to date; new taxonomic arrangement of major and minor subdivisions in each class has resulted in numerous changes. The class Ciliata has completely been reclassified, following Kahl's admirable work on free-living ciliates (1930-1935); however, unlike the latter, all parasitic ciliates have also been considered in the present work. The author continues to believe that good illustrations are in- dispensable in this kind of work, since they are far more easily comprehended than lengthy descriptions. Therefore, many old illustrations have been replaced by more suitable ones and nu- merous new illustrations have further been added. All illustrations were especially prepared for this work and in the case of those which have been redrawn from illustrations found in published papers, the indebtedness of the author is indicated by mention- ing the names of the investigators from whose works the illustra- viii PR?:FACK tions were taken. In order to increase the reference value, all figures are accompanied by scales of magnification which are uni- formly somewhat greater than those of Handbook of Protozool- ogy, since the microscope now used in the class-room has been improved upon in recent years. The list of references appended to the end of each chapter has been enlarged and is meant to aid those who wish to obtain fuller information than that w^hich is given in this volume. Since com- prehensive monographs on various groups of Protozoa are widely scattered and ordinarily not easily accessible, the author has en- deavored to provide for each group as complete an information as possible for general reference purpose within the limited space, and hopes that the present work has reference value for teachers of biology, field workers in pure and applied biological sciences, veterinarians, physicians, public health workers, laboratory tech- nicians, and others. The author is under obligation to numerous writers for their valuable contributions which have been incorporated in the text. Special thanks are due Professor L. R. Cleveland, Harvard Uni- versity; Professor R. P. Hall, New York University; Professor H. Kirby, Jr., University of California; Professor L. E. Noland, University of Wisconsin; Professor H. J. Van Cleave, University of Illinois; Professor D. H. Wenrich, University of Pennsylvania; and Professor L. L. Woodruff, Yale University, for their valued criticisms and suggestions. The author further wishes to express his appreciation to Mr. Charles C Thomas, for his patient and kind cooperation which has aided greatly in the completion and ap- pearance of the present work. R.R.K. Urbana, Illinois, U.S.A. July, 1939 Contents Preface ^^ Chapter 1 Introduction ^ Relationship of protozoology to other fields of biological science, p. 5; the history of proto- zoology, p. 9. 2 Ecology 1^ The free-living Protozoa, p. 16; the parasitic Protozoa, p. 23. 3 Morphology ^1 The nucleus, p. 32; the cytosome, p. 36; loco- motor organellae, p. 40; fibrillar structures, p. 51 ;protective or supportive organellae,p. 59; hold-fast organellae, p. 65; the parabasal ap- paratus, p. 66; the blepharoplast or centriole, p. 67; the Golgi apparatus, p. 69; the chon- driosomes, p. 71; the contractile and other vacuoles, p. 73; the chromatophore and asso- ciated organellae, p. 78. 4 Physiology ^^ Nutrition, p. 84; the reserve food matter, p. 94; respiration, p. 96; excretion and secretion, p. 98; movements, p. 101; irritability, p. 109; regeneration, p. 114. 5 Reproduction 11° Nuclear division, p. 118; cytosomic division, p. 137; colony formation, p. 140; asexual re- production, p. 142; sexual reproduction and life-cycles, p. 143. 6 Variation and heredity 162 7 Phylum Protozoa 1^1 Subphylum 1 Plasmodroma 171 Class 1 Mastigophora 171 Subclass 1 Phytomastigina 173 Order 1 Chrysomonadina 173 ix CONTENTS 8 Order 2 Cryptomonadina 184 9 Order 3 Phytomonadina 188 10 Order 4 Euglenoidina 203 Order 5 Chloromonadina 213 11 Order 6 Dinoflagellata 216 12 Subclass 2 Zoomastigina 235 Order 1 Rhizomastigina 235 13 Order 2 Protomonadina 239 14 Order 3 Polymastigina 260 15 Order 4 Hypermastigina 277 16 Class 2 Sarcodina 288 Subclass 1 Rhizopoda 289 Order 1 Proteomyxa 289 17 Order 2 Mycetozoa 296 18 Order 3 Amoebina 304 19 Order 4 Testacea 323 20 Order 5 Foraminifera 344 21 Subclass 2 Actinopoda 356 Order 1 Heliozoa 356 22 Order 2 Radiolaria 367 23 Class 3 Sporozoa 377 Subclass 1 Telosporidia 378 Order 1 Gregarinida 378 24 Order 2 Coccidia 415 25 Order 3 Haemosporidia 434 26 Subclass 2 Acnidosporidia 446 Order 1 Sarcosporidia 446 Order 2 Haplosporidia 448 27 Subclass 3 Cnidosporidia 453 Order 1 Myxosporidia 454 Order 2 Actinomyxidia 468 28 Order 3 Microsporidia 472 Order 4 Helicosporidia 479 29 Subphylum 2 Ciliophora 481 Class 1 Ciliata 481 Subclass 1 Protociliata 483 30 Subclass 2 Euciliata 487 Order 1 Holotricha 487 Suborder 1 Astomata 488 31 Suborder 2 Gymnostomata 496 CONTENTS xi Tribe 1 Prostomata 496 32 Tribe 2 Pleurostomata 517 Tribe 3 Hypostomata 522 33 Suborder 3 Trichostomata 531 34 Suborder 4 Hymenostomata 547 35 Suborder 5 Thigmotricha 560 36 Suborder 6 Apostomea 567 37 Order 2 Spirotricha 573 Suborder 1 Heterotricha 573 38 Suborder 2 Oligotricha 587 39 Suborder 3 Ctenostomata 600 40 Suborder 4 Hypotricha 603 41 Order 3 Chonotricha 614 42 Order 4 Peritricha 616 43 Class 2 Suctoria 628 Author and Subject Index 643 PROTOZOOLOGY Chapter 1 Introduction PROTOZOA are unicellular animals. The body of a protozoan is morphologically a single cell and manifests all character- istics common to the Hving thing. The various activities which make up the phenomena of life are carried on by parts within the body or cell. These parts are comparable with the organs of a metazoan which are composed of a large number of cells grouped into tissues and are called organellae or cell-organs. Thus one sees that the one-celled protozoan is a complete organism some- what unhke the cell of a metazoan, each of which is dependent upon other cells and cannot live independently. From this view- point, certain students of protozoology maintain that the Proto- zoa are non-cellular, and not unicellular, organisms. Dobell (1911) for example, points out that the "cell" is employed to designate 1) the whole protozoan body, 2) a part of an organism and, 3) a potential whole organism (a fertilized egg) which consequently re- sulted 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 organism, differently organized as compared with cellular organisms, the Metazoa and Metaphyta. The great majority of protozoologists, however, con- tinue to consider the Protozoa as unicellular animals. Through the processes of organic evolution, they have undergone cyto- logical differentiation and the Metazoa histological differentia- tion. In being unicellular, the Protozoa and the Protophyta are alike. The majority of the Protozoa are quite clearly distinguish- able from the majority of the Protophyta on the basis of nuclear condition, method of nutrition, direction of division-plane, etc. While numerous Protophyta appear to possess scattered nuclear material or none at all, the Protozoa contain at least one nu- cleus. It is generally considered that the binary fission of the Protozoa and the Protophyta is longitudinal and transverse, re- spectively. A great majority of CiUata, however, multiply by transverse division. In general the nutrition of Protozoa is holo- 4 I'ROTOZOOLOGY zoic and of Protophyta, holophytic; but there are large numbers of Protozoa which nourish themselves by holo})hytic method. Thus an absolute and clean-cut separation of the two groups of unicellular organisms is not possible. Haeckel coined the name Protista to include these organisms in a single group, but this is not generally adopted, since it includes undoubted animals and plants, thus creating an equal amount of confusion between it and the animal or the plant. Recently Calkins (1933) excluded chromatophore-bearing Mastigophora from his treatment of Pro- tozoa, thus placing organisms similar in every way, except the presence or absence of chromatophores, in two different groups. This intermingling of characteristics between the two groups of microorganisms shows clearly their close interrelationship and suggests strongly their common ancestry. Although the majority of Protozoa are solitary and the body is composed of a single cell, there are a few forms in which the body is made up of more than one cell. These forms, which are called colonial Protozoa, are well represented by the members 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 several genera there may be differentiation of the individuals into reproductive and vegetative cells. Unlike the cells in a metazoan which form tissues, these vegetative cells of colonial Protozoa are not dependent upon other cells; therefore, they do not form any tissue. The reproductive cells produce zygotes through sexual fusion, which subsequently undergo repeated division and may produce a stage comparable with the blastula stage of a meta- zoan, but never reaching the gastrula stage. Thus colonial Pro- tozoa are'only cell-aggregates without histological differentiation and may thus be distinguished from the Metazoa. Between 15,000 and 20,000 species of Protozoa are known to man. From comparatively simple forms such as Amoeba, up to highly complicated organisms as represented by numerous cili- ates, the Protozoa vary exceedingly in their body organization, morphological characteristics, behavior, habitat, etc., which ne- cessitates a taxonomic arrangement for proper consideration as set forth in detail in chapters 7 to 43. INTRODUCTION 5 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 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 simphcity of unicellular organisms has proved to be offset by the complexity of their cell-structure, nevertheless any discussion of biological principles today must take into account the information obtained from studies of Pro- tozoa. It is now commonly recognized that adequate information on various types of Protozoa is a prerequisite to a thorough com- prehension of biology and to proper application of biological prin- ciples. Practically all students agree in holding that the higher types of animals have been derived from organisms which existed in the remote past and which probably were somewhat similar to the Protozoa of the present day. Since there is no sharp distinction between the Protozoa and the Protophyta or between the Pro- tozoa and the Metazoa, and since there are intermediate forms between the major classes of the Protozoa themselves, progress in protozoology contributes toward the advancement of our knowledge of the 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 varia- tion we get a generation a day, and we may keep unlimited num- bers of pedigreed stock in a watch glass that can be placed under the microscope." Morphological variations are encountered com- monly in all forms. Whether variation is due to germinal or en- vironmental conditions, is often difficult to determine. The recent discovery of the sex reaction types in Paramecium aurelia (Son- / 6 PROTOZOOLOGY neborn; Kimbell) and in P. hursaria (Jennings) will probably assist in bringing to light many genetic problems of Protozoa which have remained obscure in the past. Parasitic Protozoa are limited to one or more specific hosts. Through studies of the forms belonging to one and the same genus or species, the phylogenetic relation among the host ani- mals 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 in- stance, Thelohania legeri has been found widely in many species of anopheline mosquitoes only; T. opacita has, on the other hand, been found in culicine mosquitoes, although the larvae of the species belonging to these two genera live frequently in the same body of water. By observing certain intestinal Protozoa in some monkeys, Hegner obtained evidence of the probable phylogenetic relationship between them and other higher mammals. The re- lation of various Protozoa of the wood-roach to those of the ter- mite, as revealed by Cleveland and his associates, gives further proof that the Blattidae and the Isoptera are of the common origin. Study of a particular group of parasitic Protozoa and their hosts may throw light on the geographic condition of the earth in the remote past. The members of the genus Zelleriella are usually found in the colon of the frogs belonging to the family Leptodactylidae. Through an extensive study of these amphibi- ans from South America and Australia, Metcalf 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 con- vergent 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 Zelleri- ella, 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 gradations of nuclear division ranging from a simple amitotic division to a complex process comparable in every detail with the typical metazoan mitosis, so that a great part of our knowledge of cytology is based upon studies of pro- tozoan cells. INTRODUCTION 7 Through the studies of various investigators in the past forty years, it has now become known that numerous parasitic Protozoa occur in man. Entamoeba histolytica, Balantidium coli, and three species of Plasmodium, all of which are pathogenic to man, are ^\'idely distributed throughout the world. In certain restricted areas are found other pathogenic forms, such as Trypanosoma and Leishmania. Since all parasitic Protozoa presumably have originated in free-living forms and since our knowledge of the morphology, physiology and reproduction of the parasitic forms has largely been obtained through studies of the free-living organisms, a general knowledge of the entire phylum is necessary to understand the parasitic forms. Recent studies have further revealed that almost all domestic animals are hosts to numerous parasitic Protozoa, many of which are responsible for serious infectious diseases. Many of the forms found in domestic animals are morphologically indistinguishable from those occurring in man. Balantidium coli is now generally considered as a parasite of swine, and man is its secondary host. Knowledge of protozoan parasites is useful to medical practi- tioners, just as it is essential to veterinarians inasmuch as certain diseases in animals, such as Texas fever, dourine, nagana, black- head, coccidiosis, etc., are caused by protozoans. Sanitary betterment and improvement are fundamental re- quirements in the modern civilized world. One of man's necessi- ties is safe drinking water. The majority of Protozoa live freely in various bodies of water and some of them seem to be responsi- ble, if present in sufficiently large numbers, for giving certain odors to the waters of reservoirs or ponds (p. 95). But these Protozoa which are occasionally harmful are relatively small in number compared with those which are beneficial to man. It is generally understood that bacteria feed on various waste materi- als present in polluted water, but that upon reaching a certain population, they would cease to multiply and would allow the excess organic substances to undergo decomposition. Numerous holozoic Protozoa, however, feed on the bacteria and prevent them from reaching the saturation population. Protozoa thus seem to help indirectly in the purification of the water. Proto- zoology therefore must be considered as an important part of modern sanitary science. Young fish feed extensively on small aquatic organisms, such 8 PROTOZOOLOGY as larvae of insects, small crustaceans, annelids, etc., all of which depend largely upon Protozoa and Protophyta as sources of food supply. 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 often cause death to large num- bers of commercially important fishes. Success in fish-culture, therefore, requires among other things a thorough knowledge of Protozoa. Since Russel and Hutchinson suggested some thirty years ago that Protozoa are probably a cause of limitation of the numbers, and therefore the activities of bacteria in the soil and thus tend to 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 climates Protozoa are present commonly and active throughout the year. The exact relation between specific protozoans and bacteria in the soil is a matter which still awaits future investigations, although numerous ex- periments and observations have already been made. All soil in- vestigators should be acquainted with the biology and taxonomy of free-living protozoans. It is a matter of common knowledge that the silkworm and the honey bee suffer from protozoan infection known as micro- sporidiosis. Sericulture in southern Europe suffered great damages in the middle of the nineteenth century because of the "pebrine" disease, caused by the microsporidian, Nosema hombycis. During the first decade of the present century, another microsporidian, Nosema apis, was found to destroy a large number of honey bees. Methods of control have been developed and put into practice so that these microsporidian infections are at present not serious, even though they still occur. On the other hand, other Micro- sporidia are now known to infect certain insects, such as mosqui- toes and lepidopterous pests, which, when heavily infected, die sooner or later. Methods of destruction of these insects by means of chemicals are more and more used, but attention should also be given to utilization of the parasitic Protozoa and Protophyta for this purpose. While the majority of Protozoa lack permanent skeletal struc- tures and their fossil forms are unknown, there are at least two large groups in the Sarcodina which possess conspicuous shells INTRODUCTION 9 and which are found as fossils. They are Foraminifera and Ra- diolaria. From early palaeozoic times 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. Al- though there is no distinctive foraminiferan fauna characteristic of a given geologic period, there are certain peculiarities of fossil Foraminifera which distinguish one formation from the other. From this fact one can understand that knowledge of foraminifer- ous 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 abun- dantly in siliceous rocks of the palaeozoic and the mesozoic, and are also identified with the clays and other formations of the miocene. 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. Pro- tozoa are unobservable with the naked eye, so that we can easily understand why they were unknown prior to the invention of the microscope. Antony van Leeuwenhoek (1632-1723) is commonly recognized as the father of protozoology. Grinding lenses himself, Leeuwenhoek made more than four hundred microscopes, includ- ing one which, it is said, had a magnification of 270 times (Hart- ing). Among the many things he discovered were various Proto- zoa. According to Dobell (1932), Leeuwenhoek saw for the first time in history, free-living Protozoa in fresh water in 1674. Among them, he observed bodies "green in the middle, and before and behind white," w^hich Dobell interprets were Euglena. Between 1674 and 1716 he apparently observed numerous microscopic or- ganisms which he communicated to the Royal Society of London and which, as Dobell considered, were Vorticella, Stylonychia, Carchesium, Volvox, Haematococcus, Coleps, Kerona, Antho- physa, Elphidium, Polytoma, etc. According to Dobell, Huy- gens gave in 1678 "unmistakable descriptions of Chilodon(ella), Paramecium, Astasia and Vorticella, all found in infusions." Colpoda was seen by Bouonanni (1691) and Harris (1696) re- discovered Euglena. In 1718 there appeared the first treatise on microscopic organisms, particularly of Protozoa, by Joblot who emphasized the non-existence of abiogenesis by using boiled hay- 10 PROTOZOOLOGY infusions in which no Infusoria developed without exposure to the atmosphere. This experiment confirmed that of Redi who, twenty years before, 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. Trembly (1715) studied division in some ciliates, including probably Paramecium, which generic name was coined by Hill in 1752. Noctiluca was first described by Baker (1753). Rosel (1755) observed an amoeba, possibly Amoeba proteus or an allied form, which he called "der kleine Proteus," and also Vorticella, Stentor, and Volvox. Ledermiiller is said to have coined the term "Infusoria" in 1763 (Biitschli). By using the juice of geranium, Ellis (1770) caused the extrusion of the 'fins' (trichocysts) in Paramecium. Eichhorn (1783) observed the helio- zoan, Actinosphaerium, which now bears his name. O. F. Miiller described Ceratium a little later and published two works on the Infusoria (1786). Although he included unavoidably some Meta- zoa and Protophyta in his monographs, some of his descriptions and figures of Ciliata were so well done that they are of value even at the present time. At the beginning of the nineteenth century the cyclosis in Paramecium was brought to light by Gruithuisen. Goldfuss (1817) coined the term "Protozoa," including in it the coelente- rates. Ten years later there appeared d'Orbigny's systematic study of the Foraminifera, which he considered as microscopical cephalopods. In 1828 Ehrenberg began publishing his observa- tions on Protozoa and 1838 he summarized his contributions in Die Infusionsthierchen als vollkommene Organismen, in which he diagnosed genera and species so well that many of them still hold good. Ehrenberg excluded Rotatoria and Cercaria from Infu- soria. Through the studies of Ehrenberg the number of known Protozoa increased greatly; he, however, proposed the term "Polygastricha," under which he placed Mastigophora, Rhizo- poda, Ciliata, Suctoria, desmids, etc., since he believed that the food vacuoles present in them were stomachs. This hypothesis became immediately the center of controversy, which inciden- tally, together with the then-propounded cell theory and improve- ments in microscopy, stimulated researches on Protozoa. Dujardin (1835) took pains in studying the protoplasm of various Protozoa and found it ahke in all. He named it "sarcode." INTRODUCTION 11 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 "proto- plasm" was coined by Purkinje in 1840. 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 einfache Organization sich auf eine Zelle reduzieren lassen." Siebold sub- divided Protozoa into Infusoria and Rhizopoda. The sharp dif- ferentiation of Protozoa as a group certainly inspired numerous microscopists. As a result, various students brought forward several group names, such as Radiolaria (J. Miiller, 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 CiUata and Mastigophora (1854, 1859-1883) contain concise descriptions and excellent illustrations of numerous species. Haeckel (1873), who went a step further than Siebold by distinguishing between Pro- tozoa and Metazoa, devoted ten years to his study of Radiolaria, especially those of the Challenger collection, and described in his celebrated monographs more than 4000 species. In 1879 the first comprehensive monograph on the Protozoa of North America was put forw^ard by Leidy under the title of Freshwater Rhizopods of North America, which showed the wide distribution of many known forms of Europe and revealed a number of new and interesting forms. This w^ork was followed by Stokes' The freshwater Infusoria of the United States, which ap- peared in 1888. Blitschli (1880-1889) estabHshed 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 general protozoology. The painstaking researches by Maupas, on the conjugation of ciliates, corrected erroneous interpretation of the phenomenon observed by Balbi- ani some thirty years before and gave impetus to a renewed cyto- logical study of Protozoa. The variety in form and structure of the protozoan nuclei became the subject of intensive studies by several cytologists. Weismann (1881) put into words the immor- tality of the Protozoa. Schaudinn contributed much toward the cytological and developmental studies of Protozoa. 12 PROTOZOOLOGY In the first year of the present century, Calkins in the United States and Doflein in Germany wrote modern textbooks on pro- tozoology deahng with the biology as well as the taxonomy. Cal- kins initiated the so-called isolation pedigree culture of ciHates in order to study the physiology of conjugation and other phe- nomena connected with the life-history of the ciHates. The appli- cation of this method has been found very popular in recent years. 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 comprehension of the morphology, physiology, and develop- ment of the latter group obviously is 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 expected, 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 ob- serve a parasitic protozoan, for he observed, according to Dobell, 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 intestinalis in his own diarrhcsic stools; and in 1683, Opalina and Nyctotherus in the gut contents of frogs. There is no record of anyone having seen Protozoa living in other organ- isms until 1828, when Dufour's account of the gregarine from the intestine of coleopterous insects appeared. Some ten years later, Hake rediscovered the oocysts of Eimeria stiedae. A flagellate was observed in the blood of salmon by Valentin in 1841, and the frog trypanosome was discovered by Gluge and Gruby (1842), the latter author creating the genus Trypanosoma for it. The gregarines were a httle later given attention by Siebold (1839), 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 flagel- lates (Trichomonas and Chilomastix). Kloss in 1855 observed the INTRODUCTION 13 coccidian, Klossia helicina, in the excretory organ of Helix and Eimer (]870) made an extensive study of Coccidia occurring in various animals. Balantidium coli was discovered by Malm- sten in 1857. Lewis in 1870 observed Entamoeba coli in India, and Losch in 1875 found Entamoeba histolytica in Russia. At the be- ginning of the last century, an epidemic disease, pebrine, of the silkworm appeared in Italy and France, and a number of biolo- gists became engaged in its investigation. Foremost of all, Pas- teur (1870) made an extensive report on the nature of the causa- tive 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 to result in an effective prac- tical 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 coccidi- ans. The groups under Sporozoa were soon definitely designated. They are Myxosporidia (Biitschli, 1881), Microsporidia (Balbi- ani, 1882) and Sarcosporidia (Balbiani, 1882). Parasitic protozoology received a far-reaching stimulus when Laveran (1880) discovered the malarial parasite in the human blood. Smith and Kilborne (1893) demonstrated that the Babe- sia of the Texas fever of cattle in the southern United States was transmitted by cattle ticks from host to host, and thus brought to light for the first time the close relationship which exists be- tween an arthropod and a parasitic protozoan. Two years later, Bruce discovered Trypanosoma brucei in the blood of horses and cattle suffering from "nagana" disease in Africa, and in the fol- lowing year he showed by experiments that the tsetse fly trans- mits the trypanosome from host to host. Studies of malarial dis- eases 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 two types of fever. MacCallum (1897-1898) found in the United States the union of a microgamete and a macrogamete of Haemoproteus of birds. Almost at the same time, Schaudinn and Siedlecki (1897) showed that anisogamy results in the production of zygotes in Coccidia. The latter author published later correct observations on the Ufe-cycle of Coccidia (1898, 1899). Ross (1898) showed how Plasmodium praecox was carried by 14 PROTOZOOLOGY Culex fatigans and described its life-cycle. Since that time several investigators have brought to light imi)ortant observations con- cerning the biology and development of these organisms and their relation to man. In the present century, Forde and Dutton (1901) observed that the sleeping sickness in Africa is due to an infection by Trypanosoma gamhiense. In 1903 Leishman and Donovan recognized Leishmania of "kala-azar." Artificial cultivation of bacteria had contributed toward a very rapid advancemtnt in bacteriology, and it was natural, as the number of known parasitic Protozoa rapidly increased, that at- tempts to cultivate them in vitro should be made. Musgrave and Clegg (1904) cultivated, on bouillon-agar, small free-living amoe- bae from old fecal matter. In 1905 Novy and McNeal cultivated successfully the trypanosome of birds in blood-agar medium, which remained free from bacterial contamination and in which the organisms underwent multiplication. Almost all species of Trypanosoma and Leishmania have since been cultivated in a similar manner. This serves for detection of a mild infection and also identification of the species involved. It was found, further, that the changes which these organisms underwent in the culture media were imitative of those that took place in the invertebrate host, thus contributing toward the life-cycle studies of them. Bass (1911), and Bass and Johns (1912) demonstrated that Plasmodium of man could be cultivated in vitro for a few genera- tions. During and since the World War, it became known that numerous intestinal Protozoa of man are widely present through- out the tropical, subtropical and temperate zones. Taxonomic, morphological and developmental studies on these forms have therefore appeared in an enormous number. Cutler (1918) seems to have succeeded in cultivating Entamoeba histolytica, though his experiment was not repeated by others. Barret and Yarborough (1921) cultivated Balantidium coli and Boeck (1921) also culti- vated Chilomastix mesnili. Boeck and Drbohlav (1925) succeeded in cultivating Entamoeba histolytica, and their work was repeated and improved upon by several investigators. While the cultiva- tion has not yet thrown much light on this and similar amoebae, it reveals certain evidences that there is no sexual reproduction in these amoebae. Since that time, almost all intestinal Protozoa of both vertebrates and invertebrates have been cultivated by nu- merous investigators. INTRODUCTION 15 References BiJTSCHLi, O. 1887-1889 Bronn's Klassen und Ordnungen des Thier-reichs. Vol. 1, Part 3. Calkins, G. N. 1933 The biology of the Protozoa. 2 ed. Phila- delphia. Cole, F. J. 1926 The history of protozoology . London. DoBELL, C. 1911 The principles of protistology. Arch. f. Protis- tenk., Vol. 23. 1932 Antony van Leeuwenhoek and his "little animals." New York. DoFLEiN, F. and E, Reichenow. 1929 Lehrbuch der Protozoen- kunde. 5 ed. Jena. NoEDENSKiOLD, E. 1928 The history of biology. New York. Woodruff, L. L. 1937 Louis Joblot and the Protozoa. Sci. Monthly, Vol. 94. Chapter 2 Ecology WITH regard to their habitats, the Protozoa may be divided into free-Uving forms and those Hving on (epizoic) or in (cndozoic) other organisms. The free-living Protozoa The vegetative or trophic stage of free-hving Protozoa have been found in every type of fresh and salt water, soil and decay- ing organic matter. 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 giv- en body of water, are temperature, light, chemical composition, acidity, kind and amount of food, and degree of adaptabihty of the individual protozoans to various environmental changes. Their early appearance as living organisms, their adaptability to various habitats and their capacity to remain viable in encysted condition possibly account for the wide distribution of the Pro- tozoa throughout the world. The common free-living amoebae, numerous testaceans and others, to mention a few, of fresh wa- ters, have been observed in innumerable parts of the world. Temperature. The majority of Protozoa are able to live only within a small range of temperature variation, although in the encysted state they can withstand a far greater temperature fluctuation. The lower Hmit of the temperature is marked by the freezing of the protoplasm, and the upper limit by the destructive chemical change within the body. The temperature toleration seems to vary among different species of Protozoa; and even in the same species under different conditions. For example. Chalk- ley (1930) placed Paramecium caudatum in 4 culture media (bal- anced 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 re- sistance varies with the hydrogen-ion concentration, maxima ap- pearing in the alkaline and acid ranges, and a minimum at or near about 7.0; 2) in a balanced saline, and in saline with an excess of sodium or potassium, the alkaline maximum is the higher, while 16 ECOLOGY 17 in saline with an excess of calcium, the acid maximum is the higher; 3) in general acidity decreases and alkalinity increases re- sistance; and 4) between pH 6.6 and 7.6, excess of potassium de- creases resistance and excess of calcium increases resistance. Glaser and Coria (1933) cultivated Paramecium 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. multimicronucleata 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 living organisms including Protozoa. Glaser and Coria obtained from the thermal springs of Virginia, several species of Mastigo- phora, Ciliata, and an amoeba which were living in the water, the temperature of which was 34-36°C., but did not notice any pro- tozoan in the water which showed 39-4 1°C. Uyemura and his co-workers made a series of studies on Protozoa living in various thermal waters of Japan, and reported that many species lived at unexpectedly high temperatures. Some of the Protozoa ob- served 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, Chilodonella 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 tem- perature if the change be made gradually. Dallinger and Drysdale showed a long time ago that Tetramitus rostratus and two other species of flagellates could be cultivated in temperatures ranging from 16° to 70°C. In nature, 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 ones. 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." Efimoff (1924) demonstrated that Paramecium, Frontonia, Colpidium and other ciliates die quickly at — 4°C., but by a quick and short 18 PROTOZOOLOGY overcooling (not lower than — 9°C.) no injury is brought about. At 0°C., Paramecium was able to multiply once in about 13 days. Wolfson (1935) studied Paramecium sp. in gradually descending subzero-temperature, and observed that as the temperature de- creases the organisms often swim backward, its bodily move- ments cease and its cilia finally stop beating. If the low tempera- ture exposure has not been of sufficient intensity or duration, warming induces a resumption of movement. Kept for 10-15 minutes at 10°C., the organism increases its body volume and be- comes rounded, from which condition it may recover if the tem- perature rises, but which otherwise is followed rapidly by a com- plete disintegration. When the water in which the ciliates are kept freezes, the organisms do not survive. Light. In the Phytomastigina which include chromatophore- bearing flagellates, the sun light is essential to photosynthesis (p. 92). 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 an- other factor concerned with the distribution of free-living pro- tozoans in the water. Chemical composition of water. The chemical nature of the water is another important factor which influences the very exist- ence of Protozoa in a given body of water. Different Protozoa show different morphological as well as physiological character- istics. As numerous cultural experiments indicate that individual protozoan species requires a certain chemical composition of the water in which it is cultivated under experimental conditions, al- though this may be more or less variable among different forms (Needham et al.). 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, oligosapro- bic, mesosaprobic, 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. Oligosa- probic forms are those that inhabit waters which are rich in min- eral matter, but in which no purification processes are taking place. Many Phytomastigina, various testaceans and many cih- ECOLOGY 19 ates, such as Frontonia, Lacrymaria, Oxytricha, Stylonychia, Vorticella, etc., inhabit such waters. Mesosaprobic protozoans live in waters in which active oxidation and decomposition of organic matter are taking place. The majority of freshwater pro- tozoans belong to this group : namely, numerous Phytomastigina, Hehozoa, Zoomastigina, and all orders of Cihata. Finally poly- saprobic 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 shme contains usually an abundance of ferrous sul- phide and other sulphurous substances. Lauterborn (1901) called this sapropelic. Examples of polysaprobic protozoans are Pelo- myxa palustris, Eughjpha alveolata, Pamphagus armatus, Mastig- amoeba, Trepomonas agilis, Hexamita inflata, Rhynchomonas nasuta, Heteronema acus, Bodo, Cercomonas, Dactylochlamys, Ctenostomata, etc. The so-called "sewage organisms" abound in such habitat (Lackey). Certain free-hving Protozoa which inhabit waters rich in de- composing organic matter are frequently found in the fecal mat- ter of various animals. Their cysts either pass through the aU- mentary canal of the animal unharmed or are introduced after the feces are voided, and undergo development and multiplica- tion in the fecal infusion. Such forms are collectively called copro- zoic Protozoa. The coprozoic protozoans grow easily in suspension of old fecal matter which are rich in decomposed organic matter and thus show a strikingly 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, Trepomonas agilis, Dimastig amoeba gruheri, Hartmanella hyalina, Chlamydophrys stercorea and Tilli- na magna. As a rule, the presence of sodium chloride in the sea water pre- vents the occurrence of the large number of fresh-water inhabi- tants. Certain 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 oc- cur in both fresh and salt waters: Mastigophora: Amphidinium 20 PROTOZOOLOGY lacustris, Ceratium hirundinella; Sarcodina: Lieberkuhnia wag- neri] Ciliata: Mesodinium pulex, Prorodon discolor, Lacrymaria olor, Amphileptus claparedei, Lionotus fasciola, Nassula aurea, Trochilioides recta, Chilodonella cucullulus, Trimyema compressum, Paramecium calkinsi, Colpidium campylum, Platynematum sociale, Cinetochilum margaritaceum, Pleuronema coronatum, Caenomorpha medusula, Spirostomum minus, S. teres, Climacostomum virens, and Thuricola folliculata; Suctoria: Metacineta mystacina, En- dosphaera engelmanni. It seems probable that many other protozoans are able to live in both fresh and salt water, judging from the observations such as that made by Finley (1930) who subjected some fifty species of freshwater Protozoa of Wisconsin to various concentrations of sea water, either by direct transfer or by gradual addition of the sea water. He found that Bodo uncinatus, Uronema marina, Pleu- ronema jaculans and Colpoda aspera are able to live and reproduce even when directly transferred to sea water, that Amoeba verru- cosa, Euglena, Phacus, Monas, Cyclidium, Euplotes, Lionotus, Paramecium, Stylonychia, etc., tolerate only a low salinity when directly transferred, but, if the sahnity is gradually increased, they live in 100 per cent sea water, and that Arcella, Cyphoderia, Aspidisca, Blepharisma, Colpoda cucullus, Halteria, etc., could not tolerate 10 per cent sea water even when the change was gradual. Finley noted no morphological changes in the experi- mental 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 systol, 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 Euplotes patella 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 amhigu- ECOLOGY 21 um were less adaptive to salt water, rarely living in 60 per cent sea water. Hydrogen-ion concentration. Closely related to the chemical composition is the hydrogen-ion concentration (pH) of the water which influences the distribution of Protozoa. The hydrogen-ion concentration of freshwater bodies vary a great deal between highly acid bog waters in which various testaceans may frequent- ly 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 decomposing organic matter, while the surface water is less acid or slightly alkaline due to the photosynthesis of green plants which utilize carbon dioxide. Several investigators have recently made obser- vations on the pH range of the water or medium in which certain protozoans live, grow, and multiply, which data are collected in a table on page 22. Seemingly various Protozoa require a definite pH value in order to carry on maximum metabolic activities. As a matter of fact, Pringsheim, Hall, Loefer, Johnson, and others, found that sodium acetate may increase or decrease the growth rate of various Phytomastigina subject to the hydrogen-ion concentration of the culture media. Food. The kind and amount of food available in a given body of water also controls the distribution of Protozoa. The food is ordinarily one of the deciding factors of the number of Protozoa in a natural habitat. Species of Paramecium and many other holozoic protozoans cannot live in waters in which bacteria or mi- nute protozoans do not occur. If other conditions are favorable, then the greater the number of food bacteria, the greater the number of these protozoans. Didinium nasutum feeds almost ex- clusively on Paramecium, hence it cannot live in the absence of the latter ciliate. Euryphagous protozoans are widely distributed and stenophagous forms are limited in their distribution. Some protozoans inhabit soil of various types and localities. Under ordinary circumstances, they occur near the surface, their maximum 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 distribu- tion of apparently one and the same species. For example, San- don found Amoeba proteus in samples of soil collected from Green- 22 PROTOZOOLOGY pH range of Protozoa medium in which Optimum Observers growth occurs range In bacteria-free cultures Euglena gracilis 3.5-9.0 — Dusi 3.0-7.7 6.7 Alexander 3.9-9.9 6.6 Jahn E. deses 6.5-8.0 7.0 Dusi 5.3-8.0 7.0 Hall E. pisciformis 6.0-8.0 6.5-7.5 Dusi 5.4-7.5 6.8 Hall Chilomonas Paramecium 4.1-8.4 4.9;7.0 Loefer Chlorogonium euchlorutn 4.8-8.7 7.1-7.5 Loefer C. elongaturn Colpidium striatum 4.0-8.9 5.5-5.7 Elliott C. campylum 4.0-8.9 6.5 Elliott Glaucoma pyriformis 4.0-8.9 4.8-5.3 Johnson G. ficaria 4.0-9.5 5.1-6.0 Johnson Paramecium bursaria 5.3-8.0 6.7-6.8 Loefer In cultures containing bacteria Carteria obtusa — 3.5-4.5 Wermel Acanthocystis aculeata 7 .4 or above 8.1 Stern Paramecium caudatum 5.3-8.2 7.0 Darby 6.0-9.5 7.0 Morea P. aurelia 5.7-7.8 6.7 Morea 5.9-8.2 — Phelps P. multimicronucleata 4.8-8.3 7.0 Jones P. sp. — 7.8-8.0 Saunders 7.0-8.5 7.8-8.0 Pruthi Colpidiuvi sp. 6.0-8.5 — Pruthi Colpoda cucullus 5.5-9.5 6.5;7.5 Morea Holophyra sp. 6.5-7.4 — Pruthi Plagiopyla sp. 6.9-7.5 — Pruthi Amphilepius sp. 6.8-7.5 7.1-7.3 Pruthi Spirostomum ambiguum 6.8-7.5 7.4 Saunders 5. sp. 6.5-8.0 7.5 Morea Blepharisma xmdulans — 6.5 Moore Gastrostyla sp. 6.0-8.5 — Pruthi Stylonychia pustulata 6.0-8.0 6.7;8.0 Darby land, Tristan da Cimha, Goiigh 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, ECOLOGY 23 Greenland, England, Japan, Australia, St. Helena, Barbados, Mauritius, Africa, and Argentina. The parasitic Protozoa Some Protozoa belonging to all groups live on or in other or- ganisms. The Sporozoa are made up exclusively of such forms. The relationships between the host and the protozoan differ in various ways, which make the basis for distinguishing the associa- tions into three types as follows : commensalism, symbiosis, and parasitism. The commensalism is an association in which an organism, the commensal, is benefited, while the host is neither injured nor benefited. Depending upon the location of the commensal in the host body, the ectocommensalism or endocommensalism is used. The ectocommensalism is often represented by Protozoa which may attach themselves to any aquatic animals that inhabit the same body of water, as shown by various species of Chonotricha, Peritricha, and Suctoria. In other cases, there is a definite rela- tionship between the commensal and the host. For example, Kerona polyporum is found on various species of Hydra, and the ciliates placed in Thigmotricha (p. 560) are inseparably associated with certain species of the mussels. The endocommensalism is often difficult to distinguish from the endoparasitism, since the effect of the presence of the com- mensal upon the host cannot be easily understood. On the whole, the protozoans which live in the lumen of the alimentary canal of the host may be looked upon as endocommensals. These proto- zoans use undoubtedly part of the food material which could be used by the host, but they do not invade the host tissue. As examples of endocommensals may be mentioned: Endamoeba hlattae, Lophomonas hlattaru7n, L. striata, Nyctotherus ovalis, etc., of the cockroach; Entamoeba coli, lodamoeba butschlii, Endolimax nana, Dientamoeba fragilis, Chilomastix mesnili, Giardia intesti- nalis, etc., of the human intestine; numerous species of Proto- ciliata of Anura, etc. Because of the difficulties mentioned above, the term parasitic Protozoa, in its broad sense, includes the commensals also. The symbiosis on the other hand is an association of two species of organisms which is of mutual benefit. The cryptomonads be- longing to Chrysidella ("zooxanthellae") containing yellow or 24 PROTOZOOLOGY brown chromatophores, which Uvc in Foraminifera and Radio- laria, and certain algae belonging to Chlorella ("zoochlorellae") containing green chromatophores, which occur in some fresh- water protozoans, such as Paramecium bursaria, Stentor amethys- tinus, etc., are looked upon as holding symbiotic relationship with the respective protozoan host. Several species of the highly in- teresting Hypermastigina, which are present commonly and abundantly in various species of the termite and the woodroach Cryptocercus,have been demonstrated by Cleveland to digest the cellulose material which makes up the bulk of wood-chips the host animals take in and to transform it into glycogenous sub- stances which 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 lose the flagellates and die, even though the intestine is filled with wood-chips. If removed from the gut of the termite, the flagellates die. Thus the associa- tion here may be said to be an absolute symbiosis. The 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. Hydramoeha hydroxena (p. 321) feeds on ectodermal cells of Hydra which, according to Reynolds and Looper, die on an average in 6.8 days as a result of the infection and the amoebae disappear in from 4 to 10 days if removed from a host Hydra. Costia necatrix (p. 264) often occurs in an enormous number, attached to various freshwater fishes especially in an aquarium, by piercing through the epidermal cells and ajipears to disturb the normal functions of the host tissue. Ichthyophthirius multifiliis (p. 504), another ectoparasite of freshwater fishes, goes further by completely 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 abnormal 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 ECOLOGY 25 was stated before, are without exception coelozoic, histozoic, or cytozoic parasites. Because of their modes of Uving, the endoparasitic Protozoa cause certain morphological changes in the cells, tissues, or organs of the host. The active growth of Entamoeba histolytica in the glands of the colon of the victim, produces slightly raised nodules first which develop into abscesses and the ulcers formed by the rupture of abscesses, may reach 2 cm. or more in diameter, com- pletely destroying the tissues of the colon wall. Similar patho- logical changes are also noticed in the case of infection by Bala?itidium coli. In Leishmania donovani, the victim shows an increase in number of the large macrophages and mononuclears and also an extreme enlargement of the spleen. Trypanosoma cruzi brings about the degeneration of the infected host cells and an abundance of leucocytes in the infected tissues, followed by an increase of fibrous tissue. T. gamhiense, the causative organ- ism of African sleeping sickness, causes enlargement of lymphatic glands and spleen, followed by changes in meninges and an in- crease of cerebro-spinal fluid. Its most characteristic changes are the thickening of the arterial coat and the round-celled infiltra- tion around the blood vessels of the central nervous system. Von Brand's (1938) summary of the carbohydrate metabolism of the pathogenic trypanosomes tends to show that the sugar is only partially oxidized in the presence of oxygen and that the carbo- hydrate metabolism of the infected host is disturbed, as shown mainly by the unbalanced condition of the blood sugar, by lower- ing of the glycogen reserves, and by reduced ability to build glycogen from sugar. Malarial infection is invariably accom- panied by an enormous enlargement of the spleen ("spleen index"); the blood becomes watery; the erythrocytes decrease in number; the leucocytes, subnormal; but mononuclear cells in- crease in number; pigment granules which are set free in the blood plasma at the time of merozoite-liberation are engulfed by leucocytes; and enlarged spleen contains large amount of pig- ments which are lodged in leucocytes and endothelial cells. In Plamodium falciparum, the blood capillaries of brain, spleen and other viscera may completely be blocked by infected erythro- cytes. In Myxosporidia which are either histozoic or coelozoic para- sites of fishes, the tissue cells that are in direct contact with highly 26 PROTOZOOLOGY enlarged parasites, undergo various morphological changes. For example, the circular muscle fibers of the small intestine of Pomoxis sparoides, which surround Myxobolus intestinalis, a myxosporidian, become modified a great deal and turn about 90° from the original direction, due undoubtedly to the stimulation exercised by the myxosporidian parasite (Fig. 1, a). In the case of another myxosporidian, Thelohanellus notatus, the connective tissue cells of the host fish surrounding the protozoan body, trans- ?^fl^^^S^SS!f:^^. ks^-yy--- Fig. 1. Histological changes in host fish caused by myxosporidian, infection, X1920 (Kudo), a, portion of a cyst of Mijxoholus intestinalis, surrounded 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. form themselves into "epithelial cells" (Fig. 1, 6), a state com- parable to the formation of the ciliated epithelium from a layer of fibroblasts lining a cyst formed around a piece of ovary in- planted into the adductor muscle of Pecten as observed by Drew (1911). Practically all Microsporidia are cytozoic, and the infected cells become hypertrophied enormously, producing in one genus the so-called Glugea cysts (Fig. 220). In many cases, the hyper- trophy of the nucleus of the infected cell is far more conspicuous than that of the cytoplasm (Fig. 218). Information concerning ECOLOGY 27 toxic substances produced by parasitic Protozoa is meager. Sarcosporidia appear to produce a certain toxic substance which, when injected in the blood vessel, is highly toxic to experimental animals. This was named sarcocystine (Laveran and Mesnil) or sarcosporidiotoxin (Teichmann and Braun). As in bacterial in- fection, the reaction and resistance of the host to protozoan in- fection apparently differ among different individuals. Taliaferro demonstrated that there occur in the blood of animals suffering from trypanosomiasis or malaria, certain agents which would either inhibit the rate of multiplication of the parasites or destroy the parasites themselves. 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 themselves 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 ob- tain a continued supply of food material. Examples of such ectocommensals abound everywhere. The ectocommensalism may next lead into ectoparasitism as in the case of Costia or Hydra- moeba, and then again instead of confining themselves to the body surface, the Protozoa may bore into the body wall from out- side and actually acquire the habit of feeding on tissue cells of the attached animals as in the case of Ichthyophthirius. The next step in the evolution of parasitism must have been reached when Protozoa, accidentally or passively, were taken into the digestive system of the Metazoa. Such a sudden change in habitat appears to be fatal to most protozoans. But certain others possess extraordinary capacity to adapt themselves to an entirely different environment. For example, Dobell (1918) ob- served in the tad-pole gut, a typical free-living limax amoeba, with characteristic nucleus, contractile vacuoles, etc., which was found in numbers in the water containing the fecal matter of the tadpole. Glaucoma pyriformis (p. 548), a free-living ciliate, was found to occur in the body cavity of the larvae of Theohaldia annulata (after MacArthur) and in the larvae of Chironomus plumosus (after Treillard and Lwoff). Lwoff successfully inocu- lated this ciliate into the larvae of Galleria niellonella which died 28 I'ROTOZOOLOGY later from the infection. Recently Janda and Jlrovec (1937) in- jected 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 in- fected 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 injected, died from the in- fection in a few days. The course of development 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 in- fected 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 Tri- trichomonas fecalis in feces of a single human subject for three years which grew well in feces diluted with tap water, in hay in- fusions with or without 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 further be speculated that Vahlkampfia, Hydramoeba, Schizamoeba, and Endamoeba, are the different stages of the course the intestinal amoebae might have taken during their evolution. Obviously endocommensalism in the alimentary canal was the initial phase of endoparasitism. When these endocom- mensals began to consume an excessive amount of food or to feed on the tissue cells of the host gut, they became the true endo- parasities. Destroying or penetrating through the intestinal wall, they became first established in body cavities or organ cavities and then invaded tissues, cells or even nuclei, thus developing ECOLOGY 29 into pathogenic Protozoa. The endoparasites developing in in- vertebrates which feed upon the blood of vertebrates as source of food supply, will have opportunities to establish themselves in the higher animals. References Chalkley, H. W. 1930 Resistance of Paramecium to heat as affected by changes in hydrogen-ion concentration and in inorganic salt balance in surrounding medium. U. S. Publ. Health, Rep. Vol. 45. Cleveland, L. R. 1926 Symbiosis among animals with special reference to termites and their intestinal flagellates. Quart. Rev. Biol., Vol. 1. 1928 Tritrichomonas fecalis nov. sp. of man; its ability to grow and multiply indefinitely in faeces diluted with tap water and in frogs and tadpoles. Amer. Jour. Hyg., Vol. 8. DoBELL, C. 1918 Are Entamoeba histolytica and Entamoeba ranarum the same species? Parasitology, Vol. 10. DouDOROFF, M. 1936 Studies in thermal death in Paramecium. Jour. Exp. ZooL, Vol. 72. Efimoff, W. W. 1924 Ueber Ausfrieren und Ueberkaltung der Protozoen. Arch. f. Protistenk., Vol. 49. FiNLEY, H. E. 1930 Toleration of freshwater Protozoa to in- creased salinity. Ecology, Vol. 11. Glaser, R. W. and N. A. Coria 1933 The culture of Parame- cium caudatum free from living microorganisms. Jour. Parasit. Vol. 20. Janda, V. and O. JIrovec 1937 Ueber kiinstlich hervorgerufenen Parasitismus eines freilebenden Ciliaten Glaucoma piriformis und Infektionsversuche mit Euglena gracilis und Spirochaeta biflexa. Mem. soc. zool. tehee, de Prague, Vol. 5. KoLKWiTZ, R. and M. Marsson 1909 Oekologie der tierischen Saprobien. Intern. Rev. Ges. Hydrobiol. u. Hydrogr., Vol. 2. Kudo, R. R. 1929 Histozoic Myxosporidia found in freshwater fishes of Illinois, U.S.A. Arch. f. Protistenk., Vol. 65. Lackey, J. B. 1925 The fauna of Imhof tanks. Bull. New Jersey Agr. Exp. Stat., No. 417. Lauterborn, R. 1901 Die "sapropelische" Lebewelt. Zool. Anz., Vol. 24. Needhum, J. G., P. S. Galtsoff, F. E. Lutz and P. S. Welch. 1937 Culture methods for invertebrate animals. Ithaca. NoLAND, L. E. 1925 Factors influencing the distribution of freshwater ciliates. Ecology, Vol. 6. Reynolds, B. D. 1936 Colpoda steini, a facultative parasite of the land slug, Agriolimax agrestis. Jour. Parasit., Vol. 22. and J. B, Looper 1928 Infection experiments with Hydramoeba hydroxena nov. gen. Ibid., Vol. 15. 30 PROTOZOOLOGY Sandon, H. 1927 The composition and distribution of the proto- zoan fauna of the soil. Edinburgh, Taliaferro, W. H. 1926 Host resistance and types of infections in trypanosomiasis and malaria. Quart. Rev. Biol., Vol. 1. VON Brand, T. 1938 The metabolism of pathogenic trypano- somes and the carbohydrate metabolism of their hosts. Ibid., Vol. 13. Wenyon, C, M. 1926 Protozoology. 2 vols. London and New York. WoLFSON, C. 1935 Observations on Paramecium during ex- posure to sub-zero temperatures. Ecology, Vol. 16. YocoM, H. B. 1934 Observations on the experimental adapta- tion of certain freshwater ciliates to sea water. Biol. Bull., Vol. 67. Chapter 3 Morphology PROTOZOA range in size from ultramicroscopic to macro- scopic, 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 dimensions. Noctiluca, Foraminifera, Radiolaria, many ciliates such as Stentor, Bursaria, etc., represent larger forms. Colonial protozoans such as Carchesium, Zoothamnium, Ophrydium, etc., are even greater than the solitary forms. Plas- modium, Leishmania, and microsporidian spores may be men- tioned as examples of the smallest forms. The unit of measure- ment employed in protozoology is, as in general microscopy, 1 micron (/x) which is equal to 0.001 mm. The body forms of Protozoa are even more varied, and fre- quently, because of its extreme plasticity it does not remain constant. 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 pos- sess a definite symmetry. Thus bilateral symmetry is noted in all members of Diplomonadina (p. 272); radial symmetry in Gon- ium, Cyclonexis, etc.; and universal symmetry, in certain Helio- zoa, Volvox, etc. The fundamental component of the protozoan body is the pro- toplasm which is without exception difTerentiated into the nucleus and the cytosome. Haeckel's monera are now considered as nonexistent, since improved microscopic technique failed in recent years to reveal any anucleated protozoans. The nucleus and the cytosome are inseparably important to the well-being of a protozoan, as has been shown by numerous investigators since Verworn's pioneer work. In all cases, successful regenera- tion of the body is only accomplished by the nucleus-bearing portions and enucleate parts degenerate soon or later. On the other hand, when the nucleus is taken out of a cell, both the nucleus and cytosome degenerate, which indicate their intimate association in carrying on the activities of the body. It appears certain that the nucleus controls the assimilative phase of metab- 31 32 PROTOZOOLOGY olism which takes place in the cytosome in normal animals, while the cytosome is capable of carrying on catabolic phase of the metabolism. Aside from the importance as the controlling center of metabolism, evidences point to the conclusion that the nucleus contains the genes or hereditary factors which character- ize each species of protozoans from generation to generation, as in the cells of multicellular animals and plants. The nucleus Because of a great variety of external body forms and of con- sequent body organizations, 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 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 pos- sesses two similar nuclei, as in Pelomyxa hinucleata, Arcella vulgaris, Diplomonadina, Protoopalina and Zelleriella. In Eucil- iata and Suctoria, two dissimilar nuclei, a macronucleus and a micronucleus, are typically present. The macronucleus is always larger than the micronucleus, and controls the trophic activities of the organism, while the micronucleus is concerned with the reproductive acti\dty. Certain Protozoa possess numerous nuclei of similar structure, as for example, in Mycetozoa, Actino- sphaerium, Opalina, Cepedea, Myxosporidia, Microsporidia, etc. Dileptus anser contains many small macronuclei, a condition not observed in other euciliates. The essential components of the protozoan nucleus are the nuclear membrane, chromatin, plastin and nucleoplasm. Their interrelationship varies sometimes from one developmental 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) consists of a nuclear mem- brane which is sometimes very delicate, but distinct, nucleo- plasm and chromatin. Besides there is an intranuclear body which is, as a rule, more or less spherical and which appears to be of dif- ferent make-ups, as judged by its staining reactions among dif- ferent nuclei. It may be composed of chromatin, of plastin, or of a mixture of both. The first type is sometimes called karyosome MORPHOLOGY 33 and the second, nucleolus or plasmosome. Absolute distinction between these two terms cannot be made as they are based upon the difference in affinity to nuclear stains which cannot be stand- ardized and hence do not give uniformly the same result. Fol- lowing Minchin and others, the term endosome is advocated here to designate one or more conspicuous bodies other than the chromatin granules, present within the nuclear membrane. When viewed in life, the nucleoplasm is ordinarily homo- geneous and structureless. But, upon fixation, there appear in- variably plastin strands or networks which seem to connect the Nuclear membrane Endosome Achromatic strand Chromatin granules a b Fig. 2. a, vesicular nucleus; b, compact nucleus (diagrams). endosome and the nuclear membrane. Some investigators hold that these strands or networks exist naturally in life, but due to the similarity of refractive indices of the strands and of the nucleoplasm, they are not visible and that, when fixed, they be- come readily recognizable because of a change in these indices. In some nuclei, however, certain strands have been observed in life, as for example in the nucleus of the species of Barbu- lanympha (Fig. 131, c), according to Cleveland and his associates (1934). Others maintain that the achromatic structures promi- nent in fixed vesicular nuclei are mere artifacts brought about by fixation and do not exist in life and that the nucleoplasm is a homogeneous liquid matrix of the nucleus. The chromatin substance is ordinarily present as small granules although at times they may be in block forms. Precise knowledge of chromatin is still lacking. At present the determination of the chromatin depends upon the following tests: 1) artificial digestion which does not destroy this substance, while non-chromatinic parts of the nucleus are completely dissolved; 2) acidified methyl green which stains the chromatin bright green; 3) 10 per cent sodium chloride solution which dissolves, or causes swelling of, chromatin granules, while nuclear membrane and achromatic substances remain unattacked; and 4) in the fixed condition 34 PROTOZOOLOGY Feulgen's niicleal reaction. The vesicular nucleus is most com- monly present in various orders of the Sarcodina and Mastig- ophora. The compact nucleus (Fig. 2, b), on the other hand, contains a large amount of chromatin substance and a comparatively small amount of nucleoplasm, and is thus massive. The macronucleus of the 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 root-like. The nuclear membrane is always distinct, and the chromatin substance is usually spheroidal, varying in size among different species and often even in the same nucleus. In the majority of species, the chromatin granules are small and compact, though in some forms, such as Nydotherus ovalis (Fig. 3), they may reach 20^ or more in diame- ter, and while the smaller chromatin granules seem to be solid, larger forms contain alveoli of different sizes in which smaller chromatin granules are suspended (Kudo, 1936). There is no sharp demarcation between the vesicular and compact nuclei, since there are numerous nuclei the structures of which are intermediate between the two. Moreover what appears to be a vesicular nucleus in hfe, 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 endosomes in the vesicular nucleus, and the amount and arrangement of the chromatin in the compact nu- cleus, vary according to the physiological state of the protozoan concerned. The macronucleus may be divided into two or more parts with or without connections among them and in Dileptiis anser into more than 200 small nuclei, each of which is "composed of a plastin core and a chromatin cortex" (Calkins; Hayes). In general, the chromatin granules or spherules fill the intra- nuclear space compactly, in which one or more endosomes may occur. In many nuclei these chromatin granules appear to be suspended freely, while in others a reticulum appears to make the background. The chromatin of compact nuclei gives a strong posi- tive Feulgen's nucleal reaction. The macronuclear and micro- nuclear chromatin substance responds 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 MORPHOLOGY 35 caudatum, P. aiirelia, Chilodonella, Nydotherus ovalis, etc., the macronuclear chromatin is colored more deeply than the micro- nuclear chromatin, while in Colpoda, Urostyla, Euplotes, Sty- lonychia, and others, the reverse seems to be the case, which may support the validity of assumption that the two types of the Fig. 3. Four macronuclei of Nydotherus ovalis, s^liuwing chromatin spherules of different sizes, X650 (Kudo). nuclei of Euciliata and Suctoria are made up of different chroma- tin substances — idiochromatin in the micronucleus and tropho- chromatin in the macronucleus — and in other classes of Protozoa, the two kinds of chromatin are present together in a single nucleus. Chromidia. Since the detection of chromatin had solely de- pended on its affinity to nuclear stains, several investigators 36 PROTOZOOLOGY found extranuclear chromatin granules in many protozoans. Finding such granules in the cytosome of Actinosyhaerium eich- horni, Arcella vulgaris, and others, Hertwig (1902) called them chromidia, and maintained that under certain circumstances, such as lack of food material, the nuclei disappear and the chro- matin granules become scattered throughout the cytosome. In the case of Arcella vulgaris, the two nuclei break down completely to produce a chromidial-net which later reforms into smaller secondary nuclei. It has, however, been found by Belaf that the lack of food caused the encystment rather than chromidia- formation in Actinosphaerium and, according to Reichenow, Jollos observed that in Arcella the nuclei persisted, but were thickly covered by chromidial-net which could be cleared away by artificial digestion to reveal the two nuclei. In Difflugia, the chromidial-net is vacuolated or alveolated in the fall and in each alveolus appear glycogen granules which seem to serve as reserve food material for the reproduction that takes place during that season (Zuelzer), and the chromidia occurring in Actinosphaerium appear to be of a combination of a carbohydrate and a protein (Rumjantzew and Wermel). Apparently the widely distributed volutin (p. 95), and many inclusions or cytozoic parasites, such as Sphaerita, 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 are chromatin. Thus at present the real nature of chromidia is still not clearly known, although many protozoologists are inclined to think that the substance is not chromatinic, but, in some way, is connected with the metabolism of the protozoan. The cytosome The extranuclear part of the protozoan body is the cytosome. It is composed of the cytoplasm, a colloidal system, which may be homogenous, granulated, vacuolated, reticulated, or fibrillar in optical texture, and is almost ahvays colorless. The chromato- MORPHOLOGY 37 phore-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 various colorations. In several forms pigments are diffused throughout the cytoplasm. For ex- ample, many dinoflagellates are beautifully colored which, ac- cording to Kofoid and Swezy, is due to a thorough diffusion of pigment in the cytoplasm. Stentor coeruleus is ordinarily blue- colored, the pigment responsible for which was called Stentorin (Lankester) and is lodged in granules between the surface striae; and rose- or purple-coloration of several species of Blepharisma appears to be due to a special pigment, zoopurpurin (Arci- chovskij) which is lodged in the ectoplasmic granules often called protrichocysts (p. 65). The development of zoopurpurin is definitely correlated with the sun-light, as shown by Giese. Deeply pink specimens will lose color completely in a few hours when exposed to strong sun-light and the recoloration takes place in darkness very slowly. The extent and nature of the cytosomic differentiation differs greatly among various groups. In the majority of Protozoa, the cytoplasm is differentiated into the ectoplasm and the endo- plasm. The ectoplasm is the cortical zone which is hyaline and homogeneous. In the Ciliophora, it is a permanent and distinct part of the body and contains several organellae; in the Sarcodina and the Sporozoa, it is more or less a temporarily differentiated zone and hence varies greatly at different times and, in the Mastigophora, it seems to be more or less permanent. The endo- plasm is more voluminous and fluid. It is granulated or alveo- lated 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 gran- ulated or vacuolated cytoplasm indicates invariably the degen- eration of the protozoan body. In numerous Sarcodina and certain Mastigophora, the body surface is naked and not protected by any form-giving organella. According to observations by Kite, Howland, and others, the surface layer is not only elastic, but solid, and therefore the name plasma-membrane may be applied to it. Such forms are capable of undergoing amoeboid movement by formation of pseudopodia and by continuous change of form due to the movement of the 38 PROTOZOOLOGY cytoplasm which is more fluid. However, the majority of Proto- zoa possess a characteristic and constant body form due to the development of a special envelope, the pellicle. In Amoeba striata and A. verrucosa, there is an extremely thin pellicle. The same is true with some flagellates, such as certain species of Euglena, Peranema, and Astasia, in which it is elastic and expansible so that the organisms possess 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 parallel to the longitudinal axis of the body as four girdles. In certain peritrichous ciliates, such as Vorticella moni- lata, Carchesium granulatum, etc., the pellicle may possess nodu- lar 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 Proto- zoa which may cover the body rather loosely. These are the shell, test, lorica or envelope. The shell of various Phytomastigina is mainly made up of cellulose, a carbohydrate, which is widely dis- tributed among 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 sub- stance may in many forms be produced to surround the shelled body or in the members of Volvocidae to form the matrix of the entire colony in which the individuals are imbedded. In the dino- flagellates, the shell is highly developed, and often composed of numerous plates which are variously sculptured. 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 w^ays which char- acterize the different genera. Newly formed shell is colorless, but older ones become brownish, because of the presence of iron oxide. Difflugia and related genera form shells by glueing together small sand-grains, diatom-shells, debris, etc., with chitinous or pseudo- chitinous substances which they secrete. Many foraminiferans seem to possess a remarkable selective power in the use of foreign MORPHOLOGY 39 material for the construction of their shells. According to Cush- man, Psammos'phaera 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. howmanni, on the other hand, uses only mica flakes which are found in a comparatively small amount, and P. rustica uses acerose sponge spicules for the Fig. 4. Diagram of the shell of Peneroplis pertusus, X about 35 (Carpenter), ep, external pore; s, septum; sc, stolon canal. framework of the shell, skillfully fitting smaller broken pieces into polygonal areas. Other foraminiferans combine chitinous secre- tion with calcium carbonate and produce beautifully complicated shells (Fig. 4) with one or numerous pores. In the Coccolithidae, variously shaped platelets of calcium carbonate ornament the shell. The silica is present further in the shells of various Protozoa. In Euglypha and related testaceans, siliceous scales or platelets are produced in the endoplasm and compose a new shell at the time of fission or of encystment together with the chitinous secre- tion. In many heliozoans, siliceous substance forms spicules, platelets, or combination of both which are embedded in the mucilaginous envelope which surrounds the body and, in some 40 PROTOZOOLOGY cases, a special clathrate shell composed of silica, is to be found. In some Radiolaria, isolated siliceous spicules occur as in Helio- zoa, while in others the lateral development of the spines results in production of highly complex and most beautiful shells with various ornamentations or incorporation of foreign material. Many pelagic radiolarians possess numerous conspicuous radiat- ing spines in connection with the skeleton, which apparently aid the organisms to maintain their existence in the open sea. Some flagellates may be encased in a chitinous lorica or house and in addition there is occasionally a collar developed at one end. The lorica found in the Ciliophora is mostly composed of chitinous substance alone, especially in Peritricha, although some produce a house made up of gelatinous secretion containing foreign material as in Stentor (p. 581). In the Tintinnidiidae, the loricae are either solely chitinous in numerous marine forms not mentioned in the present work or composed of sand-grains or coccoliths cemented together by chitinous secretion. 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 able also to produce them. According to their form and structure, four kinds of pseudopodia are distinguished. 1). The lobopodium is formed by an extension of the ecto- plasm and by a flow of endoplasm as is commonly found in Amoeba proteus (Figs. 42; 140). It is finger- or tongue-like, some- times 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, MORPHOLOGY 41 Vahlkampfia Umax, Pyxidicula operculata, etc., in which case it is a broadly tongue-like extension of the body in one direction and the progressive movement of the organisms is comparatively rapid. The lobopodia may occasionally be conical in general shape, as in Amoeba spumosa. Although ordinarily the formation of lobopodia is by general flow of the cytoplasm, in some it is sudden and "eruptive," as in Endamoeha hlattae or Entamoeba histolytica in which the flow of the endoplasm presses against the inner zone of the ectoplasm and the accumulated pressure finally causes breaks through the line, resulting in a sudden ex- tension of the endoplasmic flow at that point. 2). The 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 Aynoeba radiosa may be considered as approaching this type more than the lobopodia. 3). The rhizopodium is also filamentous, but branching and anastomosing. It is found in numerous Foraminifera, such as Elphidium, Peneroplis (Fig. 5), etc., and in certain testaceans, such as Lieberkuhnia, Myxotheca, etc. The abundantly branch- ing and anastomosing rhizopodia often produce a large network which serve almost exclusively for capturing prey. 4). The axopodium is, unlike the other three types, a more or less semi-permanent structure and composed of axial rod and cytoplasmic envelope. The axopodia are found in many Heliozoa, such as Actinophrys, Actinosphaerium, Camptonema, Sphaera- strum, and Acanthocystis. The axial rod, which is composed of fibrils (Doflein; Roskin), arises from the central body or the nucleus located in the approximate center of the body, from each of the nuclei in multinucleate forms, or from the zone between the ectoplasm and endoplasm (Fig. 6). Although semipermanent in structure, the axial rod is easily absorbed and reformed. In the genera of Heliozoa, not mentioned above and in numerous radio- larians, the radiating filamentous pseudopodia are so extremely delicate that it is difficult to determine whether an axial rod exists in each or not, although they resemble axopodia in general ap- pearance. There is no sharp demarcation between the four types of 42 PROTOZOOLOGY psciidopodia, as there are transitional pseiidopodia between any two of them. For example, the pseudopodia formed by Arcella, Lesquereusia, Hyalosphaenia, etc., resemble more lobopodia '■t'V^'>'^"'' V 'i" ,'.,:•-'. ,•:■■. '.'■ 'I'M', ' > ;V/' ^^/< 7,'/ /./•■•/T / '(■/.<.i./.,-^' V/ii nil i \ ■ / ■■ ' /ill ' • ^ //J /^h \ \ Fig. 5. Pseudopodia of Elphidium strigilata, X about 50 (Schulze from Kiihn). than filopodia, though composed of the ectoplasm only. The pseudopodia of Actinomonas, Elaeorhanis, Clathrulina, etc., may be looked upon as transitional between rhizopodia and axopodia. MORPHOLOGY 43 While the pseudopodia formed by an individual are usually of characteristic form and appearance, they may show an entirely different appearance under different circumstances. According to the often-quoted experiment of Verworn, limax amoebae change into radiosa amoebae upon addition of potassium hydrate to the water (Fig. 7). Mast has recently shown that when Amoeba proteus or A. dubia is transferred into pure water, the amoeba produced radiating pseudopodia, and when transferred to a salt Fig. 6. Portion of Actinosphaerium eichhorni, X800 (Kiihn). ar, axial rod; cv, contractile vacuole; ec, ectoplasm; en, endoplasm; n, nucleus. medium, it changed into monopodial form, which change, he was inclined to attribute to the difference in the water contents of the amoeba. In some cases during and after certain internal changes, an amoeba may show conspicuous differences in pseudopodia (Neresheimer). As was stated before, pseudopodia occur widely in forms which are placed under classes other than Sarcodina during a part of their life-cycle. Care, therefore, should be exer- cised in using them for taxonomic consideration of the Protozoa. Flagella. The flagellum is a filamentous extension of the cyto- plasm and is ordinarily extremely fine and highly vibratile, so 44 PROTOZOOLOGY that it is difficult to recognize it in life under the microscope with a moderate magnification. In a number of species, the flagellum, however, can be seen in life as a long filament, as for example in Peranema. As a rule, the number of flagella present in a single individual is small, varying from one to eight, but in Hyper- mastigina there are numerous flagella. A flagellum appears to be composed of at least two parts (Fig. 8, a, h). An axial filament which is elastic, takes its origin directly, or indirectly through d e f Fig. 7. Form-change in a limax-amoeba (Verworn). a, b, contracted forms; c, individual showing typical form; d-f, radiosa-forms, after addition of KOH solution to the water. basal granule, in the blepharoplast. Surrounding this filament there is a sheath of contractile cytoplasm which varies in thick- ness alternately on the opposite sides of the filament. The flagellum ordinarily tapers toward its distal end where the axial filament is said to be frequently exposed. Recently Vlk found, besides the kind above mentioned which he called the whip-flagellum, another form named by him as the ciliary flagellum. The latter is said to be uniformly thick, but possesses dense ciliary projections which are arranged on a flagellum in one or two spiral rows (Fig. 8, c, d). Vlk found the whip-flagellum in Chlamydomonas, Polytoma uvella(e), Cercomonas MORPHOLOGY 45 axial filament cytoplasmic sheath \ / Fig. 8. Diagrams of flagella. a, flagellum of Euglena (Biitschli); b, flagellum of Trachelomonas (Plenge); c, ciliary flagellum with one row of cilia; d, a ciliary flagellum with two rows of cilia; e, whip- flagella of Pohjtoma uvella; f, ciliary flagellum of Urceolus cyclostomus; g, the flagella of Monas socialis (Vlk) . 46 PROTOZOOLOGY crassicauda, Trcpomonas rotans, T. agilis, Hexamita injlata, Urophagus rostratus, etc.; the ciliary flagcllum, in JMallomonas, Chromulina, Trachclomonas, Urccolus (/), Phaciis, Euglena, Astasia, Distigma, etc.; and both kinds in Syniira, Uroglena, Dinobryon, Monas (g), etc. The flagcllum is most frequently inserted near the anterior end of the body and directed forward, its movement pulling the organism forward. Combined with this, there may be a trailing flagellum which is directed i)osteriorly and which serves to steer Fliigellum Undulatinji membrane Nucleus Basal granule Blepharoplast Anterior flagellum Basal granule Blepharoplast Rhizoplast Nucleus Parabasal body Posterior flagellum Fig. 9. Diagrams of two flagellates, showing their structures (Kiihn). a, Trypanosoma brucei; b, Proteromonas lacertae. the course of movement or to push the body forward to a certain extent. In a comparatively small niunber of flagellates, the flagel- lum is inserted near the posterior end of the body and would push the body forward by its -snbration. Lankester coined tractella and pulsella for pulling and pushing flagella respectively. In certain parasitic Mastigophora, such as Trjrpanosoma (Fig. 9, o), 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 rotaforiutn of the frog, and is called the undulating membrane. In many of the dinoflagellates, the transverse flagel- lum seems to be similarly constructed (Kofoid and Swezy) (Fig. 101, d, /). MORPHOLOGY 47 Cilia. The cilia are the organella of locomotion and food- capturing found in the Ciliophora. They seem to serve often as a tactile organella. The cilia are fine and more or less short proc- esses of ectoplasm and occur in large numbers in the majority of the Holotricha. They may be uniformly long, as in Protocihata, or may be of different lengths, being longer at the extremities, on certain surfaces, in peristome or in circumoral areas. Ordinarily the cilia are arranged in longitudinal, oblique, or spiral rows, being inserted on either the ridges or the furrows. Again the cilia may be confined to certain parts or zones of the body. Each cilium originates in a basal granule situated in the deeper part of the ectoplasm and, in a few species, a cilium is found to be made up of an elastic axial filament arising from the basal granule and contractile sheath. Gelei observed in flagella and cilia, lipoid substance in granular or rod-like forms which differed even among different individuals of the same species; and Klein found in many cilia of Colpidium colpoda, an argentophilous substance in granular form much resembling the lipoid structure of Gelei and called them "cross-striation" of the contractile component (Fig. 10). The cilia are often present in a certain area more densely than in other parts of body and, consequently, such an area stands out conspicuously, and is sometimes referred to as a cihary 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 trans- verse bands of close-set cilia. In a number of forms, such as Coleps Stentor, etc., there occur, mingled among the vibratile cilia, immobile stiff cilia which are apparently solely tactile in function. In the Hypotricha, the ciHa are largely replaced by cirri, al- though in some species both may occur. A cirrus is composed of a number of cilia arranged in 2 to 3 rows which fused into one structure completely (Figs. 11, 12), which was demonstrated by Taylor. Klein also showed by desiccation that each marginal cirrus of Stylonychia was composed of 7 to 8 ciha. In some in- stances, the distal portion of a cirrus may show two or more branches. The cirri are confined to the ventral surface in Hypo- tricha, and called frontal, ventral, anal, caudal, and marginal cirri, according to their location (Fig. 11). Unhke the cilia, the cirri may beat in any direction so that the organisms bearing them, show various ways of locomotion. Oxytricha, Stylonychia, 48 PROTOZOOLOGY etc., walk on frontals, ventrals, and anals, while swimming move- ment by other species is of different types. In all euciliates except Holotricha, there are adoral membranel- lae. A membranella is composed of a double ciliary lamella, fused completely into a plate (Fig. 12). A number of these membranel- lae occur on a margin of the peristome, forming the adoral zone Fig. 10. Diagrams of cilia (Klein), a, Coleps; b, Cyclidium glaucoma; c, Colpidiu7n colpoda. af, axial filament; bg, basal granule; of, circular fibril; cs, cross-striation; sg, secondary granule. of membranellae, which serves for bringing the food particles to the cytostome. The frontal portion of the zone, the so-called frontal membrane, appears to serve for locomotion and Kahl considers that it is probably made up of three lamellae. The membranes which are often found in Holotricha and Hetero- tricha, are transparent thin membranous structures composed of one or two rows of cilia, which are more or less strongly fused. MORPHOLOGY 49 Cirrus fiber Ectoplasmic granules Basal plate of the cirrus Basal granules 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 patella (Taylor); b, schematic ventral view of Stylonychia to show the distribution of the cirri. 50 PROTOZOOLOGY The membranes, located in the lower end of the peristome, are sometimes called perioral membranes, and those in the cyto- pharynx, undulating membranes. In Suctoria, cilia are present only during the developmental stages, and, as the organisms become mature, tentacles are de- veloped. The tentacles are concerned with food-capturing, and are either prehensile or usually suctorial. In a few instances the cpg Fig. 12. Diagrams of cirrus and membranella of Euplotes pateUoi X1450 (Taylor), a, an anal cirrus in side view; b, a membranella; bg- basal granule; cpg, coagulated protoplasmic granules; cr, ciliary root; fp, fiber plate. tentacles are tubular and this type is interpreted by Collin as possibly derived from a cytostome and cytopharynx of the ciliate (Fig. 13). Although the vast majority of Protozoa possess only one of the three organellae of locomotion mentioned above, a few may possess pseudopodia in one phase and fiagella in another phase during their life-cycle. Among many examples, may be men- tioned Dimastigamoebidae (Fig. 139), Tetramitus rostratus (Fig. 122), etc. Furthermore, there are some protozoans which possess two types of organellae at the same time. Flagellum or fiagella MORPHOLOGY 51 and pseudopodia occur in many Phytomastigina and Rhizomasti- gina, and a flagellum and cilia are present in Ileonema (Fig. 235, b, c). In the cytosome of Protozoa there occur various organellae, each of which will be considered briefly here. Fibrillar structures One of the 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 organella, and the whole body reacts. But in certain other Protozoa, there Fig. 13. Diagrams showing the possible development of a suctorian tentacle from a cytostome and cytopharynx of a ciliate (Collin). are special organellae of contraction. Many Ciliophora are able to contract instantaneously when subjected to mechanical pres- sure, 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 was Lieberkiihn (1857) who noted "muscle fibers" in the ectoplasm of Stentor which were later named myonemes (Haeckel) or Neurophanes (Neresheimer). The myonemes of Stentor have been studied by several in- vestigators. According to Schroder (1906), there is a canal be- tween each two longitudinal striae and in it occurs a long banded myoneme which measures in cross-section 3-7 fx high by about 1m wide and which appears cross-striated (Fig. 14). Roskin (1923) considers that the myoneme is a homogeneous cytoplasm (kino- plasm) and the wall of the canal is highly elastic and counteracts 52 PROTOZOOLOGY the contraction of the myonemes. All observers agree that the myoneme is a highly contractile organella. Many stalked peritrichous ciliates have well-developed myo- nemes not only in the body proper, but also in the stalk. Koltzoff's studies show that the stalk is a pseudochitinous tube, enclosing an inner tube filled with granulated thecoplasm, surrounding a cl m mc m bg gis Fig. 14. Myonemes in Stentor coeruleus (Schroder), a, cross-section of ectoplasm; b, surface view of three myonemes; c, two isolated myonemes; bg, basal granules; cl, cilium; gis, granules between striae; m, myonemes; mc, myoneme canal. central rod, composed of kinoplasm, on the surface of which are arranged skeletal fibrils (Fig. 15). The contraction of the stalk is brought about by the action of kinoplasm and walls, while elastic rods will lead to extension of the stalk. Myonemes present in the cihates aid in the contraction of body, but those which occur in many Gregarinida aid apparently in locomotion, being arranged longitudinally, transversely and probably spirally (Fig. 15). In certain Radiolaria, such as Acanthometron elasticum MORPHOLOGY 53 (Fig. 168, c), etc., each axial spine is connected with 10-30 myo- nemes (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 Isoiricha prostoma and /. iritestinalis, Schuberg (1888) observed that the nucleus is suspended by ectoplasmic fibrils and Fig. 15. a, b, fibrillar structures of the stalk of Zoothamnium (Kolt- zoff); 0, myonemes in Gregarina (Schneider), ef, elastic fiber; ie, inner envelope; k, kinoplasm; oe, outer envelope; t, thecoplasm. called the apparatus karyophores. In some forms these fibrils are replaced by ectoplasmic membranes as in Nyctotherus ovalis (Zuluta; Kudo), ten Kate (1927) studied fibrillar systems in Opalina, Nyctotherus, Ichthyophthirius, Didinium, and Balantid- ium, and found that there are numerous fibrils, each of which originates in a basal granule of a cilium and takes a transverse or oblique course through the endoplasm, ending in a basal granule located on the other side of body. He further noted that the cyto- 54 PROTOZOOLOGY Fig 16. A composite drawing from three median sagittal sections of Evidinium ecaudatum, fixed in Zenker and stained with Ma lory s connective 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, encio- plasm; m, motorium; oc, oral cilia; od, oral disk; oef, oesophageal fibers; of, opercular fibers; p, pellicle; prs, pharyngeal retractor strands; si, skeletal laminae; vs, ventral skeletal area. MORPHOLOGY 55 pharynx 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 {Diplodiniutn) ecaudatum (Fig. 16). Sharp recognized in this ciHate a complicated fibrillar system, connecting all the motor organellae of the cyto- stomal region, and thinking that it was "probably nervous in function," as its size, arrangement 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 stain- ing), located very deeply 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-oesophageal ring and oesophageal fibers); and several strands into the ecto- plasm of the operculum (opercular fibers). A similar apparatus has since been observed in many other ciliates: Euplotes (Yocom; Taylor), Balantidium (McDonald), Paramecium (Rees; Brown; Lund), Tintinnopsis (Cambell), Boveria (Pickard), Dileptus (Visscher), Chlamydodon (MacDougall), Entorhipidium and Lechriopyla (Lynch), Eupoterion (MacLennan and Connell), Metopus (Lucas), Troglodytella (Robertson), Oxytricha (Lund), Ancistruma and Conchophthirus (Kidder), etc. Euplotes patella, a common free-living hypotrichous ciliate, has been known for nearly 50 years to possess definite fibrils connecting the anal cirri with the anterior part of the body. Engelmann suggested that their function was somewhat nerve- like, while others maintained that they were supporting or con- tracting in function. Yocom (1918) traced the fibrils to the mo- torium, a very small bilobed body (about S^t by 2yu) located close to the right anterior corner of the triangular cytostome (Fig. 17). Joining with its left end are five long fibers from the anal cirri which converge and appear to unite with the motorium as a single strand. From the right end of the motorium extends the mem- 56 PROTOZOOLOGY ^ — mfp Fig. 17. Diagrams showing the neuromotor apparatus of Euplotes patella (Taylor), a, diagrammatic dorsal view of the entire apparatus, X1600; b, dissected portion of disintegrating membranella fiber plates attached to the membranella fiber; c, a dissociated fiber plate of a frontal cirrus with its attached fibers, X1450. acf, anal cirrus fiber; afp, anal fiber plate; eg, small and large ectoplasmic granules; m, motorium; mf, membranella fiber; mfp, membranella fiber plate. branella-fiber anteriorly, and then to left along the proximal bor- der of the oral lip and the bases of all membranellae. Yocom further noticed that within the lip there was a latticework struc- ture whose bases very closely approximate the cytostomal 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 contiguous with the basal plates of the frontal, ventral and marginal cirri, to each of which are attached the dissociated fibers (c). By means of microdissection needles, Taylor demonstrated that these fibers have nothing to do with the maintainance of 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 mem- branellae and anal cirri. Turner (1933) however is inclined to think that there is no motorium in this protozoan. MORPHOLOGY 57 Fig. 18. The silverline system of Ancistruma mytili, XlOOO (Kidder), a, ventral view; b, dorsal view. 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 organellae, are found the basal granules or plates to which the "nerve" fibers from the motorium are attached. Independent of the studies on the neuromotor system of American investigators, Klein (1926) introduced the silver-im- pregnation 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. K,lein (1926-1930) subjected the ciliates of numerous genera and species to this method, and observed that there was a fibrillar system in the ectoplasm at the level of the basal granules which cannot be demonstrated by other methods. Ivlein (1927) named the fibers silver lines and the whole complex, the silverline sys- tem, which is characteristic of each species (Fig. 18). Chatton 58 PROTOZOOLOGY and Lwoff, Gelci, Jirovec, Lynch, Jacobson, Kidder, Lund, and others, applied the silver-impregnation methods 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, therefore, named it infraciliature. 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 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 main- tains that the neuromotor apparatus in Euplotes patella is augmented by a distinct but connected external network of sensory fibrils. Lund (1933) also made a comparative study of the two systems in Paramecium multimicronucleata, and observed that the silver- line 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 cytostome, cytopyge, and sutures. This system of lines stains if the organisms are well dried. Usually the lines appear solid, but frequently they are interrupted to appear double at the ver- tices 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" (subpel- licular) conductile system consists essentially of the longitudinal lines connecting all basal granules in a longitudinal row of hexa- gons and of delicate transverse fibrils connecting granules of adjacent rows especially in the cytostomal region (Fig. 19). By using Sharp's technique, Lund found the neuromotor sys- tem of Paramecium multimicronucleata constructed as follows: The subpellicular portion of the system is the longitudinal fibrils which connect the basal granules. In the cytostomal region, the fibrils of right and left sides curve inward forming complete cir- cuits (the circular cytosomal fibrils) (Fig. 20). The postoral MORPHOLOGY 59 suture is separated at the point where the cytopyge is situated. Usually 40-50 fibrils radiate outward from the cytostome (the radial cytostomal fibrils). The pharyngeal portion is more com- plex and consists of 1) the oesophageal network, 2) the motorium and associated fibrils, 3) penniculus which is composed of 8 rows of basal granules, thus forming a heavy band of cilia in the Fig. 19. Diagram of the cortical region of Paramecium multimicronu- cleata, showing various organellae, x7300 (Lund), bg, basal granule; c, cilia; et, tip of trichocyst; If, longitudinal fibril; p, pellicle; t, tricho- cyst; tf, transverse fibril. 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 system, the last of which is that part of the neuromotor system that concerns with the body ciHa. ten Kate (1927) suggested that sensomotor ap- paratus is a better term than the neuromotor apparatus. Protective or supportive organellae The external structures as found among various Protozoa which serve for body protection have already been considered 60 PROTOZOOLOGY Fig. 20. The neuromotor system of Paramecium multimicronudeata (Lund), a, oral network; b, motorium, X1670. aep, anterior end of penniculus; c, cytopyge; ccf, circular cytostomal fibril; cof, circular oesophageal fibril; cpf, circular phar3aigeal fibril; ef, endoplasmic fibrils; Ibf, longitudinal body fibril; lof, longitudinal oesophageal fibrils; Ipf, longitudinal pharyngeal fibril; m, motorium; oo, opening of eosophagus; op, oesophageal process; paf, paraoesophageal fibrils; pep, posterior end of penniculus; pnc, posterior neuromotor chain; pof, postoesophageal fibrils; rcf, radial cytostomal fibril; s, suture. MORPHOLOGY 61 (p. 38). 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 membranous 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 processes, and thus the capsule is to be con- sidered as a protective organella. The endoskeletal structures of Radiolaria vary in chemical composition and forms, and are ar- ranged with a remarkable regularity (pp. 371-376). In some of the astomous 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 pecuhar organella, furcula, observed by Lynch in Lechriopyla (p. 536) is said to be concerned with either the neuromotor system or protection. The members of the family Ophryoscolecidae, which are common commensals of the stomach of ruminants, have conspicuous endoskeletal plates which arise in the oral region and extend posteriorly. Dogiel (1923) believed that the skeletal plates of Cycloposthium and Ophryoscolecidae are made up of hemicellulose, "ophryoscolecin," which was also observed by Strelkow (1929). MacLennan found that the skeletal plates of Polyplastron multivesiculatuni were composed of small, roughly prismatic blocks of glycogen, each possessing a central granule. In certain Polymastigina and Hypermastigina, there occurs a flexible structure known as the axostyle, which varies from a filamentous structure as in several Trichomonas, to a very con- spicuous rod-like structure occurring in Parajoenia, Giganto- monas, 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 which have connections with the flagella as seen in Polymonadina and certain Hypermastigina such as Lophomonas. Kirby showed that in Trichomonas tennop- sidis, the axostyle and the granules occurring in it, are of glycog- enous substance. In trichomonad flagellates there is often present along the at- tachment of the undulating membrane a rod-like structure which has been known as costa (Kunstler) and which, according to 62 PROTOZOOLOGY Kirby's extensive study, appears to be the most highly developed in Pseudotrypanosoma and Trichomonas. The staining reaction indicates that its chemical composition is different from that of fiagella, blepharoplast, parabasal body, or chromatin. In the gymnostomous cihates, the cytopharynx is often sur- rounded by rod-hke bodies, and the entire apparatus is often called oral or pharyngeal basket, which is considered as sup- portive in function. The rod-like bodies appear in most cases to Fig. 21. a, trichites in Spathidium spathula, X300 (Woodruff and Spencer); b, trichites in Enchelyodon farctus, X400 (Roux). be trichites which may have been derived from the trichocysts, but which do not explode as do the latter. For example, in Chilodonella cucullulus the oral basket is composed of 12 trichites which are so completely fused in part that the lower portion ap- pears as a smooth tube and in Enchelyodon farctus (Fig. 21, b) much longer trichites form the basket, with reserve structures scattered throughout the cytoplasm (Engelmann). In Spathidium spathula (Fig. 21, a), trichites are imbedded hke 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." MORPHOLOGY 63 Whether the numerous 12-20^ long needle-Hke endoskeletal structures which Kahl observed in Remanella (p. 522) are modi- fied trichites or not, is not known. In numerous ciliates, there is another ectoplasmic organella, the trichocyst, which is much shorter, though somewhat similar in general form. As seen in a Paramecium, the refractile fusiform trichocysts are embedded in the ectoplasm and arranged at right angles to the body surface, while in forms, such as Cyclogramma they are situated obliquely (Fig. 240, c). In Frontonia leucas (Fig. 22), Tonniges found that the trichocysts originated in the chromatinic endosomes of the macronucleus and development takes place during their migration to the ectoplasm; on the other hand, Brodsky believes that the trichocysts are composed of colloidal excretory substances and are first formed in the vicinity of the macronucleus, becoming fully formed during the course of their migration toward the periphery of the body. In species of Prorodon, Kriiger recently observed that the rod-like trichocysts of these ciliates are composed of a cylindrical sac containing a long filament which is arranged in a manner somewhat similar to the polar capsule of cnidosporidian spores. The end facing the body surface is filamentous and connected with the pellicle. The extrusion of the trichocysts is easily induced by means of mechanical pressure or chemical (acid or alkaline) stimulation, though the mechanism of extrusion is not well understood in all forms. Brodsky maintains that the fundamental force is not the mechanical pressure, but that the expansion of the colloidal sub- stances results under the influence of certain stimuli in the ex- trusion of the trichocysts through the pellicle. The fully extruded trichocysts are needle-like in general form. The trichocysts of Frontonia leucas are about G/j, long, but when extruded, measure 50-60/x in length, and those of Paramecium caudatum may reach 40m ill length. Dileptus anser feeds on various ciliates through the cytostome, located at the base of the proboscis, which possesses a band of long trichocysts on its ventral side. When food organisms come in contact with the ventral side of the proboscis, they give a violent jerk, and remain motionless. Visscher saw no formed elements discharged from the trichocysts, and, therefore, considered that these trichocysts contained a toxic fluid and named them toxi- cytes. Recently Hayes found that the exploded trichocysts (Fig. 64 PROTOZOOLOGY ill thr WW extr •I I ! trb P trg trb rt en Fig. 22. a, b, cortical region of Frontonia leucas, with embedded and extruded trichocysts (Tonniges) ; c, d, embedded and discharged tricho- cysts of Dileptus anser, X4200 (Hayes); e, two extruded trichocysts of Paramecium caudatutn, X1530. ci, cilium; ec, ectoplasm; en, endo- plasm; extr, extruded trichocyst; p, pellicle; rt, root of trichocyst; thr, thread of trichocyst; tr, trichocyst; trb, bulb of trichocyst; trg, trichocyst granule. MORPHOLOGY 65 22) could be distinctly seen and suggested that these trichocysts themselves may be toxic. Although the trichocyst was first discovered by Elhs (1769) and so named by Allman (1855), nothing concrete is yet known as to their function. Ordinarily the trichocysts are considered as a defensive organella as in the case of the oft-quoted example Paramecium, but, as Mast demonstrated, the extruded tricho- cysts of this ciUate do not have any effect upon Didinium other than forming a viscid mass about the former to hamper the latter. Penard considers that some trichocysts may be secretory organel- le to produce material for loricae or envelope,, with which view Kahl concurs, as granular to rod-shaped trichocysts occur in Metopus, Amphileptus, etc. Klein has called these ectoplasmic granules protrichocysts, and in Prorodon, Kriiger observed, be- sides typical tubular trichocysts, torpedo-like forms to which he applied the same name. To this group may belong the trichocysts recognized by Kidder in Conchophthirus mytUi. The trichocysts present in certain Cryptomonadina (Chilomonas and Cyatho- monas) are probably homologous with the protrichocysts. The pigments, which give a beautiful coloration to certain ciliates such as Stentor and Blepharisma, are said to be lodged in the protrichocysts. Hold-fast organellae In the Mastigophora, Ciliophora, and a few Sarcodina, there are forms which possess a stalk supporting the body or the lorica. With the stalk the organism is attached to a solid surface. In some cases, as in Anthophysa, Maryna, etc., the dendritic stalks are made up of gelatinous substances rich in iron, which gives to it a reddish color. In parasitic Protozoa, there are special organellae developed for attachment. Many genera of cephaline gregarines are provided with an epimerite of different structures (Figs. 181- 183), by w^hich the organisms are able to attach themselves to the gut epithelium of the host. In Astomata, such as Intoshellina, Maupasella, Lachmannella, etc., simple or complex protrusible chitinous structures are often present in the anterior region; or a certain area of the body may be concave and serves for adhesion to the host, as in Rhizocaryum, Perezella, etc.; or, again, there may be a distinctive sucker-hke organella near the anterior ex- tremity of the body, as in Haptophyra, Steinella, etc. A sucker is also present on the antero-ventral part of Giardia intestinalis. 66 PROTOZOOLOGY In the Myxosporidia and Actinomyxidia, there appear, during the development of s})ore, 1-4 special cells which develop into 1-4 polar capsules, each, when fully formed, enclosing a more or less long spirally coiled hollow thread, the polar filament (Fig. 221). 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. The nema- tocysts (Fig. 104, b) of certain dinoflagellates belonging to Nema- toidium 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 indicates that they are characteristic structures of these dinoflagellates and not foreign in origin as had been held by some. The function of the nemato- cysts in these protozoans is not understood. The parabasal apparatus In the cytosome of many parasitic flagellates, there is frequent- ly present a conspicuous structure known as the parabasal ap- paratus (Janicki), consisting of the parabasal body and the thread (Cleveland), which latter may be absent in some cases. This structure varies greatly among different genera and species in appearance, structure and position within the body. It is usually connected with the blepharoplast and located very close to the nucleus, though not directly connected with it. It may be single, double, or multiple, and may be pyriform, straight or curved rod-like, bandform, spirally coiled or collar-like (Fig. 23). Kofoid and Swezy considered that the parabasal body is derived from the nuclear chromatin, varies in size according to the meta- bolic demands of the organism, and is a "kinetic reservoir." On the other hand, Duboscq and Grasse maintain that this body is the Golgi apparatus, since 1) acetic acid destroys both the para- basal body and the Golgi apparatus; 2) both are demonstrable with the same technique; 3) the parabasal body is made up of chromophile and chromophobe parts as is the Golgi apparatus; and 4) there is a strong evidence that the parabasal body is secre- tory in function. According to Kirby, who has made an extensive study of this organella, the parabasal body could be stained with Delafield's haematoxylin or Mallory's triple stain after fixation with acetic acid-containing fixatives and the body does not show MORPHOLOGY 67 any evidence to indicate that it is a secretory organella. Moreover the parabasal body is discarded or absorbed at the time of divi- sion of the body and two new ones are formed. In the parabasal body of LopJwmonas hlattarum to which the name was originally applied, the structure is discarded when the organism divides and two new ones are reformed from the cen- triole or blepharoplast (Fig. 59), and its function appears to be Fig. 23. Parabasal apparatus in: a, Lophomonas hlattarum (Kudo); b, Metadevescovina debilis; c, Devescovina sp. (Kirby). af, axostylar filaments; bl, blepharoplast; f, food particles; fl, flagella; n, nucleus; pa, parabasal apparatus. supportive. Possibly not all so-called parabasal bodies are homol- ogous or analogous and a fuller comprehension of the function of the organella rests with further investigations. The blepharoplast or centriole In the jMastigophora or in other groups in which flagellate stages occur, the flagellum ends internally in a basal granule, which, in turn, is sometimes connected by a much larger body. This latter organella has been called the centriole or blepharo- 68 PROTOZOOLOGY plast. In many instances they appear to be combined in one. The blepharoplast is further connected by a fibril, the rhizoplast, with the nucleus (Fig. 24). The blepharoplast and centriole are con- l4':5 hJ Fig. 24. Flagellar attachment in Euglenoidina (Hall and Jahn). a, Euglena deses, X2025; b, E. acus, X750; c, E. spirogyra, X720; d, Menoidium incurvum, X1550. sidered synonymous by Minchin, Cleveland, and others, since this organella gives rise to the kinetic element. Woodcock and Minchin held, on the other hand, that the blepharoplast was a nucleus holding a special relation with locomotor organellae, and MORPHOLOGY 69 called it kinetonucleus. In recent years it has become known that the blepharoplast of many flagellates responds positively to Feulgen's nucleal reaction which indicates the presence of thymo- nucleic acid or chromatin in this structure. The Golgi apparatus With the discovery of a wide distribution of the so-called Golgi apparatus in metazoan cells, a number of protozoologists also re- ported a homologous structure from many protozoans. It seems impossible at present to indicate just exactly what is the Golgi Fig. 25. The Golgi bodies in Amoeba proteus (Brown). apparatus, since the so-called Golgi techniques, the important ones of which are based upon the assumption that the Golgi ma- terial is osmiophile and argentophile, and possesses a strong affin- ity to neutral red, are not specific and the results obtained by using the same method often vary a great deal. Some of the ex- amples of the Golgi apparatus reported from various Protozoa are mentioned on page 70. 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- 70 PROTOZOOLOGY Protozoa Golgi apparatus Observers Monocystis, Gregarina Spheres, rings, crescents Hirschler Endamoeba blattae Spheres, rings, crescents Hirschler Adelea Crescents, beaded grains King and Gatenby Entamoeba gingivalis Rings, crescents to network Causey Vorticella, Lionotus, The membrane of con- Nassonov Paramecium, Dogi- tractile vacuole and col- ella, Nassula, Chilo- lecting canals monas, Chilodonella Holomastigotes, Pyr- Parabasal bodies Dubocsq and sonympha, etc. Grasse Aggregata, gregarines Crescents, rings Joyet- Lavergne Euglenoidina Stigma Grass^ Chilomonas Granules, vacuoles Hall Peranema Rings, globules, granules Hall Chromulina, Astasia Rings, spherules with a dark rim Hall Amoeba proteus (Fig. 25) Rings, crescents, globules, granules Brown Pyrsonympha, Di- Rings, crescents, spherules; Brown nenympha granules break down to form network near pos- terior end Euglena gracilis Spherical, discoidal with dark rim; tend to group around or near nucleus Brown Blepharisma undulans Rings in the cytoplasm Moore quently the cortical layer is of unequal thickness, and, therefore, crescentic forms appear. Ringform apparatus was noted in Chilo- donella and Dogiella by Nassonov and network-like forms were observed by Brown in Pyrsonympha and Dinenympha. The numerous observations on the Golgi apparatus of Protozoa as well as of Metazoa, indicate that it is composed of a lipoidal ma- terial in combination with a protein. In line with the suggestion made for the metazoan cell, the Golgi apparatus of Protozoa is considered as having something to do with secretion or excretion. Nassonov considers that osmio- philic lipoidal substance, which he observed in the neighborhood of the walls of the contractile vacuole and its collecting canals in many ciliates and flagellates, is homologous with the meta- MORPHOLOGY 71 zoan 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, but fusion of minute vacu- oles associated with crescentic Golgi bodies produces the vacuole. Duboscq and Grasse who hold that the parabasal body is the Golgi apparatus, maintain that this body is a source of energy which is utilized by the motor organellae. Joyet-Lavergne pointed out that in certain sporozoans the Golgi apparatus is granular and may be the center of enzyme production. The exact morpho- logical and physiological information of the Golgi apparatus must be looked for in future observations. The chondriosomes Widely distributed in many metazoan cells, the chondriosomes have also been recognized in various Protozoa. The chondriosomes possess a low refractive index, and are composed of substances easily soluble in alcohol, acetic acid, etc. 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. Ac- cording 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 resist- ant to reagents. The chondriosomes occur as small spherical to oval granules, rod-like or filamentous bodies, and show a tendency to adhere to or remain near protoplasmic surfaces. In many cases they are dis- tributed without any definite order; in others, as in Paramecium or Opalina, they are regularly arranged between the basal gran- ules of cilia (Horning). In Peranema trichophorum (Fig. 26), ac- cording to Hall, the chondriosomes are said to be located along the spiral striae of the pellicle. Causey (1925) noticed in Leish- mania hrasiliensis usually eight spherical chondriosomes in each individual, which become rod-shaped when the organism divides. He further observed spherical and rod-like chondriosomes in Noctiluca scintillans. 72 PROTOZOOLOGY In certain Protozoa, the chondriosomcs are not always demon- strable. For example, Horning states in Monocystis the chondrio- somes present throughout the asexual Hfe-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 chondrio- somcs appear as soon as the sporozoites are set free. Thus it would appear that the chondriosomcs were reformed de novo. On the other hand, Faure-Fremiet, the first student of the chondrio- somcs in Protozoa, maintained that they reproduce by division, v» /» » .» » .'A' A VM ~ « » * N^ \' "\v>: Fig. 26. The chrondriosomes in Peranema trichophorum, X1750 (Hall)' a, b, surface views and c, optical section of a single individual. which has since been confirmed by many observers. As a matter of fact, Horning found in Opalina, the chondriosomcs are twisted filamentous structures and underwent multiple longitudinal fis- sion in asexual division phase. Before encystment, the chondrio- somcs divide repeatedly transversely and become spherical bodies which persist during encystment and in the gametes. In zygotes, these spherical bodies fuse to produce longer forms which break up into elongate filamentous structures. Richardson and Horning further succeeded in bringing about division of the chondriosomcs in Opalina by changing pH of the medium. As to the function of chondriosomcs, opinions vary. A number of observers hold that they are concerned with the digestive process. After studying the relationship between the chondrio- MORPHOLOGY 73 somes 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 sub- stance for this purpose. 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 ac- tively concerned with the development of the gametes of the Metazoa is well known. Zweibaum's observation, showing an in- crease 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 macro- nuclear material into the chondriosomes. It may be stated that the chondriosomes appear to be associated with the formation of enzymes which participate actively in the processes of catalysis or synthesis in the protozoan body. The author agrees with McBride and Hewer who wrote: "it is a remarkable thing that so Httle is known positively about one of the 'best known' proto- plasmic inclusions." The contractile and other vacuoles The majority of Protozoa possess one or more vacuoles known as pulsating or contractile vacuoles. They occur regularly in all freshwater inhabiting Sarcodina and Mastigophora, and in Cilio- phora regardless of habitat. In the Sporozoa, which are all para- sitic, and the Sarcodina and Mastigophora, which live either in salt water or in the body of other animals, there is no contractile vacuole. In various species of free-living amoebae, the contractile vacu- ole 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 con- tents to outside. The location of the vacuole is not definite in such forms and, therefore, it moves about with the cytoplasmic move- ments; and, as a rule, it is confined to the temporary posterior region of the body. Although almost spherical in form, it may oc- casionally be irregular in shape, as in Amoeba striata (Fig. 140, /). In many testaceans and heliozoans, the contractile vacuoles 74 PROTOZOOLOGY ,y X' Fig. 27. Diagrams showing the contractile vacuole, the accessory vacuoles and the aperture, during diastole and systole in Conchoph- thirus (Kidder). which are variable in number, are formed in the ectoplasm and the body surface bulges out above the vacuoles at diastole. In the Mastigophora, the contractile vacuole appears to be more or less constant in position. In Phytomastigina, they are usually located near the anterior region and, in Zoomastigina, as a rule, in the posterior half of the body. The number of the vacuoles present in an individual varies from one to several. In Euglenoidina, one or more vacuoles are sometimes arranged near the reservoir which opens to "cytopharynx." 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 contents of the vacuole are discharged to outside. In the spe- cies of Conchophthirus, 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 refrac- tile. 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 contents 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 dis- tributed without apparent order, in linear series, or along the body outline. When the contractile vacuoles are deeply seated, there is a delicate duct which connects 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 cytopyge located at the posterior extremity, through which it empties its contents. MORPHOLOGY 75 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, Disemato- stoma, 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 Climacostomum, Eschaneustyla, etc., and several in Tillina. In Peritricha, the contractile vacuole occurs near the posterior region of the peristome and its contents are discharged through a canal into the vestibule. \.y o= <=>!^3^^ <^f^'%Z> \Z7 \ vU =>o FiG. 28. Diagrams showing the successive stages in the formation of the contractile vacuole in Paramecimn rnultimicronucleata (King) ; upper figures are side views; lower figures front views; solid lines indi- cate permanent structures; dotted lines temporary structures, a, full diastole; b-d, stages of systole; e, contents of ampulla passing into injection canal; f, formation of vesicles from injection canals; g, fusion of vesicles to form contractile vacuole; h, full diastole. Of numerous observations concerning the operation of the con- tractile vacuole, that of King (1935) on Paramecimn multimi- croniicleata (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 76 PROTOZOOLOGY Fig. 29. Contractile vacuoles of Paramechivi multimicrouudeata, X1200 (King), a, early systole, side view; b, diastole, front view; c, complete systole, front view; d, systole, side view. MORPHOLOGY 77 collecting canals with strongly osmiophile walls (Fig. 29), and each canal is made up of terminal portion, a proximal injection canal, and an ampulla between them. Surrounding the distal por- tion, there is osmiophilic cytoplasm which may be granulated or finely reticulated, and which Nassonov interpreted as homologous to the Golgi apparatus of the metazoan cell. The injection canal extends up to the pore. The ampulla becomes distended first with fluid transported discontinuously down the canal and the fluid next moves into the injection canal. The fluid now is expelled into the cytoplasm just beneath the pore as a vesicle, the membrane of which is derived from a membrane which closed the end of the injection canal. These fluid vesicles coalesce presently to form the contractile vacuole in full diastole and the fluid is discharged to exterior through the pore, which becomes closed by the remains of the membrane of the discharged vacuole. The function of the contractile vacuole is considered in the following chapter (p. 98). 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 surface. These vesicles (Fig. 30, a-c) vary in diameter from 5 to 8.5/i and contain a clear fluid in which one large spherule or several small highly refractile spherules are suspended. In some, there is a filamentous connection between the spherules and the wall of the vesicle. Penard maintains that these bodies are balanc- ing cell-organs and called the vesicle, the statocyst, and the spher- ules, the statoliths. Another vacuole, known as concrement vacuole, is a character- istic organella in Butschliidae and Paraisotrichidae. As a rule, there is a single vacuole present in an individual at the anterior third of body. It is spherical to oval and its structure appears to be highly complex. According to Dogiel, the vacuole is composed of a pelhcular cap, a permanent vacuolar wall, concrement grains and two fibrillar systems (Fig. 30, d). When the organism divides, the anterior daughter individual retains it, and the posterior in- dividual develops a new one from the pellicle into which concre- ment grains enter after first appearing in the endoplasm. This vacuole shows no external pore. Dogiel believes that its function is sensory and has named the vacuole, the statocyst, and the en- closed grains, the statoliths. Food vacuoles are conspicuously present in the holozoic Pro- 78 PROTOZOOLOGY Fig. 30. a-c, Mtiller's vesicles in Loxodes (a, b) and in Remanella (c) (a, Penard; b, c, Kahl); d, concrement vacuole of Blepharoprosth- ium (Dogiel). cf, centripetal fibril; eg, concrement grains; cp, cap; fw, fibrils of wall; p, pellicle; vp, vacuolar pore; w, wall. tozoa 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 sus- pended the food matter, such as various Protophyta, other Pro- tozoa or small Metazoa. In the Sarcodina, the Mastigophora and the Suctoria, which do not possess a cytostome, the food vacuoles assume the shape of the food particles and, when these particles are large, it is difficult to make out the thin film which surrounds them. When minute food particles are taken through a cyto- stome, as is the case with the majority of euciliates, the food vacu- oles are usually spherical and of approximately the same size within a single protozoan. In the saprozoic Protozoa, which ab- sorb fluid substances through the body surface, food vacuoles containing solid food, of course, do not occur. The chromatophore and associated organellae In the Phytomastigina and certain other forms which are green-colored, one to many chromatophores (Fig. 31) or chloro- plasts containing chlorophyll occur in the cytosome. The chroma- I MORPHOLOGY 79 t ophores vary in form among different species ; namely, discoidal, ovoid, band-form, rod-like, cup-like, network or irregularly dif- fused. The color of the chromatophore depends upon the amount and kinds of pigment which envelops the underlying chlorophyll substance. Thus the chromatophores of Chrysomonadina are brown or orange, as they contain one or more accessory pigments, including phycochrysin, and those of Cryptomonadina are of various types of brown with very diverse pigmentation. In Chla- romonadina, the chromatophores are bright green, containing an excess of xanthophyll. In dinoflagellates, they are dark yellow or brown, because of the presence of pigments: carotin, phylloxan- thin, and peridinin (Kylin), the last of which is said to give the brown coloration. A few species of Gymnodinium contain blue- green chromatophores for which phycocyanin is held to be re- sponsible The chromatophores of Phytomonadina and Euglenoi- dina are free from any pigmentation, and therefore green. Aside from various pigments associated with the chromatophores, there are carotinoid pigments which occur often outside the chrom- atophores, and are collectively known as haematochrome. The haematochrome occurs in Haematococcus pluvialis, Euglena sanguinea, Chlamydomonas, etc. In Haematococcus, it increases in volume and in intensity when there is a deficiency in phosphorus and especially in nitrogen; and when nitrogen and phosphorus are sufficiently present in the culture medium, the haematochrome loses its color completely (Reichenow; Pringsheim). Steinecke also noticed that the frequent yellow coloration of phytomonads in moorland pools is due to a development of carotin in the chro- matophores as a result of deficiency in nitrogen. In association with the chromatophores are found the pyre- noids (Fig. 31) which are usually embedded in them. The pyre- noid is a viscous structureless mass of protein (Czurda), and may or may not be covered by tightly fitting starch-envelope, com- posed of several pieces or grains which appear to grow by apposi- tion 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 gen- erally agreed that the pyrenoid is concerned with the formation of the starch and allied anabolic products of photosynthesis. Chromatophore-bearing Protozoa usually possess also a stigma (Fig. 31) or eye-spot. The stigma may occur in exceptional cases in colorless forms, as in Khawkinea according to Jahn. It is ordi- 80 PROTOZOOLOGY narily situated in the anterior region and appears as a reddish or brownish red spot or rod, embedded in the cortical layer of the cytoplasm. The color of the stigma is due to the presence of drop- lets 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 division. In many species, the stigma possesses no acces- sory parts, but, according to Mast, the pigment mass in Chlamy- Flagella Stigma Pyrenoids Chromatophores — Nucleus Shell Chromatophores Pyrenoids Fig. 31. a, Trachelomonas hispida, X530 (Doflein); b, c, living and stained reproductive cells of Pleodorina illinoisensis, XlOOO (Merton) ; d-f, terminal cells of Hydrur us foetid us, showing division of chromato- phore and pyrenoid (Geitler); g-i, Chlamydomonas sp., showing the division of pyrenoid (Geitler). domonas, 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. 101,c, d,(j,h) M'hich 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 considers MORPHOLOGY 81 that the stigma in the Volvocidae is an organella which deter- mines the direction of the movement. References Belar, K. 1926 Der Formwechsel der Protistenkerne. Ergebn, u. Fortschr. Zool., Vol. 6. Brodsky, a. 1924 Die Trichocysten der Infusorien. Arch. rus. protist., Vol. 3. Brown, V. E. 1930 The Golgi apparatus of Amoeba proteus. Biol. Bull., Vol. 59. 1930 The Golgi apparatus of Pyrsonympha and Dine- nympha. Arch. f. Protistenk., Vol. 71. Chatton, E. and A. Lwoff 1935 Les cilies apostomes. Arch. zool. exp. et gen.. Vol. 77. Causey, D. 1925-1926 Mitochondria and Golgi bodies in Endamoeba gingivalis. Mitochondria in Leishmania hrasilien- sis. Mitochondria in Noctiluca scintillans. Univ. Calif. Publ. Zool., Vol. 28. Cleveland, L. R., S. R. Hall, E. P. Sanders and J. Collier 1934 The wood-feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts Sci., Vol. 17. Cushman, J. A. 1933 Foraminifera: their classification and eco- nomic use. Second edition. Sharon, Mass. Doflein, F. 1916 Studien zur Naturgeschichte der Protozoen. VII. Zool. Jahrb. Abt. Anat., Vol. 39. Dogiel, V. 1923 Cellulose als Bestandteil des Skellettes bei einigen Infusorien. Biol. Zentralbl., Vol. 43. 1929 Die sog. "Konkrementenvakuole" der Infusorien als eine Statocyste betrachtet. Arch. f. Protistenk., Vol. 68. DuBoscQ, O. and P. P. Grasse 1933 L'appareil parabasal des flagelles. Arch. zool. exp. et gen.. Vol. 63. Gelei, J. VON 1926 Zur Kenntnis des Wimperapparates. Zeitschr. f. ges. Anat., Abt. I, Vol. 81. Giese, a. C. 1938 Reversible bleaching of Blepharisma. Trans. Amer. Micr. Soc, Vol. 57. Hall, R. P. 1929 Reaction of certain cytoplasmic inclusions to vital dyes and their relation to mitochondria and Golgi ap- paratus in the flagellate Peranema trichophorum. Jour. Morph. Physiol., Vol. 48. and T. L. Jahn 1929 On the comparative cytology of certain euglenoid flagellates and the systematic position of the families Euglenidae and Astasiidae. Trans. Amer. Micro. Soc, Vol. 48. Hayes, M. L. 1938 Cytological studies on Dileptus anser. Ibid., Vol. 57. Hertwig, R. 1902 Die Protozoen und die Zelltheorie. Arch. f. Protistenk., Vol. 1. 82 PROTOZOOLOGY Horning, E. S, 1926 Observations on mitochondria. Austral. Jour. Exp. Biol. Med. Sci., Vol. 3. 1927 On the orientation of mitochondria on the surface cytoplasm of infusorians. Ibid., Vol. 4. 1929 Mitochondrial behavior during the life cycle of a sporozoan (Monocystis). Quart. Jour. Micr, Sci., Vol. 73. Janicki, C. v. 1911 Zur Kenntnis des Parabasalapparates bei parasitischen Flagellaten. Biol. Zentralbl., Vol. 31. Kidder, G. W. 1933 On the genus Ancistruma Strand (Ancis- trum Maupas). Biol. Bull., Vol. 64. 1933 Conchophthirus caryoclada sp. nov. Ibid., Vol. 65, 1934 Studies on the ciliates from freshwater mussels. Ibid., Vol. 66. King, R. L. 1935 The contractile vacuole of Paramecium multi- micronucleata. Jour. Morph., Vol. 58. KiRBY, Jr., H. 1931 The parabasal body in trichomonad flagel- lates. Trans. Amer. Micr. Soc, Vol. 50. Klein, B. M. 1926 Ergebnisse mit einer Silbermethode bei Ciliaten. Arch. f. Protistenk., Vol. 56. 1927 Die Silverliniensystem der Ciliaten. Ibid., Vol. 58. — 1929 Weitere Beitrage zur Kenntnis des Silberlinien- systems der Ciliaten. Ibid., Vol. 65. 1930 Das Silberliniensystem der Ciliaten. Ibid., Vol. 69. KoFOiD, C. A. and Olive Swezy 1921 The free-living un- armored Dinoflagellata. Mem. Univ. California. Vol. 5. KoLTZOFF, N. K. 1911 Untersuchung iiber die Kontraktilitat des Stieles von Zoothamnium alternans. Biol. Zeitschr. Mos- kau., Vol. 2. Kruger, F. 1934 Untersuchungen liber die Trichocysten einiger Prorodon-Arten. Arch. f. Protistenk., Vol. 83. Kudo, R. R. 1924 A biologic and taxonomic study of the Micro- sporidia. Illinois Biol. Monogr., Vol. 9. 1926 Observations on Lophomonas hlattarum, a flagel- late inhabiting the colon of the cockroach, Blatta orientalis. Arch. f. Protistenk., Vol. 53. 1936 Studies on Nydotherus ovalis Leidy, with special reference to its nuclear structure. Ibid., Vol. 87. Lund, E. E. 1933 A correlation of the silverline and neuromotor systems of Paramecium. Univ. Calif. Publ. ZooL, Vol. 39. Lynch, J. E. 1930 Studies on the ciliates from the intestine of Strongylocentrotus. II Lechriopylamijsta.r, gen. nov., sp. nov. Ibid. Vol. 33. Mast, S. O. 1928 Structure and function of the eye-spot in unicellular and colonial organisms. Arch. f. Protistenk., Vol. 60. Nassonov, D. 1924 Der Exkretionsapparat (kontraktile Vaku- ole) der Protozoen als Homologen des Golgischen Apparatus der Metazoenzelle. Arch. mikr. Anat., Vol. 103. MORPHOLOGY 83 Penard, E. 1922 Etudes sur les infusoires d'eau douce. Geneva. PiNEY, A. 1931 Recent advances in microscopy. London. Pringsheim, E. 1914 Die Ernahrung von Haematococcus pluvi- alis. Beitr. Biol. Pflanzen, Vol. 12. Reichenow, E. 1909 Untersuchungen an Haematococcus pluvi~ alis nebst Bemerkungen liber andere Flagellaten. Arch, kaiserl, Gesundheitsamt., Vol. 33. 1928 Ergebnisse mit der Nuklealfarbung bei Protozoen. Arch. f. Protistenk., Vol. 61. Richardson, K. C. and E. S. Horning 1931 Cytoplasmic struc- tures in binucleate opalinids with special reference to the Golgi apparatus. Jour. Morph. Physiol., Vol. 52. RosKiN, G. 1923 La structure des Myonemes des infusoires. Bull. biol. France et Belg., Vol. 57. ■ 1925 Ueber die Axopodien der Heliozoa und die Greift- entakel der Ephelotidae. Arch. f. Protistenk., Vol. 52. Rumjantzew, a. and E. Wermel 1925 Untersuchungen ueber den Protoplasmabau von Actinosphaerium eichhorni. Ibid., Vol. 52. Schroder, O. 1906 Beitrage zur Kenntnis von Stentor coeruleus und St. roeselii. Ibid., Vol. 8. Schuberg, a. 1888 Die Protozoen des Wiederkauermagens. I. Zool. Jahrb. Abt. System., Vol. 3. Sharp, R. 1914 Diplodinium ecaudatum with an account of its neuromotor apparatus. Univ. Calif. Publ. Zool., Vol. 13. Strelkow, a. 1929 Morphologische Studien liber oHgotriche Infusorien aus dem Darme des Pferdes. I. Arch. f. Protist- enk., Vol. 68. Taylor, C. V. 1920 Demonstration of the function of the neuro- motor apparatus in Euplotes by the method of micro-dissec- tion. Univ. Calif. Publ. Zool., Vol. 19. ten Kate, C. G. B. 1927 Ueber das Fibrillensystem der Ciha- ten. Arch. f. Protistenk., Vol. 57. Tonniges, C. 1914 Die Trichocysten von Frontonia leucas und ihr chromidialer Ursprung. Ibid., Vol. 32. Turner, J. P. 1933 The external fibrillar system of Euplotes with notes on the neuromotor apparatus. Biol. Bull. Vol. 64. Verworn, M. 1903 Allgemeine Physiologic. Fourth edition. Jena. VisscHER, J. P. 1926 Feeding reactions in the ciliate Dileptus gigas, with special reference to the trichocysts. Biol. Bull, Vol. 45. Vlk, W. 1938 Ueber den Bau der Geissel. Arch. f. Protistenk., Vol. 90. Woodruff, L. L. and H. Spencer 1922 Studies on Spathidium spathula. I. Jour. Exp. Zool., Vol. 35. YocoM, H. B. 1918 The neuromotor apparatus of Euplotes patella. Univ. Calif. Publ. Zool, Vol. 18. Chapter 4 Physiology THE morphological consideration which has been given in the last chapter, is, though necessarily brief, indicative of the oc- currence 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 interaction of different substances which enter into the make-up of protoplasm and, for the most part, these are be- yond our powers of analysis." A full discussion of various physio- logical problems pertaining to Protozoa is out of question in the present work and, therefore, a general consideration on protozoan physiology will suffice for our purpose. Nutrition The Protozoa obtain nourishment in manifold ways, which may be placed under three types: holozoic, holophytic, and sapro- zoic. 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, the digestion and assimilation, and rejection of indigestible portions. The methods of food-capture vary among different forms. In the Sarcodina, the food organisms are captured and taken into the body at any point. The methods however vary. According to Rhumbler's oft-quotied observations, four methods of food-inges- tion occur in amoebae (Fig. 32) ; namely, 1) by "import," in which the food is taken into the body upon contact, with very little movement on the part of the amoeba (a); by "circumfluence," in which the cytoplasm flows around the food organism as soon as it comes in contact with it on all sides and engulfs it (b); 3) by "cir- 84 PHYSIOLOGY 85 cumvallation," in which the amoeba without contact with the food, forms pseuclopodia which surround the food on all sides and ingest it (c); 4) by "invagination," in which the amoeba touches and adheres to the food, and the ectoplasm in contact with it is Fig. 32. Various waj's by which amoebae capture food organisms. a, Amoeba verrucosa feeding on Oscillatoria by 'import' (Rhumbler); b, A. proteus feeding on bacterial glea by 'circumfluence'; c, on Para- mecium by 'circumvollation' (Kepner and Whitlock); d-h, A. ver- rucosa ingesting a food particle by 'invagination' (Gross- Allermann). invaginated into the endoplasm as a tube, the cytoplasmic mem- brane later liquefies and disappears {d-h). Jennings, Kepner, Schaeffer and others, have made studies with reference to the food-ingestion in amoebae. In certain testaceans, such as Gromia, several rhizopodia co- operate in engulfing the prey and, in Lieberkuhnia (Fig. 33), Ver- worn noted cihates are captured and digested in the rhizopodium. 86 PROTOZOOLOGY Fig. 33. A filopodium of Lieberkuhnia, capturing and digesting Colpidium colpoda (Verworn). Similar observation was made by Schaudinn in the heliozoan Camptonema in which several axopodia anastomose to capture a prey (Fig. 163, d). In the holozoic Mastigophora, such as Hyper- mastigina, which do not possess cytostome, the food-ingestion is by pseudopodia also. The food particles become attached to the pseudopodium and are held there on account of the viscid nature of the pseudopodi- um. The sudden immobility of active organisms upon coming in contact with pseudopodia of certain forms, such as Actinophrys, Actinosphaerium, Gromia, Elphidium, etc., suggests, however, probable discharge of poisonous substances. In the Suctoria which lack a cytostome, the tentacles serve as food-capturing organel- lae. The suctorial tentacle bears on its distal end a rounded knob which, when it comes in contact with an actively swimming cili- ate, stops the latter immediately (Parapodophrya typha, Fig. 287, a). The prehensile tentacles of Ephelotidae are said to be similar in structure to the axopodia, in that each possesses a bun- dle of axial filaments around a cytoplasmic core (Roskin). These tentacles are capable of piercing through the body of a prey. In some suctorians, such as Choanophrya (Fig. 291, a), the tentacles are said to be tubular, and both solid and liquid food materials are sucked in through the cavity. The rapidity with which a tentacle of a suctorian stops a very actively swimming ciliate is attributed to a certain substance secreted by the tentacles which paralyzes the prey. PHYSIOLOGY 87 In the cytostome-bearing Mastigophora, the lashing of flagella will aid in bringing about the food-particles to the cytostome, where it is taken into the endoplasm. In the ciliates there are nu- merous types of cytostomes and associated organellae. But food- capturing seems to be in general of two kinds. When the cytostome is permanently open, the organism ingests food-particles which are small enough to pass the cytostome and cytopharynx, as in the case of Paramecium. Another type is one, such as noted in Coleps, Didinium, etc., where the ciliate attacks other organism and sucks in the body substance of the latter through the en- larged cytostome. The ingested food-particles are always surrounded by a film of fluid which envelops the organism and the whole is known as the food vacuole (p. 77). The quantity of fluid taken in with the food varies greatly and, generally speaking, seems to be inversely proportional to the size, but proportional to the activity, of the food organisms. Food vacuoles composed entirely of surrounding liquid medium have occasionally been observed. Edwards (1925) observed ingestion of fluid-medium by an amoeba by forming food-cups under changed chemical composition. Brug (1928) re- ports seeing Entamoeba hystolytica engulf liquid culture medium by formation of lip-Uke elevation of the ectoplasm and Kirby (1932) figures ingestion of the brine containing no visible organ- isms by the cytostome of Rhopalophrya salina. Mast and Doyle (1934) stated that if Amoeba proteus, A. dubia, A. dofleini, or A. radiosa is placed in an albumin solution, a hypertonic balanced salt solution or a hypertonic solution of calcium gluconate, it rap- idly 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 indicate that the organism ingests considerable quantities of fluid in this way. The two authors consider that it is "a biological adaptation which serves to compensate 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 endoplasm. In the ciliates, the fluid endoplasm often shows a definite rotation movement. In Paramecium, the general direction is along one side up to the anterior end and down the other side, with a short cyclosis in the posterior half of the body. In Carchesium, according to Greenwood, the food-vacuoles pass 88 PROTOZOOLOGY down to one end of the macronucleus and then move close to its concave surface to near the anterior end of the nucleus where def- ecation to the vestibule takes place (Fig. 34). As stated above, in a number of species the food organisms are paralyzed or killed upon contact with pseudopodia, tentacles or exploded trichocysts. In numerous other cases, the captured or- FiG. 34. Diagram showing the digestion within the food vacuoles in Carchesium polypimim (Greenwood), a, digestion area; b, region of little change; c, region of acid reaction; d, region of neutral reaction; e, defecation area. ganism is taken into the food-vacuole alive, as will easily be noted by observing Chilomonas taken in by Amoeba proteus or actively moving bacteria ingested by Paramecium. But the prey ceases to move in a very short time. Apparently 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. Engel- mann (1878) demonstrated that the granules of blue litmus, when ingested by Paramecium or Amoeba, became red in a few minutes. PHYSIOLOGY 89 Brandt (1881) examined the staining reactions of amoebae by means of haematoxylin, and found that the watery vacuoles con- tained acid. Metschnikoff (1889) also showed that there appears an acid secretion around the ingested litmus grains in Myceto- zoa. Greenwood and Saunders (1884) found in Carchesium that ingestion of food particles stimulated the cytoplasm to secrete a cv cv Fig. 35. Diagram showing changes in reactions in food-vacuoks of Paramecium caudatum, after ingesting litmus (Shapiro), b, blue; cv, contractile vacuole; lb, light blue; Ir, light red; r, red. mineral acid (Fig. 34). 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 sec- ond 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 acid during the entire period of protein digestion, and becomes neutral to finally alkaline when the solution of the food substance is ended. Metalnikoff (1912) 90 PROTOZOOLOGY found in tlie food vacuoles of Paramecium, besides acid-alkaline reaction change, that some vacuoles never show acid reaction and others occasionally show sustained acid reaction. According to Shapiro (1927), who observed reaction change of the food vacu- oles in Paramecium caudatum (Fig. 35) by using phenol red, neu- tral red, Congo red, and litmus, 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 poste- rior half of the body. Later, the vacuoles show a decreased acidity, finally reaching pH 7.0 prior to excretion. In Vorticella sp. and Stylonychia pustulata, the range of pH observed in the food vacu- oles was said to be 4.5-7.0 and 4.8-7.0 respectively. The food vacuoles of Actinosphaerium, according to Rowland (1928), pos- sess 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. Just exactly what processes take place in the food vacuole have been observed only in a few cases. Nirenstein noticed the appear- ance of numerous neutral red-stainable granules around the food vacuole which pass into the inside of the vacuole, and regarded them as carriers of a tryptic ferment, while Roskin and Levinsohn demonstrated the oxidase reaction in these granules. A number of enzymes have been reported in the Protozoa, some of which are mentioned on page 91. 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 di- gested and decomposed 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 reported in Mycetozoa, Foraminifera, Pelomyxa, Amoeba, Entamoeba, Ophryoscolecidae and other ciliates by sev- eral investigators. In Pelomyxa, Stole (1900) found that the so- called refractile bodies are intimately associated with the carbo- hydrate metabolism in that they are filled with glycogen which amount is proportionate to the food matter the organism ob- tains. The members of Vampyrella (p. 291) are known to dissolve the cellulose wall of algae, especially Spirogyra in order to feed on PHYSIOLOGY 91 Protozoa Enzymes Observers Aethalium septicum Pelomyxa palustris Soil amoebae Balantidium coli Glaucoma pyriformis Colpidium striatum Poly- and Hyper- mastigina in wood roach Pepsin-like enzyme, dis- solving albumins in acid medium Pepsin-like and diastatic enzymes "Amoebodiastase": tryp- sin-like, active in neutral or slightly alkaline me- dium, liquefies gelatin, coagulates albumin, in- active at 60°C. Diastatic enzyme Proteolytic enzyme, ca- pable of hj^drolyzing casein Proteolytic enzyme, ca- pable of hydrolyzing casein Cellulase; Cellobiase Krukenberg (1886) Hartog and Dixon (1893) Mouton (1902) Glaessner (19081 Lwoff (1932) Elliott (1933) Cleveland et al. (1934) their contents. Pelomyxa (Stole), Foraminifera (Schaudinn), Amoeba (Rhumbler), Hypermastigina, Polymastigina (Cleve- land), etc., have also been known for possessing the power of cellulose digestion. Many of the Hypermastigina and Polymasti- gina which lead symbiotic life in the intestine of the termite and the wood roach, as demonstrated by Cleveland and his cowork- ers, 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 ciliate commensals inhabiting the stomach of ruminants also apparently digest the cellulose, since the fecal matter as a rule does not contain this substance. The digestion of fat by Protozoa had not been known, although oils and fat have been observed in numerous Protozoa, until Dawson and Belkin (1928) injected different oils into Amoeba duhia and found that from 1.4 to 8.3 per cent of the injected oil was digested. The indigestible residue of the food is extruded from the body. The extrusion may take place at any point on the surface in many Sarcodina by a reverse process of ingestion of food. But in pelli- 92 PROTOZOOLOGY cle-bearing forms, the defecation takes place either through the cytopyge located in the posterior region of the body or through an aperture to the vestibule (in Carchesium). Permanent cyto- pyge is lacking in some forms. In Fabrea salina, Kirby (1934) no- ticed that a large opening is formed at the posterior end, the con- tents of food vacuoles discharged, and the opening closes over. At first the margin of the body is left uneven, but soon the evenly rounded outline is restored. The same seems to be the case with Spirostomum (Fig. 36), Blepharisma, etc. Fig. 36. Outline sketches showing the defecation process in Spirostoni^un ambiguum (Blattner). Holophytic (autotrophic, phytotrophic) nutrition. This is the type of nutrition in which the Protozoa are able to decompose carbon dioxide by means of chlorophyll contained in chromato- phores (p. 78) in the presence of the sunUght, liberating the oxy- gen and combining the carbon with other elements derived from water and inorganic salts. The pyrenoids (p. 79) are inseparably connected with the reserve carbohydrate formation in this nutri- tion. Aside from the Phytomastigina, chromatophores were defi- nitely observed in Cyclotnchium meunieri (Fig. 230, o) by Powers. In a number of other cases, the organism itself is without chro- matophores but is apparently not holozoic, because of the presence of chlorophyll-bearing organisms within it. For example, in the testacean Paulinella (Fig. 155, c) in which occur no food vacuoles, chromatophores of peculiar shape are always present. The latter appear to be a species of algae which holds a symbiotic relation- ship with the testacean, and perhaps it acts for the sarcodinan as the chromatophores of the Phytomastigina. Saprozoic (saprophytic) nutrition. In this nutrition, the Proto- zoa obtain nourishment by diffusion through the body surface. This is accomplished without any special organellae. Perhaps the PHYSIOLOGY 93 only instance in which the saprozoic nutrition is accompHshed through a special organella is the pusules (Figs. 101, 102) in ma- rine dinoflagellates which, according to Kofoid and Swezy, ap- pear to contain decomposed organic matter and aid the organ- isms in carrying on this process. The dissolved food matters are simpler compounds which have originated in animal or vegetable matter due to the decomposing activities of bacterial organisms. Numerous free-living Zoomastigina nourish themselves with this method. Recently a number of investigators found that saprozoic Protozoa could be cultivated in bacteria-free media of known compositions. For example, Pringsheim observed in Polytoma uvella (Fig. 91, h) that sodium acetate is needed from which the starch among others is produced, and carbohydrates have no di- rect bearing upon the nutrition, but fatty acids derived from them participate in the metabolism. Hall, Jahn, Loefer and oth- ers are following the same line of work which may lead to a better understanding of saprozoic nutrition as found in Protozoa. The Protozoa which live within the body of another organism are able to nourish themselves by absorbing the digested or de- composed substances of the host and could be considered as sap- rozoic though parasitic has sometimes been used. Coelozoic Pro- tozoa belong to this group, as for example, Protociliata, astomous ciliates, Trypanosomidae, etc. In the case of cytozoic or certain histozoic forms, such as Cnidosporidia, the host cytoplasm is ap- parently liquefied or hydrolyzed by enzymes (?) before being ab- sorbed by the latter. The parasitic Protozoa, which actually feed on host tissue cells, such as Entamoeba histolytica, Balantidium coli, etc., or endocommensals, employ, of course, the holozoic nu- trition. Many Protozoa nourish themselves by more than one method at the same or different times, subject to a change in external conditions. This is sometimes referred to as mixo trophic nutrition (Pfeiffer). For example, Euglena gracilis, according to Zumstein (1889) and Lwoff (1932) loses its green coloration and becomes Astasia-like in the dark, or even in the light when the culture me- dium is very abundant in decomposed organic substances, which would indicate that this organism is capable of carrying on both holophytic and saprozoic nutrition. On the other hand Chloro- gonium euchlorum and C. elongatum are said, according to Loefer (1934), to retain their green coloration after a year of cultivation 94 PROTOZOOLOGY in total darkness, although the chromatophores appear somewhat modified. The reserve food matter The anabolic activities of Protozoa result in the growth and in- crease in 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, ether; become swollen and later dissolved in boiling water; and are reduced to a sugar by boiHng in dilute sulphuric acid. This substance which composes the refractile bodies is called paraglycogen (Biitschli) or zooamylum. The abun- dant glycogen bodies of Pelomyxa have already been mentioned (p. 90). Rumjantzew and Wermel demonstrated glycogen in Actinosphaerium. In lodamoeba, glycogen body is conspicuously present and is taken as a characteristic feature of the organism. The iodinophile vacuole of the spores of Myxobolidae is a con- spicuously well-defined vacuole containing glycogenous substance and is also considered as possessing a taxonomic value. In many cihates, both free-living (Paramecium, Glaucoma, Vorticella, etc.) and endozoic (Ophryoscolecidae, Nyctotherus, Balantidi- um, etc.), glycogenous bodies are always present. 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 characteristic 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. 37) is spirally coiled which confirms BiitschU's observation. The paramylon appears to be a polysaccharide which is insoluble in boiling water, but dis- solves in concentrated sulphuric acid, potassium hydroxide, and slowly in formaldehyde. It does not stain with either iodine or chlor-zinc-iodide and when treated with a dilute potassium hy- droxide, the paramylon bodies become enlarged and frequently exhibit a concentric stratification. In the Chrysomonadina, the reserve food material is in the form of refractile bodies which are collectively called leucosin, PHYSIOLOGY 95 probably a carbohydrate. Oils occur in various Protozoa and when there is a sufficient number of oil producing forms in a body of water, the water may develop various odors. Whipple lists the following Protozoa, each of which if present in large numbers, may produce an offensive odor: 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), Pando- rina (faintly fishy), Volvox (fishy), Ceratium (vile stench, rusty Fig. 37. a-d, two types of paramylon present in Euglena gracilis (Biitschli); e-h, paramylon of E. sanguined, XllOO (Heidt). e, natural appearance; dried forms; h, strongly pressed bodies. brown color), Glenodinium (fishy), Peridinium (fishy, like clam- shells), and Bursaria (Irish moss, salt marsh, fishy). Fats have also been detected in many Protozoa, such as Myxo- sporidia, Protociliata, certain Euciliata, Trypanosoma, etc. Ac- cording to Panzer, the fat contents of Eimeria gadi was 3.55 per cent and Pratje reports that 12 per cent of the dry matter of Noc- tiluca scintillans appeared to be the fatty substance present in granular forms and which are said to give phosphorescence upon mechanical or chemical stimulation. A number of other dinoflag- ellates, such as Peridinium, Ceratium, Gonyaulax, Gymnodini- um, etc., also emit phosphorescence. In other forms the fats may be hydrostatic in function, as is the case with a number of pelagic Radiolaria. Another reserve food-stuff which occurs widely in Protozoa, excepting Ciliophora, is the so-called volutin or metachromatic granules. It is apparently equally widely present in Protophyta. In fact it was first discovered in the protophytan Spirillum volu- tans. Meyer coined the name and held it to be made up of a nu- 96 PROTOZOOLOGY cleic acid. It stains deeply with nuclear dyes. Reichenow (1909) demonstrated that if Haematococais pluvialis (Fig. 38) was culti- vated in phosphorus-free medium the volutin is quickly used up and does not reappear. If however, the organisms are cultivated in a medium rich in phosphorus, the volutin increases greatly in volume and, as the culture becomes old, it gradually breaks down. In Polytomella agilis (Fig. 92, c, r/), Doflein showed that an addi- tion of sodium phosphate resulted in an increase of volutin. Reichenow, Schumacher, and others, hold that the volutin appears Fig. 38. Haematococcus phivialis, showing the development of volu- tin in the medium rich in phosphorus and its disintegration in ex- hausted medium, X570 (Reichenow). a, second daj^; b, third day; c, fourth da}'; d, e, sixth day; f, eighth day. to be a free nucleic acid, and is a special reserve food material for the nuclear substance. Recently Sassuchin (1935) studied the volutin in Spirillu?n volutans and Sarcina flava and found that the volutin appears during the period of strong growth, nourishment and multiplication, disappears in unfavorable condition of nour- ishment and gives a series of characteristic carbohydrate reac- tions. Sassuchin considers that the volutin is not related to the nucleus, but is reserve food material of the cell, which is compos- ed of glykoproteid. 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 in- volved in this process appears to be brought into contact with the substances in two ways in Protozoa. The great majority of free- living, epizoic and certain endozoic 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. PHYSIOLOGY 97 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. It is considered that endozoic Protozoa of metazoan digestive systems live also in a medium containing no dissolved oxygen. All these forms appear to possess capacity of splitting complex oxy- gen-bearing substances present in the body to produce necessary oxygen. The liberation of energy is accompanied by production of water and carbon dioxide. Several investigators studied the influence of abundance or lack of oxygen upon different Protozoa. For example. Putter dem- onstrated that several ciliates reacted differently when subjected to anaerobic condition, some perishing rapidly, others living for a considerable length of time. Death is said by Lohner to be brought about by a volume-increase due to accumulation of the waste products. 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, sup- posed that the difference in longevity of aerobic Protozoa in ana- erobic conditions was correlated with that of the amount of reserve food material such as protein, glycogen and paraglycogen present in the body. Noting Paramecium is less affected by anaerobic con- ditions than Spirostomum in a small amount of water, Putter maintained that the smaller the size of Protozoa and the more elaborate the contractile vacuole system, they suffer the less lack of oxygen in the water, since the removal of catabolic waste de- pends upon these factors. The variety of habitats and results of artificial cultivations of various Protozoa clearly indicate 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 consumption of Paramecium is said, according to Lund (1918) and Amberson (1928), to be fairly constant over a wide range of oxygen concentration. Specht (1934) considers the meas- urements of the oxygen consumption and carbon dioxide produc- tion in Spirostomum amhiguum vary because of the presence of a base produced by the organism. Soule (1925) observed in the cul- 98 PROTOZOOLOGY tural tubes of Trypanosoma lewisi and Leishmania tropica, the oxygen contained in about 100 c.e. of air of the test tube is used up in about 12 and 6 days respectively. A single Paramecium caudatum is said to consume in one hour at 21°C. from 0.0052 c.c. (Kalmus) to 0.00049 c.c. (Rowland and Bernstein) of oxygen. 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 of Spirostomum. The Hypermastigina of the termite are killed, ac- cording to Cleveland, when the host animals are kept in an excess of oxygen. Jahn (1935) found that Chilomonas Paramecium in bacteria-free cultures in heavily buffered peptone-phosphate media at pH 6.0 required for rapid growth carbon dioxide which apparently brings about a favorable intracellular hydrogen-ion concentration. Excretion and secretion The catabolic waste material composed of w^ater, carbon diox- ide, urea and other nitrogenous compounds, all of which are solu- ble, pass out of the body by diffusion through the surface or by means of the contractile vacuole (p. 73). The protoplasm of the Protozoa is generally considered to possess a molecular make-up which appears to be similar among those living in various habi- tats. In the freshwater Protozoa, the water diffuses through the body surface and so increases the water contents of the body pro- toplasm 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 endozoic. Protozoa live in isotonic media and there is no excess of water entering the body, hence the con- tractile vacuoles are not found in them. Just exactly why all eucili- ates and suctorians possess the contractile vacuole regardless of habitat, has not been explained. There are accumulating evidences to indicate that the pellicle of the ciliate is impermeable to water and salts, and that the water enters the ciliate body through the cytostome and cytopharynx only. Frisch (1937) observed recently such is the case in Paramecium vmltimicronucleata. If this is true in all ciliates, it is quite easy to understand the universal occur- rence of the contractile vacuole in the cytostome-bearing ciliates. However, it does not explain all cases, as a number of astomous ciliates with a definite peUicle possess contractile vacuoles (p. 488). PHYSIOLOGY 99 That the ehmination of excess amount of water from the body is one of the functions of the contractile vacuole appears to be beyond doubt judging from the observations of Zuelzer (1907), Finley (1930) and others, on Amoeba verrucosa which lost gradu- ally its contractile vacuole as sodium chloride was added to the water, losing the organella completely in the seawater concentra- tion. Herf (1922) studied the pulsation of the contractile vacuoles of Paramecium caudatum in fresh water as well as various salt concentrations, and obtained the following measurements : Per cent NaCl in water . 25 0.5 . 75 1 . 00 Contraction period in second 6.2 9.3 18.4 24.8 163.0 Excretion per hour in body volumes 4.8 2.82 1.38 1.08 0.16 The contractile vacuole also serves to remove from the body part of soluble catabolic wastes, judged by numerous observa- tions. Weatherby (1929) showed that the contractile vacuoles of Paramecium and Spirostomum contain urea, and that of Didini- um contains ammonia and occasionally trace of uric acid. The number of the contractile vacuoles present in a given species as in various species of Paramecium, is not always constant. Nor is its size constant. According to Taylor (1920) the average size of the contractile vacuole of Euplotes patella is 29/i at maximum dias- tole, but may become 45-50^ in diameter upon disturbance or after incision. The rate of pulsation is subject to changes with temperature, physiological state of the organism, amount of food substances present in the water, etc. For example, Rossbach ob- served in the three ciliates mentioned below that the pulsation of the contractile vacuole increased first rapidly and then more slowly with the rise of the temperature of the water: Time in seconds between two systoles at different temperature (C.) 5° 10° 15° 20° 25° 30° 61 48 31 28 22 23 18 14 10-11 6-8 5-6 4 9 7 5 4 4 — Euplotes char on Stylonychia pustulata Chilodonella cuculhdus Aside from the soluble forms, there often occur in the protozoan body insoluble catabolic products in the forms of crystals and granules of various kinds. Schewiakoff (1893) first noticed that Paramecium often contains crystals (Fig. 39) composed of calcium 100 PROTOZOOLOGY 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 con- sidered that these crystals were first dissolved and excreted by the contractile vacuoles, as they were seen collected around the vacuoles. In Amoeba proteus, Schubotz (1905) noted that the crys- tals were of similar chemical composition and of usually bipyram- idal or rhombic form, and that they measure about 2-5^ in length and doubly refractile. Schaeffer (1920) observed calcium phosphate crystals in three species of Amoeba and was inclined to think that the form and dimensions of these crystals were Fig. 39. Examples of crystals present in Protozoa, a-e, in Parame- cium caudatum (Schewiakoff), (a-d, XlOOO, e, X2600); f, in Amoeba proteus; g, in A. discoides; h, in A. dubia (Schaeffer). characteristic of each species. Thus in Amoeba proteus, they are truncate bipyramids, rarely flat plates, up to 4.5/i long; in A. dis- coides, abundant, truncate bipyramids, up to 2.5At long; and in A. dubia, variously shaped (4 kinds) few, but large, up to IOjjl, 12/x, 30m long (Fig. 39). Rowland detected uric acid in Paramecium caudatum and Amoeba verrucosa. Luce and Pohl (1935) noticed that at certain times amoebae 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 to transmitted light. These crystals are tubular and six-sided, and vary in length from 0.5 to 3.5)U. They consid- ered the crystals were composed of calcium chlorphosphate. 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 Ifj, in length and which are suspended in alkaline fluid to viscous vacuoles. These two authors believe that the plate-like PHYSIOLOGY 101 crystals are probably leucine, while the bipyramidal crystals con- sist of a magnesium salt of a substituted glycine. Other crystals are said to be composed of urate, carbonate, oxalate, etc. Another catabolic product is the melanin grains which occur in many haemosporidians and which appear to be composed of a derivative of the haemoglobin of the infected erythrocyte. In cer- tain Radiolaria, there occurs a brownish amorphous mass which is considered as catabolic waste material and, in Foraminifera, the cytoplasm is frequently loaded with masses of brown granules which appear also to be catabolic waste and are extruded from body periodically. While intracellular secretions are usually difficult to recognize, because the majority remain in fluid form except those which pro- duce endoskeletal structures occurring in Heliozoa, Radiolaria, certain parasitic ciliates, etc., the extracellular secretions are eas- ily recognizable as loricae, shells, envelopes, stalks, collars, mu- cous substance, pigments which give the body a characteristic coloration (p. 37), 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 The Protozoa move about by means of the pseudopodia, flagel- la, or cilia, which may be combined with internal contractile or- ganellae. Movement by pseudopodia. The amoeboid movements have long been studied by numerous observers. The first attempt to explain the movement was by Berthold (1886), who held that the difference in the surface tension was the cause of amoeboid move- ments, which view was supported by the observations and ex- periments of Btitschli (1894) and Rhumbler (1898). According to this view, when an amoeba forms a pseudopodium, there prob- ably occurs a diminution of the surface tension of the cytoplasm at that point, due to certain internal changes which are continu- ously going on within the body and possibly to external causes, and the internal pressure of the cytoplasm will then cause the stream- ing of the cytoplasm. This results in the formation of a pseudo- podium which becomes attached to the substratum and an in- crease in tension of the plasma-membrane draws up the posterior 102 PROTOZOOLOGY -fe Fig. 40. 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, fiuid ectoplasm; ge, gelated ectoplasm. end of the amoeba, thus bringing about the movement of the whole body. Jennings (1904) found that the movement of Amoeba verrucosa (Fig. 40, 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. Bellinger (1906) studied the movement of Amoeba proteus, A. verrucosa and Difflugia spiralis. Studying in side view, he found that the amoeba (Fig. 41) extends a pseudopod, "swings it about, brings it into the line of advance, and attached it" to the substratum and that there is then a concentration of the sub- stance back of this point and a flow of the substance toward the anterior end. Bellinger held thus that "the movements of amoe- bae are due to the presence of a contractile substance," which was said to be located in the endoplasm as a coarse reticulum. PHYSIOLOGY 103 > Fig. 41. Outline sketches of photomicrographs of Amoeba proteus dur- ing locomotion, as viewed from side (Bellinger). In the face of advancement of our knowledge on the nature of protoplasm, Rhumbler realized the difficulties of the surface ten- sion theory and later suggested that the conversion of the ecto- plasm to endoplasm and vice versa were the cause of the cyto- plasmic movements, which was much extended by Hyman (1917). Hyman considered that: 1) a gradient in susceptibility to potas- sium cyanide exists in each pseudopodium, being the greatest at the distal end, and the most recent pseudopodium, the most sus- ceptible; 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 colloidal state. Solation, which is brought about by the metabolic change, is regarded as the cause of the ex- tension of a pseudopodium, and gelation of the withdrawal of pseudopodia and of active contraction. Schaeffer (1920) mentions the importance of the surface layer which is a true surface tension film, the ectoplasm, and the streaming of endoplasm in the amoe- boid movement. Pantin (1923) studied a marine hmax-type amoeba (Fig. 40, h) and came to recognize acid secretion and absorption of water at the place where the pseudopodium was formed. This results in swelling of the cytoplasm and the pseudopodium is formed. Because of the acidity, the surface tension increases and to lower or reduce this, concentration of substances in the "wall" of the pseudopodium follows. This leads to the formation of a gelatinous ectoplasmic tube which, as the pseudopodium, ex- tends moves toward the posterior region where the acid condition is lost, gives up water and contracts, finally becoming trans- formed into endoplasm near the posterior end. The contraction of 104 PROTOZOOLOGY Fig. 42. Diagram of Amoeba proteus, showing the solation and gela- tion of the cytoplasm during amoeboid movement (Mast), c, crystal; cv, contractile vacuole; f, food vacuole; he, hyaline cap; n, nucleus; pg, plasmagel; pgs, plasmagel sheet; pi, plasmalemma; ps, plasmasol. PHYSIOLOGY 105 the ectoplasmic tube forces the endoplasmic streaming to the front. This observation is in agreement vnth 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 plas- magel at the posterior end" (Fig. 42). As to how these processes work, Mast states: "The gelation of the plasmasol at the anterior Fig. 43. 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. end extends ordinarily the plasmagel tube forward as rapidly as it is broken down at the posterior end by solation and the contrac- tion of the plasmagel tube at the posterior end drives the plas- masol forward. The plasmagel tube is sometimes open at the anterior end and the plasmasol extends forward and comes in con- tact with the plasmalemma at this end (Fig. 43, a), but at other times it is closed by a thin sheet of gel which prevents the plas- masol from reaching the anterior end (6). This gel sheet at times persists intact for considerable periods, being built up by gelation as rapidly as it is broken down by stretching, owing to the pres- sure 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 gelates 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 106 PROTOZOOLOGY 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 inter- mediate 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 plasmasol is immediately reversed, and a new hyaline cap is formed at the posterior end which is thus changed into a new an- terior end." Flagellar movement. The flagellar movement is only in a few instances observable as in Peranema, but in most cases it is very difficult to observe in hfe. Since there is difference in the number, location, size, and probably structure (p. 45) of fliagella occurring in Protozoa, it is supposed that there are varieties of flagellar movements. The first explanation was advanced by Biitschli, who observed that the flagellum undergoes a series of lateral move- ments and, in so doing, a pressure is exerted on the water at right angles to its surface. This pressure 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 move- ment. There can be little doubt that this condition is satisfied in flagellated organisms 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 direc- tion of the transmission is always the same. A stationary wave, as apparently contemplated by Biitschli, could not effect propul- sion since the forces acting on the water are equal and opposite during the two phases of the movement. 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 process has often not been observed. Verworn observed long ago that in Peranema trichoyhoruni the undulation of the distal por- PHYSIOLOGY 107 tion of flagellum is accomplished by a slow forward movement, while undulation along the entire length by a rapid forward move- ment. Recently Krijgsman (1925) studied Monas sp. (Fig. 44) which he found in soil cultures, under the darkfield microscope and stated: 1) when the organism moves forward with the maxi- mum speed, the flagellum starting from cl , with the wave begin- ning at the base, stretches back (c 1-6), and then waves back {d, e), which brings about the forward movement. Another type is Fig. 44. Diagrams illustrating flagellar niovemengs 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, undulator}' movement of the flagellum in backward movement; p, lateral movement; q, turning movement. 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 or- ganism moves forward with moderate speed, the tip of the flagel- lum 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 passes along it from its base to tip (p); and 5) when the organism under- goes a slight lateral movement, the distal end of the flagellum only undulates (q). 108 PROTOZOOLOGY Ciliary movement. The cilia are the locomotor organella pres- ent liermanently in the ciliates and vary in size and distribution among different species. Just as flagellates show various types of movements, so do the ciliates. Individual cilium on a progressing ciliate bends throughout its length and strike 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. 45, a-d). In the Protociliata and the majority of holotrich- ous and heterotrichous ciliates, the cilia are arranged in longi- ..i f /"\ :a 9 Fig. 45. 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 movements of cilia in a longitudinal row. tudinal, or oblique rows and it is clearly noticeable that the ciUa are not beating in the same phase, although they are moving at the same rate. A cilium (Fig. 45, 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 trans- verse row beat synchronously, the condition clearly being recog- nizable on Opalina among others, which is much like the waves passing over a wheat field on a windy day. The organized move- ments of cilia, cirri, membranellae and undulating membranes are probably controlled by the neuromotor system (p. 55) which PHYSIOLOGY 109 appears to be condiictile as judged by the results of micro-dissec- tion experiments of Taylor (p. 56). The Protozoa which possess myonemes are able to move by contraction of the body or of stalk, and others combine this with the secretion of mucous substance as was found in Haemogrega- rina and Gregarinida. Irritability Under natural conditions, the Protozoa do not behave always in the same manner, because several stimuli act upon them usu- ally in combination and predominating stimulus or stimuli vary under different circumstances. Many investigators have, up to the present time, studied the reactions of various Protozoa to external stimulations, full discussion of which is beyond the scope of the present work. Here one or two examples in connection with the reactions to each of the various stimuli will only be men- tioned. Of various responses expressed by a protozoan against a stimulus, movement is the most clearly recognizable one and, therefore, free-swimming forms, particularly ciliates, have been the favorite objects of study. We consider the reaction to a stimu- lus in protozoans as the movement response, and this appears in one of the two directions: namely, toward, or away from, the source of the stimulus. Here we speak of positive or negative re- action. In forms such as Amoeba, the external stimulation is first received by the body surface and then by the whole protoplasmic body. In flagellated or ciliated Protozoa, these processes act in part sensory, in fact in a number of ciliates are found non-vibra- tile ciha which appear to be sensory in function. In a compara- tively small number of forms, there are sensory organellae such as stigma (p. 79), ocellus (p. 80), statocysts (p. 77), concretion vacuoles (p. 77), 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 dif- ferently. 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 110 PROTOZOOLOGY of a doad algal filament at the middle of its anterior surface (Fig. 46, a), the amoeboid movements proceeded on both sides of the filament (b), but soon motion ceased on one side, while it contin- ued on the other, and the organism avoided the obstacle by re- versing a part of the current and flowing in another direction (c). When an amoeba is stimulated mechanically by the tip of a glass rod (d), it turns away from the side touched, by changing endo- plasmic streaming and forming new pseudopodia (e). Positive re- FiG. 46. Reactions of amoebae to mechanical stimuli (Jennings), a-c, an amoeba avoiding an obstacle; d, e, negative reaction to mechan- ical stimulation; f-h, positive reaction of a floating amoeba. actions are also often noted, when a suspended amoeba (/) comes in contact with a sohd surface with the tip of a pseudopodium, the latter adheres to it by spreading out (g). Streaming of the cytoplasm follows and it becomes a creeping form (h). Positive reactions toward solid bodies account of course for the ingestion of food particles. 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. 47, c) follows and in the latter case, the organism rests with its anterior PHYSIOLOGY 111 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 or- ganellae, etc. Amoebae, Testacea and others which are usually found attached to the bottom of the container, react as a rule positively toward gravity, while others manifest negative reac- FiG. 47. Reactions of Paramecium (Jennings), a, collecting in a drop of 0.02% acetic acid; b, ring-formation around a drop of a stronger solution of the acid; c, avoiding reaction. tion as in the case of Paramecium (Jensen; Jennings), which ex- plains in part why Paramecium in a culture jar are found just be- low the surface film en mass, although, according to Dembowski (1929) the vertical movement of Paramecium caudatum is influ- enced by various factors. Reaction to current. Free-swimming Protozoa appear to move or orientate themselves against the current of water. In the case of Paramecium, Jennings observed the majority place them- selves in line with the current, with anterior end upstream. The myoetozoan is said to exhibit also a well-marked positive reaction. 112 PROTOZOOLOGY Reaction to chemical stimuli. When methylgreen, methylene blue, or sodium chloride is brought in contact with an advancing amoeba, the latter organism reacts negatively (Jennings). Jen- nings further observed various reactions of Paramecium against chemical stimulation. This ciliate shows positive reaction to weak solutions of many acids and negative reactions above certain con- centrations. For example, Paramecium enters and stays within the area of a drop of 0.02 per cent acetic acid introduced to the preparation (Fig. 47, a); and if stronger acid is used, the organ- isms collect about its periphery where the acid is diluted by the surrounding water (Fig. 47, h). The reaction to chemical stimuli is probably of the greatest importance for the existence of Proto- zoa, 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 re- sults in their finding specific host animals and their distribution in different organs and tissues within the host body. Reaction to light stimuli. Most Protozoa seem to be indifferent to the ordinary fight, but when the fight intensity is suddenly in- creased, there is usually a negative reaction. Verworn saw the direction of movements of an amoeba reversed when its anterior end was given a sudden ilfiimination ; 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 illumination remains constant, the amoeba begins to move. Ac- cording to Jennings, Stentor coeruleus reacts negatively against fight. The positive reaction to light is most clearly shown in stigma- bearing Mastigophora, as is well demonstrated by a jar contain- ing Euglena, Phacus, etc., in which the organisms collect at the place where the strongest light reaches. If the light is excluded completely, the organisms become scattered throughout the con- tainer, inactive and sometimes encysted, although the mixotroph- ic forms would continue activities by saprozoic methods. The positive reaction to light by chromatophore-bearing forms en- ables 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. In- PHYSIOLOGY 113 man found that amoebae show a greater reaction to the rays and Hertel observed that Paramecium which were indifferent to an ordinary hght, showed an immediate response (negative reac- tion) to the rays. MacDougall brought about mutations in Chilo- donella by means of these rays (p. 164). When ciUates are vitally stained with eosin, erythrosin, etc., they react sometimes posi- tively or negatively, as in Paramecium (Metzner), or always neg- atively, as in Spirostomum (Blattner). According to Efimoff, this "induced phototaxis" is not limited to fluorescent dyes, but also is possessed by all vital-staining dyes. Zuelzer (1905) studied the effects of radium rays upon various Protozoa and found that the effect was not the same among different species. For example, limax amoeba was more resistant than others. In all cases, how- ever, long exposure to the rays was fatal to Protozoa, the first ef- fect of exposure being shown by accelerated movement. Halber- staedter and Luntz (1929) studied injuries and death of Eudorina elegans by exposure to radium rays. Joseph and Prowazek (1902) found Paramecium and Volvox gave negative response to the rontgen-ray. Reaction to temperature stimuli. As was stated before, there seems to be an optimum temperature range for each protozoan, 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 repro- duction. 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 environ- ment. In the case of parasitic Protozoa which pass their life-cycle in warm-blooded and cold-blooded host animals, such as Plasmo- dium and mammalian trypanosomes, the change in body tem- perature 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 reaction to the anode and moves toward the cathode either by reversing 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 114 PROTOZOOLOGY position (Spirostomiim) or swim to the anode (Paramecium, Stentor, etc.). Of flagellates, Verworn noticed that Trachelo- monas and Peridinium moved to the cathode, while Chilomonas Cryptomonas, and Polytomella, swam to the anode. Regeneration The power of regenerating the lost parts of the body is char- acteristic of all Protozoa from simple forms to those with highly complex organization, as shown by observations of numerous investigators. The general procedure of the experiment is to cut the body of a protozoan into two or more parts and observe how far each part regenerates. It is now well established that only the parts which contain the whole or part of the nucleus are able to regenerate completely under favorable circumstances. A re- markably small portion of a protozoan is known to regenerate completely. For example, Sokoloff found 1/53-1/69 of Spiro- stomum and 1/70-1/75 of Dileptus were able to regenerate. Ac- cording to Philps, portions down to 1/80 of an amoeba are able to regenerate. Burnside (1929) cut 27 specimens of Stentor coeruleus 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 dimensional differences resulted from the different amounts of nuclear material present in the cut specimens. Apparently regula- tory processes took place and in all cases normal size was restored, no matter what was the amount of the nuclear material in an- cestral pieces. Thus, in this ciliate, biotypes of diverse size are not produced by causing inequalities in the proportions of nuclear material in different individuals. The parts which do not contain nuclear material, may continue to show certain activities, such as locomotion, contraction of the contractile vacuole, etc., for some time. For example, Penard observed enucleated amoebae lived eight days, Stole and Gruber found amoebae without nuclear material were able to live up to 30 days, and enucleated pieces of A. verrucosa were seen to remain alive for 20 to 25 days (Grosse-Allermann). At the time of reproduction of all Protozoa, the various organel- lae, such as cilia, flagella, cytostome, contractile vacuole, etc., are completely regenerated before the separation of body occurs. PHYSIOLOGY 115 References Berthold, C. 1886 Studien iiber Protoplasmamechanik. Leipzig. Brug, S. L. 1928 Observations on a culture of Entamoeba histo- lytica. Med. Dienst Volksges. Ned. Indie. BuRNSiDE, L. H. 1929 Relation of body size to nuclear size in Stentor coeruleus. Jour. Exper. Zool., Vol. 54. Cleveland, L. R. 1925 Toxicity of oxygen for Protozoa in vivo and in vitro. Animals defaunated without injury. Biol. Bull., Vol. 48. , S. R. Hall, E. P. Sanders and J. Collier 1934 The wood-feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts and Sci., Vol. 17. Dawson, J. A. and M. Belkin 1928 The digestion of oil by Amoeba dubia. Proc. Soc. Exp. Biol, and Med., Vol. 25. Bellinger, O. P. 1906 Locomotion of amoebae and allied forms. Jour. Exp. Zool., Vol. 3. Dembowski, J. 1929 Die Vertikalbewegungen von Paramecium caudatum. IL Arch. f. Protistenk., Vol. 68. DoFLEiN, F. 1918 Ueber Polytomella agilis Aragao. Zool. Jahrb. Abt. Anat., Vol. 41. Frisch, J. A. 1937 The rate of pulsation and the function of the contractile vacuole in Paramecium multimicronucleatum. Arch. f. Protistenk., Vol. 90. Gray, J. 1928 Ciliary movement. Cambridge. Greenwood, M. 1894 Constitution and formation of "food- vacuoles" in Infusoria. Phil. Trans. (B). Vol. 185, and E. R. Saunders 1884 The role of acid in protozoan digestion. Jour. Physiol., Vol. 16. Heidt, K. 1937 Form und Struktur der Paramylonkorner von Euglena sanguinea. Arch. f. Protistenk., Vol. 88. Herfs, a. 1922 Die pulsierende Vakuole der Protozoen, ein Schutzorgan gegen Ausslissung. Ibid., Vol. 44. Rowland, R. B. 1928 The pH of gastric vacuoles. Protoplasma, Vol. 5. and A. Bernstein 1931 A method for determining the oxygen consumption of a single cell. Jour. Gen. Physiol., Vol. 14. HuLPiEu, H. R. 1930 The effect of oxygen on Amoeba proteus. Jour. Exp. Zool., Vol. 56, Hyman, L. H. 1917 Metabolic gradients in amoeba and their relation to the mechanism of amoeboid movement. Jour. Exp. Zool., Vol. 24. Jennings, H. S. 1904 Contributions to the study of the be- havior of the lower organisms. Publ. Carnegie Inst. Washing- ton, No. 16. 1906 Behavior of the lower organisms. New York. Khainsky, a. 1910 Zur Morphologie und Physiologic einiger 116 PROTOZOOLOGY Infusorien {Paramecium caudatum) auf Grund einer neuen histologischen Methode, Arch. f. Protistenk., Vol. 21. KiRBY, H. Jr., 1934 Some ciliates from salt marshes in Cali- fornia. Ibid., Vol. 82. KoFOiD, C. A. and 0. Swezy 1921 The free-living unarmored Dinofiagellata. Mem. Univ. Calif., Vol. 5. KoRSCHELT, E. 1927 Regeneration und Transplantation. Vol. 1. Berlin. Krijgsman, B. J. 1925 Beitrage zum Problem der Geisselbe- wegung. Arch. f. Protistenk., Vol. 52. Kudo, R. R. 1921 On the nature of structures characteristic of cnidosporidan spores. Trans. Amer. Micr. Soc, Vol. 40. Mast, S. O. 1923 Mechanics of locomotion in amoeba. Proc. Nat. Acad. Sci. Vol. 9. 1926 Structure, movement, locomotion, and stimulation in amoeba. Jour. Morph. Physiol., Vol. 41. 1931 Locomotion in Amoeba proteus. Protoplasma, Vol. 14. and W. L. Doyle 1934 Ingestion of fluid by amoeba. Ibid., Vol. 20. 1935 Structure, origin and function of cytoplas- mic constituents in Amoeba proteus with special reference to mitochondria and Golgi substance. Arch. f. Protistenk., Vol. 86. Meldrum, N. U. 1934 Cellular respiration. London. Metalnikoff, S. 1912 Contribution a I'etude de la digestion intracellulaire chez les protozoaires. Arch. zool. exp. (ser. 5), Vol. 9. Mouton, H. 1902 Recherches sur la digestion chez les amibes et sur leur diastase intracellulaire. Ann. Inst. Pasteur. Vol. 16. Nirenstein, E. 1925 Ueber die Natur und Starke der Saure- bildung in den Nahrungsvakuolen von Paramecium cauda- tum. Zeitschr. wiss. Zool., Vol. 125. NoLAND, L. E. 1927 Conjugation in the ciliate Metopus sig- moides. Jour. Morph. Physiol., Vol. 44. Pantin, C. F. a. 1923 On the physiology of amoeboid move- ment I. Marine Biol. Ass. Plymouth, N. S., Vol. 13. Panzer, T. 1913 Beitrag zur Biochemie der Protozoen. Hoppe- Seylers Zeitschr. phys. Chemie, Vol. 86. Powers, P. B. A. 1932 Cyclotrichium meunieri sp. nov.; cause of red water in the gulf of Maine. Biol. Bull., Vol, 63. Pratje, a. 1921 Makrochemische, quantitative Bestimmung des Fettes und Cholesterins, sowie ihrer Kennzahlen bei Noctiluca miliaris. Biol. Zentralbl., Vol. 21. Pringsheim, E. G. 1923 Zur Physiologie saprophytischer Flagellaten. Beitr. allg. Bot., Vol. 2. Putter, A. 1905 Die Atmung der Protozoen. Zeitschr. allg. Physiol., Vol. 5. PHYSIOLOGY 117 1908 Methoden zur Erforschung des Lebens der Pro- tistenk. Tigerstedt's Handb. physiol. Methodik, Vol. 1. RosKiN, G. and L, Levinsohn 1926 Die Oxydasen und Per- oxydasen bei Protozoen. Arch. f. Protistenk., Vol. 56. Rhumbler, L. 1910 Die verschiedenartigen Nahrungsaiifnah- men bei Amoeben als Folge verschiedener Colloidalzustande ihrer Oberflachen. Arch. Entw. Organism., Vol. 30. Sassuchin, D. N, 1935 Zum Studium der Protisten- und Bak- terien-kerne. Arch. f. Protistenk., Vol. 84. ScHAEFFER, A. A. 1920 Amoehoid movement. Princeton. ScHEWiAKOFF, W. 1894 Ueber die Natur der sogenannten Ex- kretkorner der Infusorien. Zeitschr, wiss. Zool., Vol. 57. Shapiro, N. N. 1927 The cycle of hydrogen-ion concentration in the food vacuoles of Paramecium, Vorticella, and Stylony- chia. Trans. Amer. Micro. Soc, Vol. 46. SoKOLOFF, B. 1924. Das Regenerationsproblem bei Protozoen. Arch. f. Protistenk., Vol. 47. SouLE, M. H. 1925 Respiration of Ti^ypanosoma lewisi and Leishmania tropica. Jour. Inf. Dis., Vol. 36. Stolc, a. 1900 Beobachtungen und Versuche ueber die Ver- dauung und Bildung der Kohlenhydrate bei einen amoeben- artigen Organismen, Pelomyxa palustris. Zeitschr. wiss. Zool. Vol. 68. Verworn, M. 1889 Psycho-physiologische Protisten-studien. Jena. 1903 Allgemeine Physiologie. 4te Aufi. Jena. Wetherby, J. H. 1929 Excretion of nitrogenous substances in Protozoa. Physiol. Zool., Vol. 2, Whipple, G. C. 1927 The microscopy of drinking water. 4 ed. New York, Zuelzer, M. 1907 Ueber den Einfluss des Meerwassers auf die pulsierende Vacuole. Berlin. Sitz.-Ber. Ges. naturf. Freunde. ZuMSTEiN, H. 1899 Zur Morphologie und Physiologie der Eu- glena gracilis Klebs. Pringsheims Jahrb. wiss. Botanik Vol. 34. Chapter 5 Reproduction THE mode of reproduction in Protozoa is highly variable among different groups, although it is primarily a cell divi- sion. The reproduction is initiated by the nuclear division in all cases, which will therefore be considered first. Nuclear division Between a simple direct division on the one hand and a com- plicated indirect division which is comparable with the typical metazoan mitosis on the other hand, all types of nuclear division are to be encountered. Direct nuclear division. While not so widely found as it was thought to be in former years, amitosis occurs normally and regu- larly in many forms. The macronuclear division of the Ciliophora is without exception direct. The macronucleus elongates itself without any particular changes in its internal structure and be- comes divided through the middle, resulting in formation of two daughter nuclei as seen commonly in Paramecium (Fig. 48). It is assumed that the nuclear components undergo solation during division, since the formed particles of nucleus which are stationary in the resting stage, manifest a very active Brownian movement as was observed m vivo in Endamoeha hlattac (Fig. 49). Furthermore, in some cases the nuclear components may undergo phase reversal, that is to say, the chromatin granules which are dispersed phase in the non-staining fiuid dispersion medium in the resting nucleus, become dispersion medium in which the latter is suspended as dispersed phase. By using Feulgen's nucleal reaction, Reichenow (1928) demonstrated this reversal phenome- non in the division of the macronucleus of Chilodonella cucullulus (Fig. 50). When the macronucleus is elongated as in Spirostomum, Stentor, Euplotes, etc., the nucleus becomes condensed into a rounded form prior to its division. When the macronuclear ma- terial is distributed throughout the cytoplasm as numerous grains as in Dileptus anser (Fig. 239, c), "each granule divides where it happens to be and with the majority of granules both halves 118 REPRODUCTION 119 remain in one daughter cell after division" (Calkins). Hayes noticed a similar division, but at the time of simultaneous divi- sion prior to cell division, each macronucleus become elongated and breaks into several small nuclei. Fig. 48. Nuclear and cj'tosomic division of Paramecium caudatum as seen in stained smears, X260 (Kudo). The macronucleus becomes at the time of its division somewhat enlarged and its chromatin granules are more deeply stained than before. Since the number of chromatin granules appear ap- proximately the same in the macronuclei of different generations 120 PROTOZOOLOGY of a given species, the reduced number of chromatin granules 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 chromo- FiG. 49. Division of Endamoeha blaltae as seen in life, X250 (Kudo). The entire process took one hour and seven minutes. meres." Reichenow (1928) found that in Chilodonella cucullulus the lightly Feulgen positive endosome appeared to form chroma- tin granules and Kudo (1936) maintained that the large chroma- tin spherules of the macronucleus of Nyctotherus ovalis probably produce smaller spherules in their alveoli. In the elongated or miniliform macronuclei of a number of ciliates, there occur, prior to and during division, 1-2 character- istic zones which have been called by various names, such as nuclear clefts, reconstruction bands, reorganization bands, etc. In Euplotes patella, Turner (1930) observed before division, a reorganization band consisting of an unstained zone ("reconstruc- REPRODUCTION 121 Fig. 50. The solation of chromatin during the macronuclear division of Chilodonella cuculhilus, positive to Feulgen's nucleal reaction, X1800 (Reichenow). tion plane") and a stained zone (''solution plane"), appears at each end of the macronucleus (Fig. 51, a) and as each moves toward the middle, a more chromatinic area is left behind (h-d). According to Summers (1935), a similar change occurs in Dio- FiG. 51. Macronuclear reorganization before division in Euplotes patella, X240 (Turner), a, reorganization band appearing at a tip of the macronucleus; b-d, later stages. 122 PROTOZOOLOGY phrys appendtculata and Styloyiychia pustulata; but in Aspidisca lynceus (P'ig. 52) a reorganization band ai)peared first near the middle region of the macronucleus (b), divided into two and each moved toward an end, leaving between them a greater chroma- tinic contents of the reticula (c-i). Summers suggested that "the Fig. 52. 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. reorganization bands are local regions of karyolysis and re- synthesis of macronuclear materials with the possibility of an elimination of physically or possibly chemically modified non- staining substances into the cytoplasm." The discarding of a certain portion of the macronuclear material during division has been observed in a number of species. REPRODUCTION 123 In Uroleptus 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 di\'ides into two nuclei. In the more or less rounded macronucleus which is commonly found in many ciHates, no reorganization band has been recognized. A number of observers have however noted during the nuclear division there appears and persists a small body within the nuclear figures, ■mMim^mmf^m Fig. 53. Macronuclear division in Conchophthirus mytili, X440 (Kidder). located at the division plane as in the case of Loxocephalus (Behrend), Eupoterion (MacLennan and Connell) and even in the widely different protozoan, Endamoeba hlattae (Kudo) (Fig. 49). We owe Kidder for a careful comparative study of this body. Kidder (1933) observed that during the division of the macro- nucleus of Conchophthirus mytili (Fig. 53), the nucleus ''casts out a part of its chromatin at every vegetative division," which "is broken down and disappears in the cytoplasm of either daughter organism." A similar phenomenon has since been found 124 PROTOZOOLOGY further in C. anodontae, C. curtus, C. magna (Kidder), Uro- centrum turbo, Colpidium colpoda, C. campylum, Glaucoma scintillans (Kidder and Diller), and Allosphaerium convexa (Kidder and Summers). Kidder and his associates beheve that the process is probably eHmination of waste substances of the prolonged cell-division, since chromatin extrusion does not take place during a few divisions subsequent to reorganization after conjugation in Conchophthirus mytili and since in Colpidium and Glaucoma, the chromatin elimination appears to be the cause of high division rate and infrequency of conjugation. Other examples of amitosis are found in the vesicular nuclei in the trophozoite of Myxosporidia, as for example, Myxosoma catostomi (Fig. 54), Thelohanellus notatus (Debaisieux), etc., in which the endosome divides first, followed by the nuclear con- striction. In Streblomastix strix, the compact elongated nucleus was found to undergo a simple division by Kofoid and Swezy. tf^^-j/^^ W^'^W^ f^^^^^H ffr'M^'^0 "A^S^tW a b c d e Fig. 54. Amitosis in the trophozoite of Myxosoma catostomi, X2250 (Kudo). 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 varia- tions, the change is of a uniform pattern. Chatton, Alexeieff and others, have proposed several terms to designate the various types of indirect nuclear division, but no one of these types is sharply defined. For our purpose, mentioning of examples will suffice. A veritable mitosis was noted by Dobell in the heliozoan Oxnerella maritima (Fig. 55), which possesses an eccentrically situated nucleus containing a large endosome and a central centroplast or centriole, from which radiate many axopodia (a). The first sign of the nuclear division is the shght enlargement, and migration toward the centriole, of the nucleus. The centriole first divides into two (c, d) and the nucleus becomes located be- tween the two centrioles (e). Presently spindle fibers are formed REPRODUCTION 125 and the nuclear membrane disappears (/, g). After passing through an equatorial-plate stage, the two groups of the chromo- somes move toward the opposite poles {g-i). As the spindle fibers become indistinct, radiation around the centrioles becomes con- spicuous and the two daughter nuclei are completely recon- •■#i«^fe Fig. 55. Nuclear and cytosomic division in Oxnerella maritima, X about 1000 (Dobell). a, a living individual; b, stained specimen; c-g, prophase; h, metaphase; i, anaphase; j, k, telophase; 1, division completed. structed to assume the resting phase (j-l). The mitosis of another heliozoan Acanthocystis aculeata is, according to Schaudinn and Stern, very similar to the above. Aside from these two species, 126 PROTOZOOLOGY the centriolc has been r('])()rte(l in many others, such as Hart- mannella (Arndt), Euglypha, Monocystis (Belaf), Aggregata (Dobell, Belaf, and Naville), various Hyi)ermastigina (Kofoid; Duboscq, Grasse; Kirby; Cleveland and his associates). In numerous species the division of the centriole (or blepharo- ])last) and a connecting strand between them, which have been called desmose, centrodesmose or paradesmose, have been ob- served. According to Kofoid and Swezy (1919), in Trichonympha campanula (Fig. 56), 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, be- tw^een which develops a desmose. From the posterior end of each centriole, astral rays extend out and the split chromosomes form loops, pass through "tangled skein" stage, and emerge as 26 chromosomes. In the metaphase, the equatorial plate is made up of V-shaped chromosomes as each of the split chromosomes are still connected at one end, which finally becomes separate in anaphase, followed by formation of two daughter nuclei. As to the origin and development of the achromatic figure, various observations and interpretations have been advanced. Certain Hypermastigina possess very large filiform centrioles and a large rounded nucleus. In Barbulanympha (Fig. 57), Cleveland (1938) found that the centrioles vary from 15 to 30/x in length in the four species of the genus which he studied. They can be seen, according to Cleveland, in life as made up of a dense hyahne 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^ 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 overlap to form the central spindle (d). In the resting nucleus, there are large irregular chromatin granules which are connected by fibrils with one an- other and also with the nuclear membrane. As the achromatic 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 REPRODUCTION 127 Fig. 56. Mitosis in Trichonyinpha campanula, XSOO (Kofoid and Swezy). a, resting nucleus; b-g, prophase; h, metaphase; i, j, anaphase; k, telophase; 1, a daughter nucleus being reconstructed. central spindle begins to depress the nuclear membrane and the chromosomes become shorter and move apart. The intra- and 128 PROTOZOOLOGY Fig. 57. Development of spindle and astray rays during the mitosis in Barbulanympha, x930 (Cleveland), a, interphase centrioles and centrosomes; b, prophase centrioles with astral rays developing from their distal ends through the centrosomes; c, meeting of astral rays from two centrioles; d, astral rays developing into the early central spindle; e, a later stage showing the entire nuclear figure. extranuclear 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 becomes branched, thus assuming conditions seen in the resting nucleus. REPRODUCTION 129 In the unique resting nucleus of Spirotrichonympha polygyra (Fig. 58), Cleveland (1938) found four chromosomes, each of which contains a distinct coil within a sheath and its one end con- nected with the anterior margin of the nuclear membrane by an Fig. 58. Mitosis in Spirotrichonympha polygyra (Cleveland), a, resting nucleus with 4 chromosomes; b, c, prophase; d, chromosomes mo\ang apart; e, elongation of nucleus; f, telophase; g, a daughter nucleus in which the chromosomes are splitting, a-e, X3800; f, g, X2400. intranuclear chromosomal fiber, and the other with a deeply staining endosome (a). The spindle fibers appear between the separating flagellar bands which come in contact with the nuclear membrane. Soon some of the astral rays become connected mth the intranuclear chromosomal fibers and one long and one short 130 'RUTOZOOLOGY chromosomes which become thicker and shorter move toward each pole. During the telophase, each chromosome spHts length- wise and forms the resting nucleus (g). In Lophotnonas blattarum, the nuclear division (Fig. 59) 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; Fig. 59. Nuclear division in Lophomonas blattarum, X1530 (Kudo), a, resting nucleus; b, c, prophase; d, metaphase; e-h, anaphase; i-k. telophase. the centriole divides and the desmose which grows, now stains very deeply, the centrioles becoming more conspicuous in the anaphase when new flagella develop from them. Chromatin granules become larger and form a spireme, from which 6-8 chromosomes are produced. Two groups of chromosomes move toward the opposite poles, and when the division is completed, each centriole becomes the center of formation of all motor organellae. REPRODUCTION 131 In some forms, such as Noctiluca (Calkins), Actinophrys (Belaf), etc., there may appear at each pole, a structureless mass of cytoplasm (centrosphere), but in a very large number of species there appear no special structures at poles and the spindle fibers become stretched seemingly between the two extremities of the elongating nuclear membrane. Such is the condition found in 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 schuhergi (Schaudinn), Euglena viridis (Tschenzoff), Oxyrrhis marina (Hall), Colacium vesiculosum (Johnson), Haplosporidium limnodrili (Granata), etc., the conspicuously staining endosome divides by elongation and constriction along with other chromat- ic elements, but in many other cases, it disappears during the early part of division and reappears when the daughter nuclei are reconstructed as observed in Monocystis, Dimorpha, Eu- glypha, Pamphagus (Belaf), Acanthocystis (Stern), Chilomonas (Doflein), Dinenympha (Kirby), etc. In the vegetative division of the micronucleus of Concho- phthirus anodontae (Fig. 60), Kidder (1934) found that prior to division the micronucleus moves out of the pocket in the macro- nucleus 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 seg- ments, 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 chromo- somes move to opposite poles and transform themselves into two compact daughter nuclei. In Zelleriella intermedia (Fig. 61), Chen (1936) saw the formation of 24 chromosomes, each of which is connected with a fiber of the intranuclear spindle and splits lengthwise in the metaphase. While in the majority of protozoan mitosis, the chromosomes split longitudinally, there are observa- tions which suggest a transverse division. 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, as, for example, in 132 PROTOZOOLOGY Peranema trichophorum in which the chromosomes were observed to divide transversely (Hartmann and Chagas) as well as longi- tudinally (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 SP^ 1^ T2 Fig. 60. Mitosis of the micronucleus of Concho'phthirus anodontae, X2640 (Kidder), a-c, prophase; d, e, metaphase; f, g, anaphase; h, i, telophase. by assuming, as Hail (1937) showed in Euglena gracilis, that the splitting is not completed at once and the pulling force acting upon them soon after division brings forth the long chromosomes still connected at one end. Thus the chromosomes remain 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 REPRODUCTION 133 similar in structure and behavior to typical metazoan chromo- somes. In many other cases, the so-called chromosomes or "pseudochromosomes" are slightly enlarged chromatin granules which differ from the ordinary chromatin granules in their time of appearance and movement only. In these cases it is of course Fig. 61. Stages in mitosis in Zelleriella intermedia, X1840 (Chen), a, early prophase; b, metaphase; c, anaphase; d, telophase. not possible at present to determine how and when their division occurs before separating to the respective division pole. In the following table are listed the number of the "chromosomes" which have been reported by various investigators in the Proto- zoa that are mentioned in the present work : Protozoa Number of chromosomes Observers Rhizochrysis scherffeli 22 Doflein Haematococnis pluvialis 20-30 Elhott Polytomella agilis 5 Doflein Chlamydomonas spp. 10 (haploid) Pascher Euglena -piscijormis 12-15(?) Dangeard E. viridis 30 or more Dangeard Phacus pyrum 30-40 Dangeard Menoidium incurvum About 12 Hall Vacuolaria virescens About 30 Fott Syndiniuvi turbo 5 Chatton Anthophytha vegetans 8-10 Dangeard Cercomonas longicauda 4-5 Dangeard Collodidyon triciliatum About 20 Belaf 134 PROTOZOOLOGY Protozoa Number of chromosomes Observers Chilomastix gallinarum About 12 Boeck and Tanabe Eutricho7nastix ser'penlis 5 Kofoid and Swezy Dinenympha fimbricata 25-30 Kirby Metadevescovina debilis About 4 Light Trichomonas elongatum 3 Hinshaw T. batrachorum 4 or 8 Kuczynski 6 Bishop T. augusta 6 Bishop Hexatnita salmonis 5 or 6 Davis Giardia mlestinalis 4 Kofoid and Swezy G. muris 4 Kofoid and Christi- ansen Calonympha grassii 4 or 5 Janicki Spirotrichonympha polygyra 2 doubles Cup 2 Cleveland Lophomonas blattarum 16 or 8 doubles Janicki 8 or 6 Kudo 12 or 6 doubles Belaf L. striata 12 or 6 doubles Belaf Barbulanymph a laurabxida 40 Cleveland B. uf alula 50 Cleveland Rhynchonympha tarda 19 Cleveland Urinympha talea 14 Cleveland Staurojoenia assimilis 24 Kirby Trichonympha campamda 52 or 26 doubles Kofoid and Swezy T. grandis 22 Cleveland Dim astigaynoeba bistadialis 16-IS Kiihn Endamoeba disparata About 12 Kirby Entamoeba histolytica 6 Kofoid and Swezy; Uribe E. coli 6 Swezy; Stabler Hydramoeba hydroxena 8 Revnolds and Threl- keld Actinophrys sol 44 (diploid) ; 22 (haploid) Belaf Oxnerella maritima About 24 Dobell Thalassicolla nucleata 4 Belaf A ulacantha scolymantha More than 1600 Borgert 4 in gamogony Belaf Zygosoma globosimi 12 (diploid); 6 (haploid) Noble Diplocystis schneideri 6 (diploid) ; 3 (haploid) Jameson REPRODUCTION 135 Protozoa Number of chromosomes Observers Nina gracilis 5 (haploid) Leger and Duboscq Aggregata eberthi 12 (diploid) ; Dobell and Jameson; 6 (haploid) Belaf; Naville Adelea ovata 8-10 (diploid); 4-5 (haploid) Greiner Orcheobius hcrpobdeUae 10-12 Kunze Chloromijxwm leydigi 4 (diploid); 2 (haploid) Naville Myxidium lieberkuhni 4 Bremer Sphaeromyxa sabrazesi 6 Debaisieux; Belaf 4 Naville S. bolbianii 4 Naville Myxobolus pfeijferi 4 Keysselitz; Mercier; Georgevitch Protoopalina intestinalis 8 (diploid) ; 4 (haploid) Metcalf Zelleriella antilliensis 2(?) Metcalf Z. intermedia 24 Chen Didinium nasidum 16 (diploid); 8 (haploid) Prandtl Chilodonella uncinata 4 (diploid); 2 (haploid) Enrique; McDougall C. uncinata (tetraploid) 8;4 McDougall ConchopJithirus anodontae 12 (diploid) Kidder C. mytili 16 (diploid); 8 (haploid) Kidder Ancistruma isseli About 5 (haploid) Kidder Paramecium aurelia 30-40 Diller Slentor coeruleus 28 (diploid) ; 14 (haploid) Mulsow Oxytricha fallax 24 (diploid); 12 (haploid) Gregory Uroleptus halseyi 24 (diploid); 12 (haploid) Calkins Pleurotricha lanceolata About 40 (dipl.); 20 (hapl.) Man well Stylonychia pustidata 6 Prowazek Euplotes patella 6 (diploid) Yocom; Ivanic 8 (diploid) ; Turner 4 (haploid) Carchesium polypinum 16 (diploid); 8 (haploid) Popoff Trichodina sp. 4-6 Diller 136 PROTOZOOLOGY In many other Protozoa, the division figure, especially the achromatic figure, suggests strongly a mitosis, but the chromatin substance which makes up the equatorial plate can hardly be called chromosomes. A typical example of this type is found in the nuclear division of Amoeba proteus (Fig, 62). According to Chalkley and Daniel (1933), the conspicuous granules present in the resting nucleus, under the membrane contain very little chromatin, while abundant chromatin is lodged in the central Fig. 62. Nuclear division in Amoeba proteus, X180 (Chalkley and Daniel), a, resting stage; b-d, prophase; e, metaphase; f, g, anaphase; h, a daughter nucleus. area. The peripheral granules appear to give rise to achromatic figure. At the beginning of division, the chromatin granules become aggregated in a zone (6); they then assume a ring-form along the periphery of the central mass of network (c); at this stage, the cytoplasm around the nucleus is much vacuolated. A little later appears a discoid equatorial plate or ring which is connected with the nuclear membrane by numerous fibrils, and the nucleus becomes markedly flattened with its membrane still intact (d), which is considered as the end of the prophase. In the metaphase, the nuclear membrane becomes extremely faint and the portion over one side of the plate is without it (e). At the REPRODUCTION 137 anaphase the membrane completely disappears, the equatorial plate sphts and each half contracts in the plane of the plate, pro- ducing two daughter-plates. In some specimens a faint spindle formation was noted. At about this time, vacuolated condition of the perinuclear cytoplasm disappears (/). In later phases of anaphase the plates are more widely separated and are slightly less in diameter as compared with earlier stages. There are dis- tinct polar caps of fibrillar material at the poles of the spindle (g), finally each plate transforms itself into a resting nucleus (h). The two investigators added that if the chromatin granules located in the equatorial plate are chromosomes, ''they must be extremely numerous." Liesche (1938) recently estimates the number of these granules which he called chromosomes as between 500 and 600. C5rtosomic division Binary fission. As in metazoan cells, the binary fission occurs very widely among the Protozoa. It is a division of the body through middle of the extended long axis into two nearly equal daughter individuals (Fig. 49). In Amoeba proteus, Chalkley and Daniel found that there is a definite correlation between the stages of nuclear division and external morphological changes (Fig. 63). During the prophase, the organism is rounded, studded with fine pseudopodia and exhibits under reflected light a clearly defined hyaline area at 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, 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 homo- geneous test also divides into two parts. In the majority of the Mastigophora, the division is longitudinal, as is shown by that of Menoidium incurvum (Fig. 64). In certain dinoflagellates, such as Cefatium, Cochliodinium, etc., the division plane is oblique, while in forms such as Oxyrrhis (Dunkerly; Hall), the fission is transverse. In Strehlomastix strix (Kofoid and Swezy), Lopho- monas striata (Kudo), Spirotrichonympha hispira (Cleveland), etc., the division takes place transversely but the polarity of the 138 PROTOZOOT-OGY 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), ob- served reversal of polarity in one of the daughter organisms at the time of division of normal individuals and also in those which regenerated after being cut into one-half the normal size. In the Ciliophora the division is as a rule transverse (Fig. 48), ^ ''4 Fig. 63. External morphological changes during division of Amoeba protetis, 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 al- most completed. in which the cytostome without any enlargement or elongation divides by constriction through the middle so that the two daughter individuals are about half as large at the end of division. Both individuals retain their polarity except in a few cases. Multiple division. In multiple division the body divides into a number of daughter individuals, with or without residua^ cyto- plasmic masses of the parent body. In this process the nucleus may undergo either simultaneous multiple division, as in Ag- gregata, or more commonly, repeated binary fission, as in Plasmo- dium (Fig. 198) to produce large numbers of nuclei, each of which REPRODUCTION 139 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 Foraminifera (Fig. 157), the Radiolaria (Fig. 167), a few Mastigophora such as Trypanosoma lewisi (Fig. 112), T. cruzi, and many Hypermastigina. It is very common among various groups of Sporozoa in which the trophozoite multiplies abundantly by this method. Fig. 64. Nuclear and cytosomic division in Menoidium incurvum, X about 1400 (Hall), a, resting stage; b, c, prophase; d, equatorial plate; e, f, anaphase; g, telophase. Budding. Multiplication by budding which occurs in the Proto- zoa is the formation of one or more smaller individuals from the parent organism. It is either exogenous or endogenous, depend- ing upon the location of the developing buds or gemmules. Exogenous budding has been reported in Acanthocystis, Nocti- luca (Fig. 101), Myxosporidia (Fig. 65, h), astomous ciliates (Fig. 228), Chonotricha, Suctoria (Fig. 289, k), etc. Endogenous budding has been found in Testacea, Gregarinida, Myxosporidia (Figs. 212, e; 214, j), and other Sporozoa as well as Suctoria (Fig. 289, h). Collin observed a unique budding in Tokophrya 140 PROTOZOOLOGY cyclopum in which the entire body, excepting the stalk and pelHcle, transforms itself into a young ciliated bud which leaves sooner or later the parent pellicle as a swarmer. Plasmotomy. Occasionally the multinucleate body of a proto- zoan divides into two or more small, multinucleate individuals, .•:-if.<-Joi-;.(>i V-.f(t«i'?»-».-. pa Mm 'l^ Fig. 65. a, b, budding in Myxidium lieberkuhni; c, d, plasmotomy in Chloromyxum leydigi; e, plasmotomy in Sphaeromyxa halbianii. the cytosomic division taking place independently of nuclear division. This has been called plasmotomy by Doflein. It has been observed in the trophozoites of several coelozoic myxo- sporidians, such as Chloromyxum leydigi (Fig. 65), Sphaeromyxa halbianii (Fig. 65), etc. It occurs further in Mycetozoa (Fig. 135), Foraminifera and ProtociHata. Colony formation When the division is repeated without a complete separation of the daughter individuals, a colonial form is produced. The REPRODUCTION 141 component indi\'idiials of a colony may either have protoplasmic connections among them or be grouped within a gelatinous enve- lope if completely separated. Or, in the case of loricate or stalked forms, these exoskeletal structures may become attached to one another. Although varied in appearance, the arrangement and relationship of the component individuals are constant, and this makes the basis for distinguishing the types of protozoan colonies, as follows: Catenoid or linear colony. The daughter individuals are at- tached endwise, forming a chain of several individuals. It is of comparatively rare occurrence. Examples : Astomous ciliates such as Radiophrya (Fig. 228), Protoradiophrya (Fig. 228) and dino- fiagellates such as Ceratium, Haplozoon (Fig. 103) and Poly- krikos(Fig. 104). 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. 87), Hyalobryon (Fig. 87), etc. (connection by lorica); Colacium (Fig. 96), many Peritricha (Figs. 280; 282), etc. (by stalk); Poteriodendron (Fig. 109), Stylobryon (Fig. 119), etc. (by lorica and stalk); Hydrurus (Fig. 88), Spongomonas (Fig. 118), Cladomonas (Fig. 118) and Anthophysa (Fig. 119) (by gelatinous secretions). Discoid colony. A small number of individuals are arranged in a single plane and grouped together by a gelatinous substance. Examples: Cyclonexis (Fig. 87), Gonium (Fig. 93), Platydorina (Fig. 94), Protospongia (Fig. 108), Bicosoeca (Fig. 109), etc. Spheroid colony. The individuals are grouped in a spherical form. Usually enveloped by a distinct gelatinous mass, the com- ponent individuals may possess protoplasmic connections among them. Examples: Uroglena (Fig. 87, c), Uroglenopsis (Fig. 87, d), Volvox (Fig. 93), Pandorina (Fig. 94, /), Eudorina (Fig. 94, h), etc. Such forms as Stephanoon (Fig. 94, a) appear to be inter- mediate between this and the discoid type. The component cells of some spheroid colonies show a distinct differentiation into somatic and reproductive individuals, the latter apparently de- veloping from certain somatic cells during the course of develop- ment. The gregaloid colony, which is sometimes spoken of, is a loose 142 PROTOZOOLOGY Fig. 66. Encystment of Lophomonas hlattarum, X1150 (Kudo). group of individuals of one species, usually of Sarcodina, which become attached to one another by means of pseudopodia in an irregular form. Asexual reproduction The Protozoa nourish themselves by certain methods, grow and multiply by the methods described in the preceding pages. This phase of the life-cycle of a protozoan is the vegetative stage or the trophozoite. The trophozoite repeats its asexual reproduc- tion process under favorable circumstances. Generally speaking, the Sporozoa increase to a much greater number by schizogony and the trophozoites are called schizonts. Under certain conditions, the trophozoite undergoes encyst- ment (Fig. 66). Prior to encystment, the trophozoites cease to ingest, and extrude remains of, food particles, resulting in some- what smaller forms which are usually rounded and inactive. This is often called the precystic stage. The organism presently secretes substances which become solidified into the cyst wall and thus the cyst is formed. In this condition, the protozoan ap- parently is able to maintain its vitality for a certain length of time under unfavorable conditions. The causes of encystment are still the matter which many investigators are attempting to com- prehend. It appears certain at least in some cases that the encyst- ment is brought about by changes in temperature, desiccation, and chemical composition, amount of food material, accumulation of catabolic wastes, etc., in the medium in which the organisms REPRODUCTION 143 Fig. 67. Encystment of Exiglypha acanthophora, X320 (Klihn). live. In some cases, the organisms encyst temporarily in order to undergo nuclear reorganization and multiplication. Because of the latter condition and also of the failure in attempting to cause certain Protozoa to encyst under experimental conditions, some suppose that certain internal factors play as great a part as do the external conditions in the phenomenon of encystment. Ordinarily a single cyst wall seems to be sufficient to protect the protoplasm against unfavorable external conditions. In some cases there may be a double cyst wall, the inner one usually being more delicate. The cyst wall is generally composed of homo- geneous substances, but it may contain calcareous scales as in Euglypha (Fig. 67). While chitin is the common material of which the cyst wall is composed, cellulose makes up the cyst envelope of numerous Phytomastigina. The capacity of Protozoa to produce the cyst is probably one of the reasons why they are so widely distributed over the surface of the globe. The minute protozoan cysts are easily carried from place to place by wind, attached to soil particles, debris, etc., by the flowing water of rivers or the current in oceans or by insects, birds, other animals to which they become readily attached. When a cyst encounters a proper environment, the living proto- plasmic contents excyst and the emerged organism once more return to its active trophic phase of existence. In Sporozoa, no encystment occurs. Here at the end of active schizogony, sexual reproduction usually initiates the production of large numbers of the spores (Fig. 68). Sexual reproduction and life-cycles Besides reproducing by the asexual method, numerous Proto- zoa reproduce themselves in a manner comparable with the 144 PROTOZOOLOGY sexual reproduction 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, which is a complete union of two gametes, has been re- ported from various groups, while the conjugation which is a Fig. 68. 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; n-x, spore-formation. temporary union of two gametes for the purpose of exchanging the nuclear material, is found almost exclusively in the Ciliophora. Sexual fusion. If the two gametes which take part in this process, are morphologically alike, they are called isogametes and the act the isogamy; but if unlike, anisogametes, and the act, anisogamy. The isogamy is typically represented by the flagellate Copromonas suhtilis (Fig. 69), in which there occurs, according to Dobell, a complete nuclear and cytoplasmic fusion between two isogametes. Each nucleus, after casting off a portion REPRODUCTION 145 of its nuclear material, fuses with the other and the zygote thus formed, encysts. In Stephanosphaera pluvialis (Fig. 70), both asexual and sexual reproductions occur, according to Hieronymus, Each individual multiplies and develops into numerous biflagel- late 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, Gregari- nida (Lankesterella, Fig. 174; Schizocystis, Fig. 185), etc. It per- Wm ^ Fig. 69. Sexual fusion in Copromonas subtilis, X1300 (Dobell). haps occurs in the Radiolaria also, although positive evidence has yet to be presented. Anisogamy seems to be more widely dis- tributed. On the whole, the differences between the micro- and macro-gametes are comparable with those which exist between the spermatozoa and ova of the Metazoa. The microgametes are motile, relatively small and usually numerous, while the macro- gametes are usually not motile, much more voluminous and fewer in number (Fig. 71). In Chlamydomonas monadina (Fig. 90), ac- cording to Goroschankin, the two gametes come in contact at the anterior end where the membranes become dissolved and the contents of the microgamete stream into the macrogamete. A new shell is then secreted around them. Later the shell becomes swollen and the organism multiplies into 2, 4, or 8 swarmers which in turn develop into the trophozoites. In Pandorina morum (Fig. 72), Pringsheim observed that each cell asexually develops into a young colony (a, h) or into anisogametes (c) which undergo sexual fusion {d-g) and encyst {h). The organism emerged from the cyst, develops into a young trophozoite {i-m). A similar life- cycle was found by Goebel in Eudorina elegans (Fig. 73). Among the Sporozoa, anisogamy is of common occurrence. In 14G PROTOZOOLOGY Fig. 70. The life-cycle of Stephanosphaera pluvialis (Hieronymus). a-e, asexual reproduction; f-m, sexual reproduction. Coccidia, the process was well studied in Eimeria schuhergi (Fig. 188), Aggregata eberthi {Fig. 190), Adelea ovata (Fig. 194), etc., and the resulting products are the oocyst (zygote) in which the spores or sporozoites develop. Similarly in Haemosporidia such as Plasmodium vivax (Fig. 197), anisogamy results in the forma- tion of the ookinete or motile zygote which gives rise to a large REPRODUCTION 147 Fig. 71. a, macrogamete, and b, microgamete of Volvox aureus, XlOOO (Klein). number of sporozoites. Among Myxosporidia, a complete infor- mation as to how the initiation of sporogony is associated with sexual reproduction, is still lacking. Na\dlle, however, states that Fig. 72. The life-cycle of Pandorina morum, X400 (Pringsheini). a, b, asexual reproduction; c-m, sexual reproduction. 148 PROTOZOOLOGY in the trophozoite of Sphaeromyxa sahrazesi (Fig. 210), micro- and macro-gametes develop, each with a haploid nucleus. Anisog- amy, however, is peculiar in that the two nuclei remain inde- pendent. The microgametic nucleus divides once and the two nuclei remain as the vegetative nuclei of the pansporoblast, while the macrogamete nucleus multiplies repeatedly and develop into two spores. Anisogamy^has been suggested to occur in some mem- bers of Amoebina, particularly in Endamoeba hlattae. Mercier (1909) believed that in this amoeba there occurs anisogamy soon Fig. 73. The life-cycle of Eudorina elegans (Goebel). a, asexual repro- duction; b, se.xual reproduction, a female colony with clustered and isolated microgametes. after excystment in the host's intestine, but this awaits confirma- tion. Cultural studies of various parasitic amoebae in recent years show no evidence of sexual reproduction in those forms. Among the Ciliophora, the sexual fusion occurs only in Proto- ciliata (Fig. 225) and the conjugation described below is the usual method of sexual reproduction. Conjugation. The conjugation is a temporary union of two individuals of one and the same species for the purpose of ex- changing part of the nuclear material and occurs almost ex- clusively in the Eucihata and Suctoria. The two individuals which participate in this process may be either isogamous or anisog- amous. In Paramecium caudatum (Fig. 74), two individuals come in contact on their oral surfaces. The micronucleus in each REPRODUCTION 149 Fig. 74. Diagram illustrating the conjugation of Paramecium caudatum. a-q, X about 130 (Calkins); r, X1200 (Dehorne). conjugant divides twice (6-e), forming four micronuclei, three of which degenerate and do not take active part during further 150 PROTOZOOLOGY changes (f-h). The remaining microniicleiis divides once more, l)rodiicing a wandering pronucleus and a stationary pronucleus (/, g). The wandering pronucleus in each of the conjugants enters the other individual and fuses with its stationary pronucleus {h, r). The two zygotes now separate from each other and become exconjugants. In each exconjugant, the synkaryon divides three times in succession (i-m) and produces eight nuclei (n), four of which remain as micronuclei, while the other four develop into new macronuclei (o). Cystosomic fission follows then, producing first, two individuals with four nuclei (p) and then, four small individuals, each containing a micronucleus and a macronucleus (a). According to Jennings, however, of the four smaller nuclei formed in the exconjugant indicated in Fig. 74, o, only one re- mains active, and the other three degenerate. This active nucleus divides prior to the cytosomic division so that in the next stage (p), there are two developing macronuclei and one micronucleus which divides once more before the second and last cytosomic division (q). During these changes the original macronucleus disintegrates, degenerates, and finally becomes absorbed in the cytoplasm. When the cihate 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. In Paramecium aurelia, of the eight micro- nuclei formed by two fissions of the two original micronuclei, according to Woodruff, only one undergoes the third division to produce two pronuclei. This is 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, Stylonychia pustulata; two to three in Oxytrichafallax and two to four in Uroleptus mobilis. This third division is always characterized by long extended nuclear membrane stretched be- tween the division products. Ordinarily the individuals which undergo conjugation appear to be morphologically similar to those that are engaged in the trophic activity, but in some species, the organism divides just prior to conjugation. According to Wichterman (1936), conjuga- tion in Nyctotherus cordiformis (Fig. 75) 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 REPRODUCTION 151 Fig. 75. The life-cycle of Nyctotherus cordiformis in Hijla 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 micronucleus and the other passes through the "spireme ball" stage before developing into a macronucleus; k-n, exconjugants found nearly exclusively in recently transformed host; o, mature trophozoite; p-s, binary fission stages; t, precystic stage. 152 PROTOZOOLOGY trophozoites, because of the preconjugation fission (d-e). The microiuiclear divisions are similar to those that have been de- scribed for Paramecium caudatum and finally two pronuclei are formed in each conjugant. Exchange and fusion of pronuclei follow. In each exconjugant, the synkaryon divides once to form the micronucleus and the macronuclear anlage (k-l) which de- velop into the "spireme ball" and finally into the macronucleus (m-o). A sexual process which is somewhat intermediate between the sexual fusion and conjugation, is noted in several instances. Ac- cording to Maupas' classical work on Vorticella riehulifera, the ordinary vegetative form divides twice, forming four small in- dividuals, 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 divides twice, forming four nuclei, of which three degenerate, the other dividing into two. During these changes the cytoplasm of the two conjugants fuse completely. The wandering nucleus of the smaller conjugant unites with the stationary nu- cleus of the larger conjugant, the other two pronuclei degenerat- ing. The synkaryon divides several times to form a number of nuclei, from some of which macronuclei are differentiated and exconjugant undergoes multiplication. Another example of this type has been observed by Noland (1927) in Metopus es (Fig. 76). According to Noland, the con- jugants fuse at the anterior end (a), and the micronucleus in each individual divides in the same way as was observed in Para- mecium caudatum (b-e). But the cytoplasm and both pronuclei of one conjugant pass into the other (/), leaving the degenerating macronucleus and a small amount of cytoplasm behind in the shrunken pellicle of the smaller conjugant which then separates from the other (j). In the larger exconjugant, two pronuclei fuse, and the other two degenerate and disappear {g, h). The synkaryon divides into two nuclei, one of which condenses into the micro- nucleus and the other grows into the macronucleus {i-m). This is followed by the loss of cilia and encystment. What is the significance of conjugation? What are the condi- tions which bring about conjugation in the ciliates? These are REPRODUCTION 153 Fig. 76. Conjugation of Metopus es (Noland). a, early stage; b, first micronuclear division; c, d, second micronuclear division; e, third micronuclear division; f, migration of pronuclei from one conjugant into the other; g, large conjugant with two pronuclei ready to fuse; h, large conjugant with the synkaryon, degenerating pronuclei and macronucleus; i, large exconjugant with newly formed micronucleus and macronucleus, showing the degenerating old macronucleus; j, small exconjugant with degenerating macronucleus; k-m, develop- ment of two nuclei, a, X290; b-j, X 250; k-m, X590. but two of the many problems which numerous investigators at- tempted to solve since the appearance of the first comprehensive study of the phenomenon by Maupas in 1889. Woodruff's ob- servation (1932) among others which showed that 5071 genera- tions produced asexually from a single individual of Paramecium 154 PROTOZOOLOGY aurelia between May 1, 1907 and May 1, 1915, did not manifest any decrease in vitality after eight years of asexual r(>])roduction, demonstrates beyond doubt that the sexual reproduction in the form of conjugation is not necessary for the well-being of Para- mecium aurelia under favorable environmental conditions. On the other hand, there is a large body of evidence to sup])ort the view expressed first by Maupas to the effect that the conjugation cor- rects an inherent tendency toward senescence under unfavorable conditions. Recently Sonneborn and Lynch (1932) demonstrated by using different clones of P. aurelia that the effects of conjuga- tion are diverse and characteristic of different races: 1) the con- jugation increases fission rate in some clones, decreases the rate in others; 2) it increases variation in some clones, but not in others; and 3) it increases mortality in some clones but not in others. Sonneborn (1937) continuing controlled observations on this ciliate, discovered that in certain races there are two classes of individuals with respect to sexual differentiation and that the members of different classes conjugate, while the members of each class do not. He further found that the individuals produced by binary fission from a single individual belong all to the sex re- action type to which the original individual belonged, and that in conjugation in which two sex reaction types participate, the four sets of progeny consist of the two types in chance combination, the ratio being identical with those for inheritance in higher organisms. Jennings (1938) found further four sex raction types in P. bursaria, in which the type behavior toward conjugation was exactly like that of the two types found in P. aurelia. 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 different cells, then paedogamy. The autogamy is of common occurrence in the myxosporidian spores. The young sporoplasm contains two nuclei which fuse together prior to or during the process of germination in the ahmentary canal of a specific host fish, as for example in Sphaeromyxa sahrazesi (Figs. 209; 210) and Myxosoma catostomi (Fig. 208). In REPRODUCTION 155 the Microsporidia, autogamy initiates the spore-formation at the end of schizogonie activity of individuals as in Thelohania legeri (Fig. 68). Recently Diller (1936) observed in solitary Paramecium aurelia (Fig. 77), certain micronuclear changes similar to those which occur in conjugating individuals. The two micronuclei divide twice, forming eight nuclei, some of which divide for the third time, producing two functional and several degenerating nuclei. The two functional nuclei then fuse in the "preoral cone" and Fig. 77. Diagram illustrating autogamy in Paramecium aurelia (Diller). a, normal animal; b, first micronuclear division; c, second micronuclear division; d, individual with 8 micronuclei and a macro- nucleus preparing for skein formation; e, some micronuclei dividing for the third time, with two functional nuclei near 'preoral cone'; f, two gamete-nuclei formed by the third division in the 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 remaining as micronuclei; k, macronuclei developing, micronuclei dividing; 1, one of the daughter individuals produced by fission. form the synkaryon which divides twice into four. 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, each dividing into two after the body divides into two. Diller is "inclined 156 PROTOZOOLOGY to feel that if an animal does not happen to meet another indi- vidual in the same physiological condition as itself, its reorga- nizing 'urge' will be expressed by autogamy, as a substitute for conjugation." The paedogamy occurs in at least two species of Myxosporidia, namely, Leptotheca ohlmacheri (Fig. 212) and Unicapsula muscularis (Fig. 213). The spores of these Myxosporidia contain two uninucleate sporoplasms which are independent at first, but prior to emergence from the spore, they undergo a complete fusion to metamorphose into a uninucleate amoebula. Perhaps the classical example of the paedogamy is that which was found by Hertwig (1898) in Actinosphaeriiim eichhorni. The organism encysts and the body divides into numerous uninucleate second- ary cysts. Each secondary cyst divides into two and remains together within a common cyst-wall. In each the nucleus divides twice, and forms four nuclei, one of which remains functional, the remaining three degenerating. The paedogamy results in formation of a zygote in place of a secondary cyst. Belaf (1922) observed a similar process in Adinophrys sol (Fig. 78). The helio- zoan withdraws its axopodia and divides into two uninucleate bodies which become surrounded by a common gelatinous en- velope. Both nuclei divide twice and produce four nuclei, three of which degenerate. The two daughter cells, each with one haploid nucleus, undergo paedogamy and the resulting individual now contains a diploid nucleus. Endomixis. Woodruff and Erdmann (1914) observed that in Paramecium aurelia (Fig. 79) at regular intervals of about 30 days, the old macronucleus breaks down and disappears, while each of the two micronuclei divide twice, forming eight nuclei. Of these, six disintegrate. At this point the organism divides into two, each daughter individual receiving one micronucleus. This nucleus soon divides twice into four, two of which develop into macronuclei, and the other two divide again. Here the organisms divide once more by binary fission, each bearing one macronucleus and two micronuclei. This process which is "a com- plete periodic nuclear reorganization without cell fusion in a pedigreed race of Paramecium" was called by the two authors endomixis. In the case of P. caudatum, they found endomixis occurs at intervals of about 60 days. Sonneborn (1937) succeeded in inducing endomixis in certain stocks of P. aurelia by placing REPRODUCTION 157 small mass cultures containing surplus animals from isolation lines at 31°C. for 1-2 days. Endomixis has since been observed more often in encysted stage of Spathidium spathula, Uroleptus mobilis, Euplotes longipes, Didinium nasutum, etc. As to its sig- nificance, the statement made for conjugation appears also to hold true. In Paramecium aurelia, Diller (1936) found simple fragmenta- Fig. 78. 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. tion of the macronucleus which was not correlated with any special micronuclear activity and which could not be stages in conjugation or autogamy. Diller suggests that if conjugation or autogamy is to create a new nuclear complex, as is generally held, it is conceivable that somewhat the same result might be achieved by 'purification act' (through fragmentation) on the part of the macronucleus itself, without involving micronuclei. He coined the term 'hemixis' to include these reorganizations. Meiosis. In the foregoing sections, references have been made 158 PROTOZOOLOGY to the divisions which the nuclei undergo prior to sexual fusion or conjugation. 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 number (haploid) of chromosomes typical to the species (diploid). In the zygote, the Fig. 79. Diagram showing the endomixis in Paramecium aurelia (Woodruff), a, normal individual; b, degeneration of macronucleus and first micronuclear division; c, second micronuclear division; d, degeneration of 6 micronuclei; e, cell division; f, g, first and second reconstruction micronuclear division; h, transformation of 2 micro- nuclei into macronuclei; i, micronuclear and cell division; j, typical nuclear condition is restored. diploid number is reestablished. In the Protozoa in which sexual reproduction occurs during their life-cycle, meiosis presumably takes place to maintain the constancy of chromosome-number, but the process is understood only in a small number of species. In conjugation, the meiosis seems to take place in the second micronuclear division, although in some, for example, Oxytricha fallax, according to Gregory, the actual reduction occurs during the first division. Prandtl (1906) was the first to note a reduction REPRODUCTION 159 in number of chromosomes in the Protozoa. In conjugating Didinium nasutum, 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 poly- piniim (Popoff), Uroleptiis halseyi (Calkins), etc. (see the ciliates in the list on p. 135). In various species of Paramecium and many- other forms, the number of chromosomes appears to be too great to allow a precise counting, but it is generally agreed that here probably reduction in the number also takes place. Information on the meiosis involved in the complete fusion of gametes is even more scanty and fragmentary. In Monocystis rostrata, a parasite of the earthworm, Mulsow, noticed that the nuclei of two gametocytes which encyst together, multiply by mitosis in which eight chromosomes are constantly present, but in the last division in gamete formation, each daughter nucleus receives only 4 chromosomes. In another species of Monocystis, Calkins and 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 Actinopkrys sol, Belaf found 44 chromo- somes in the first nuclear division, but after two meiotic divisions, the remaining functional nucleus contains only 22 chromosomes so that when paedogamy is completed the diploid number is re- stored. On the other hand, in the coccidian Aggregata eberthi (Fig. 190), according to Dobell and Jameson, Belaf, and Naville, and in the gregarine Diplocystis schneideri, according to Jameson, there is no reduction in the number of chromosomes during the gamete-formation, but the first zygotic division is meiotic, 12 to 6 and 6 to 3, respectively. A similar reduction in chromosome (12 to 6) takes place also in the gregarine Zygosoma glohosum, accord- ing to Noble's recent study (1938). Thus it appears in these cases that the zygote or oocyst is the only stage in which diploid nucleus occurs, while the nuclei in the stages in the remainder of the life- cycle are haploid. References Belar, K. 1926 Der Formwechsel der Protistenkerne. Ergebn. u. Fortschr. Zool., Vol. 6. 160 PROTOZOOLOGY Calkins, G. N. 1926 The biology of the Protozoa. Philadelphia. — and R. C. Bowling 1926 Gametic meiosis in Mono- cystis. Biol. Bull., Vol. 51. Chalkley, H. W. and G. E. Daniel 1933 The relation be- tween the form of the living cell and the nuclear phases of division in Amoeba proteus. Physiol. Zool., Vol. 6. Chen, T. T. 1936 Observations on mitosis in opalinids. I. Proc. Nat. Acad. Sci., Vol. 22. ^ Cleveland, L. R. 1938 Longitudinal and transverse division in two closely related flagellates. Biol. Bull., Vol. 74. 1938 Origin and development of the achromatic figure. Ibid. S. R. Hall, E. P. Sanders and J. Collier 1934 The wood-feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts and Sci., Vol. 17. DiLLER, W. F. 1936 Nuclear reorganization processes in Para- mecium aurelia, with descriptions of autogamy and 'hcmixis'. Jour. Morph., Vol. 59. DoBELL, C. 1908 The structure and life history of Copromonas subtilis. Quart. Jour. Micr. Sci., Vol. 52. 1917 On Oxnerella maritima, no v. gen., nov, spec, a new heliozoan, and its method of division, with some remarks on the centroplast of the Heliozoa. Ibid., Vol. 62. 1925 The life-history and chromosome cycle of Ag- gregata eberthi. Parasitology, Vol. 17. and A. P. Jameson 1915 The chromosome cycle in Coc- cidia and Gregarines. Proc. Roy. Soc. (B), Vol. 89. Hall, R. P. 1923 Morphology and binary fission of Menoidium incurvum. Univ. Calif. Publ. Zool., Vol. 20. 1925 Binary fission in Oxijrrhis marina. Ibid., Vol. 26. Jameson, A. P. 1920 The chromosome cycle of gregarines with special reference to Diplocystis schneideri. Quart. Jour. Micr. Sci., Vol. 64. Jennings, H. S. 1929 Genetics of the Protozoa. Bibliogr. Gen., Vol. 5. 1938 Sex reaction types and their interrelations in Paramecium bursaria. I. II. Proc. Nat. Acad. Sci., Vol. 24. Kidder, G. W. 1933 Studies on Conchophthirius mytili de Mor- gan. I. Arch. f. Protistenk., Vol. 79. and W. F. Diller 1934 Observations on the binary fission of four species of common free-living ciliates, with special reference to the macronuclear chromatin. Biol. Bull., Vol. 67. and F. M. Summers 1935 Taxonomic and cytological studies on the ciliates associated with the amphipod family Orchestiidae from the Woods Hole district. Ibid., Vol. 68. Kofoid, C. a. and Olive Swezy 1919 On Streblomastix strix, a polymastigote flagellate with a linear plasmodial phase. Univ. Calif. Publ. Zool., Vol. 20. REPRODUCTION 161 1919 On Trichonympha campanula sp. nov. Ibid. Kudo, R. R. 1925 Observations on Endamoeha blattae. Amer. Jour. Hyg., Vol. 6. 1926 Observations on Lophomonas blattarum, a flagel- late inhabiting the colon of the cockroach, Blalta orientalis. Arch. f. Protistenk., Vol. 53. 1926 A cytological study of Lophomonas striata. Ibid., Vol. 55. 1936 Studies on Nydotherus oralis Leidy, with special reference to its nuclear structure. Ibid., Vol. 87. LiESCHE, W. 1938 Die Kern- und Fortpflanzungsverhaltnisse von Amoeba proteus. Ibid., Vol. 91. Lund, E. J. 1917 Reversibility of morphogenetic processes in Bursuria. Jour. Exp. Zool., Vol. 24. Noble, E. R. 1938 The life-cycle of Zygosoma globosum sp. nov., a gregarine parasite of Urechis caupo. Univ. Calif. Publ. Zool., Vol. 43. NoLAND, L. E. 1927 Conjugation in the ciliate Metopus sig- moides. Jour. Morph. Physiol., Vol. 44. Prandtl, H. 1906 Die Konjugation von Didinium nasutum. Arch. f. Protistenk., Vol. 7. Reichenow, E. 1928 Ergebnisse mit der Nuclealfarbung bei Protozoen. Ibid., Vol. 61. SoNNEBORN, T. M. 1936 Factors determining conjugation in Paramecium aurelia. I. Genetics, Vol. 21. 1937 Induction of endomixis in Paramecium aurelia. Biol. Bull., Vol. 72. 1937 Sex, sex inheritance and sex determination in Paramecium aurelia. Proc. Nat. Acad. Sci., Vol. 23. and B. M. Cohn 1936 Factors determining conjugation in Paramecium aurelia. II. Genetics, Vol, 21. and R. S. Lynch 1937 III. Ibid., Vol. 22. Summers, F. M. 1935 The division and reorganization of the macronuclei of Aspidisca lynceus, Diophrys appendiculata and Stylonychia pustulata. Arch. f. Protistenk., Vol. 85. Turner, J. P. 1930 Division and conjugation in Euplotes patella with special reference to the nuclear phenomena. Univ. Calif. Publ. Zool., Vol. 33. Wichterman, R. 1936 Division and conjugation in Nyctotherus cordiformis with special reference to the nuclear phenomena. Jour. Morph., Vol. 60. Woodruff, L. L. 1932 Paramecium aurelia in pedigree culture for twenty-five years. Trans. Amer. Micr. Soc, Vol. 51. and R. Erdmann 1914 A normal periodic reorganization process without cell fusion in Paramecium. Jour. Exp. Zool., Vol. 17. Chaptp:r 6 Variation and heredity IT is generally recognized that individuals of a species of organism show a greater or less morphological variation. Pro- tozoa are no exceptions. Various Protozoa manifest a wide variation in a limited or in widely separated localities so that different groups of the same species are spoken of as races, varieties, etc. It is well-known that dinoflagellates show a great morphological variation in different localities. Schroder (1914) showed that there were at least nine varieties of Ceratium hirundinella (Fig. 80) occurring in various waters of Europe, and List found that the organisms living in shallow ponds showed 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 deep water may reach 200^ in length, while those from the surface water measure only about lOOju long. In Foraminifera, the shell varies in thickness even in one and the same species, depending upon the part of the ocean in which they live. Thus the forms which live floating in surface water have a much thinner shell than those which dwell on the bottom of the ocean. For example, according to Rhumbler, Orhulina universa inhabiting surface water has a very thin shell, 1.28- 18m thick, while individuals living on the bottom may show a thick shell, up to 24^ in thickness. According to Uyemura, Amoeba sp., occurring in the thermal waters of Japan, showed a distinct dimensional difference in different springs; namely, it varied from 10-40^t in diameter in sulphurous water, and from 45-80)u in ferrous water; in both types of water the amoebae were larger at 36-40°C. than at 51°C. Such differences in morpho- logical characteristics appear to be influenced by environmental conditions, and will continue to exist under those conditions, but when the organisms are subjected to a similar environment the differences disappear, as has been demonstrated by many observers. Evidences obtained by various investigators point to a general conclusion that when environmental influences are brought upon 162 VARIATION AND HEREDITY 163 a protozoan at the time of nuclear reorganization either by division, conjugation, or by endomixis, they may bring about long-lasting modifications (JoUos) or mutations. In Popoff's ex- periment with Stentor and in Chatton's with Glaucoma, both Fig. 80. Varieties of Ceratium hirundineUa from various European waters (Schroder), a, /?trcoiV/es-type (130-300/i by 30-45/x); b, brachy- ceroides-type (130-145)u by 30-45ju); c, silesiacurn-type (148-280/i by 28-34^i); d, carinthiacum-type (120-145/i by 45-60ju); e, gracile-tyTpe (140-200M by 60-75ju); f, aiistriacum-type (120-160/x by 45-60^); g, robustum-type (270-310/i by 45-55ju); h, scotticum-type (160-210ju by 50-60m); i, piburgense-type (180-260^ by 50-60^). conducted during the asexual division, long-lasting modifications have appeared in the experimental animals. Calkins (1924) ob- served a double-type Uroleptus mohilis which was formed by a complete fusion of two conjugants. This abnormal animal divided 367 times, living for 405 days, but reverted into normal forms 164 PROTOZOOLOGY without reversion to a double form. It is probable that the organ- ism showed a long-lasting modification, but there was no con- stitutional change in the organization of the animal. Jollos (1913-1934) observed that Paramecium, when subjected to various environmental influences, such as high temperature, arsenic acid, etc., showed variations which were gradually lost, although lasting through one or more periods of conjugation and endomixis, and that if the organisms were subjected to environ- mental changes during the late phase of conjugation, certain Fig. 81. Chilodonella uncinata (MacDougall). a, b, ventral and side view of normal individual; c, d, ventral and side view of the tailed mutant. individuals, if not all, become permanently changed. Possibly here one sees that the reorganizing nuclear material has been affected in such a way that the hereditary constitution or geno- type has become 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) tem- porary variations which disappeared by the third generation; and 3) variations which were transferred unchanged through successive generations, hence considered as mutations. The mutants were triploid, tetraploid, and tailed diploid forms (Fig. 81), which bred true for a variable length of time in pure-line cultures, either being lost or dying off finally. The tailed form dif- fered from the normal form in the body shape, the number of VARIATION AND HEREDITY 165 ciliary rows, with three contractile vacuoles, and mode of move- ment, but during conjugation showed the diploid nimiber of chro- mosomes as in the normal form. The tailed form remained true and underwent 20 conjugations during ten months. The first comprehensive study dealing with the variation in size with respect to inheritance in the uniparental reproduction of Protozoa was done by Jennings (1909). From a "wild" lot of Paramecium, Jennings isolated eight races with the relative mean lengths of 206, 200, 194, 176, 142, 125, 100, and 45// which were inherited in each race. It was found further that within each clone derived from a single parent the size of different component individuals varies extremely, which is attributable to growth, amoimt of food and other environmental conditions, any one of which may give rise to progeny of the same mean size. Jennings thus showed that selection within the pure race has no effect on the size and that differences brought about merely by environ- ment are not inherited. Jennings (1916) also studied the inheritance of size and number of spines, dimensions of tests, diameter of mouth and size and number of teeth of the testacean Difflugia corona, and found that "a population consists of many hereditarily diverse stocks, and a single stock, derived from a single progenitor, gradually differen- tiates 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 (1923) with A. polypora, obtained similar results. Jennings (1937) carried on his study on the inheritance of teeth in Difflugia corona further in normal reproduction and by altering the mouth and teeth of the parent by operation, and observed that operated normal mouth or teeth were restored in three to four generations and that three factors appeared to determine the character and number of teeth: namely, the size of the mouth, the number and arrange- ment of the teeth in the parent, and "something in the constitu- tion of the clone (its genotype) which tends toward the produc- tion of a mouth of a certain size, with teeth of a certain form, arrangement and number." In the case of biparental inheritance, two nuclei of two different individuals participate to produce new combinations which would naturally bring about a greater variation among the off- spring. For example, if two individuals from a single clone of a 16G PROTOZOOLOGY ciliate, conjugate and the exconjugants are allowed to reproduce by fission, the descendants will show a greater variation among them with respect to the dimensions, fission-rate, etc. Thus several new biotypes may appear. If conjugation takes place between individuals of different clones and the descendants of Fig. 82. Hybridization in Chlamydomonas (Pascher). a, vegetative individual of Chlamydomonas I; b, that of II; c, sexual fusion of two gametes of I; d, that of two gametes of II; e, homozygote of I; f, homozygote of II; g, sexual fusion of a gamete of I with a gamete of II; h, i, heterozygotes between I and II; j-m, four types of individuals arising from a heterozygote in culture. this pair in turn conjugate and multiply by fission, the progeny will show conditions comparable to those which one sees in Mendehan inheritance in higher animals. In nature and in mass culture, it is supposed that this process is taking place con- tinuously. Since various species of Protozoa commonly co-inhabit small confines of water in nature, it is probable that hybridization be- tween varieties or species may occur. Information on hand on VARIATION AND HEREDITY 167 experimental hybridization of Protozoa is however very meager. Pascher (1916) succeeded in producing a small number of hybrid zygotes between two species of Chlamydomonas (Fig. 82). The two possessed the following characteristics. Species I: pyriform; without a membrane-papilla; with a delicate mem- brane; flagella about twice the body length; chromatophore and pyrenoid lateral; nucleus central; stigma a narrow streak in the anterior third; with 2 contractile vacuoles (a); division into 4 zoospores ; gametes up to 8, narrowed without membrane ; zygote deeply sculptured and without spreading envelope (e). Species II: spherical; with a distinct membrane and a membrane-papilla; chromatophore and pyrenoid posterior; nucleus central; stigma more anterior and fusiform; flagella short (6); division into 4 zoospores; gametes ellipsoid, ends rounded; zygote with a smooth but spreading envelope; with discarded gamete membrane (/). The hybrid-zygotes were morphologically intermediate {h, i) between the two parent zygotes. Thirteen zygotes were reared and in five cultures the offspring were either species I or II, two of each four zoospores being similar to those of I and the other two similar to those of II. In the eight cultures, each zygote de- veloped into four different zoospores. Pascher described these four zoospores (j-m), which tended to indicate that for each of several pairs of characters, two zoospores possessed that of I and the other two that of II and that hybridization brought to- gether two diverse sets of determiners in the heterozygote, which became segregated into four new sets of determiners, because of reduction during the formation of zoospores. These zoospores were however less active and abnormal so that they finally died in the culture without further development. Strehlow (1929) at- tempted to produce hybrids from three combinations of species of Chlamydomonas, succeeding in only one. Heterozygotes were obtained from the "positive" strain of C. paradoxa and the "negative" strain of C. hotnjodes (Fig. 83). Germination of the zygotes was however not observed. Hybridization between different varieties or different species of the same genus of ciliates, was either unsuccessful or not genetically studied until quite recently. By using different clones of Paramecium aurelia which differed in fission rate, viability, and body length, Sonneborn and Lynch succeeded in following through three or four sexual generations and observed: "Groups 168 PROTOZOOLOGY of hybrid clones obtained by crossing diverse clones manifested, on the average, characteristics intermediate between those of the l)arent clones or intermediate between those of the two groups of clones obtained by inbreeding the parent clones. Hybrid clones are of two types differing in the origin of their cytoplasm and Fig. 83. Hybridization of Chlamydomonas (Strehlow). a, C. paradoxa; h, C. botryodes; c-f, heterozygotes between them. macronuclear fragments; one type derives these from one parent clone, the other type from the other parent clone entering into the cross. One hybrid clone of each type arises from each pair of hybrid exconjugants. Sets of each of the two types of hybrid clones, as well as groups including both types of sets, were inter- mediate in characteristics. When hybrids of either type of cyto- plasmic descent were inbred, the resulting F2 generation included some clones resembling one parent, some clones resembling the other parent, and some clones with intermediate characteristics. When such F2 segregates were further inbred, the resulting F3 generation showed that some F2 segregates were pure and others still mixed in genetic constitution." Sonneborn and Lynch point out further that "there is no longer ground for doubting that the nucleus carries the determiners of hereditary characters, and there is considerable evidence that the nucleus carries these determiners arranged in separable pairs like the chromosomes or genes of higher organisms. If the cytology of the chromosomes in Paramecium were better known, the exact strength of the latter point could be more precisely estimated. For P. aurelia, Hertwig (1889) described a small number of chromosomes (8-10) undergoing conventional reduction during conjugation; but other investigators of this species have not hazarded chromosome counts and have given the impression that little progress in Paramecium chromosome cytology can be ex- VARIATION AND HEREDITY 169 pected with present methods. Unless further progress can be made in cytological studies, the burden of attack must fall all the more heavily on purely genetic methods. On the basis of genetic work alone, we are led to conclude that the usual Mendelian situation, modified by certain details pecuHar to the organism, probably exists in Paramecium. In agreement with Pascher, we find the fundamental patterns of protozoan and metazoan genetics to be very nearly the same." De Garis produced monsters in P. caudatum by exposing dividing individuals either to low temperature or cyanide vapor, which were L-shaped and one or both components divided usually on the second or third day, producing free individuals. The genet- ic constitution of progeny was not altered by the experience of monster formation. By bringing about conjugation between monsters and free individuals which differed in fission rate and body length, De Garis produced hereditary diverse races from the two lines. On the other hand, the conjugation between P. aurelia and double monster of P. caudatum was found to have lethal effects on both ciliates, as the former species degenerated ("cloudy swelling") and died on the second or third day after conjugation, while the latter species manifested hyaline degenera- tion and died on the second to twelfth day after conjugation. The discovery of sex reaction types in Paramecium aurelia and P. bursaria, as was stated in the last chapter, and further researches along this line, will, it is hoped, throw a clearer light on various genetical problems in Protozoa. References Calkins, G. N, 1924 Urole-ptus mohilis. V. Jour. Exp. Zool., Vol. 41. De Garis, C. F. 1934 Genetic results expressed by fission rates following conjugation between double monsters and free individuals of Paramecium caudatum. Amer. Nat., Vol. 68. 1935 Lethal effects of conjugation between Paramecium aurelia and double monsters of P. caudatum. Ibid., Vol.69. Jennings, H. S. 1909 Heredity and variation in the simplest organisms. Ibid., Vol. 43. 1916 Heredity, variation and the results of selection in the uniparental reproduction of Difflugia corona. Genetics, Vol. 1. 1929 Genetics of the Protozoa. Bibliographia Genetica, Vol. 5. 170 PROTOZOOLOGY 1937 Formation, inheritance and variation of the teeth in Difflugia corona. Jour. Exp. ZooL, Vol. 77. JoLLOS, V, 1934 Dauermodifikationen und Mutationen bei Protozoen. Arch. f. Protistenk., Vol. 83. Hegner, R. W. 1919 Heredity, variation, and the appearance of diversities during the vegetative reproduction of Arcella dentata. Genetics, Vol. 4. MacDougall, M. S. 1929 Modifications in Chilodon uncinatus produced by ultraviolet radiations. Jour. Exp. Zool.,Vol. 54. 1931 Another mutation of Chilodon uncinatus produced by ultra-violet radiation, with a description of its maturation process. Ibid., Vol. 58. Pascher, a. 1916 Ueber die Kreuzung einzelliger, haploider Organismenl Chalmydomonas. Ber. deutsch. Bot. Ges. Vol. 34. - — 1918 Ueber die Beziehung der Reduktionsteilung zur Mendelschen Spaltung. Ibid., Vol. 36. Reynolds, B. D. 1923 Inheritance of double characteristics in Arcella polypora Penard. Genetics, Vol. 8. — 1924 Interactions of protoplasmic masses in relation to the study of heredity and environment in Arcella polypora. Biol. Bull., Vol. 46. Root, F. M. 1918 Inheritance in the asexual reproduction in Centropyxis aculeata. Genetics, Vol. 3. SoNNEBORN, T. M. and R. S. Lynch 1934 Hybridization and segregation in Paramecium aurelia. Jour. Exp. Zool.,Vol. 67, Strehlow, K. 1929 Ueber die Sexualitat einiger Volvocales. Zeitschr. Botanik, Vol. 21. Taliaferro, W. H. 1926 Variability and inheritance of size in Trypanosoma lewisi. Jour. Exp. Zool., Vol. 43. Chapter 7 Phylum Protozoa Goldfuss rriHE Protozoa are divided into two subphyla as follows: Locomotor organellae, pseudopodia or flagella, or lacking (in Sporo- zoa); nucleus of one kind Subphylum 1 Plasmodroma Locomotor organellae, cilia or cirri; nuclei of two kinds Subphylum 2 Ciliophora (p. 481) Subphylum 1 Plasmodroma Doflein Class 1 Mastigophora Diesing The Mastigophora includes those Protozoa which possess one to several flagella. Aside from this common characteristic, this class makes a very heterogeneous assemblage and seems to pre- vent a sharp distinction between the Protozoa and the Proto- phyta, as it includes Phytomastigina which are often dealt with by botanists. In the majority of Mastigophora, each individual possesses 1-4 flagella during the vegetative stage, although species of Polymastigina may possess up to 8 or more flagella and of Hyper- mastigina a greater number of flagella. The palmella stage (Fig. 84) is common among the Phytomastigina and, unlike the encysted stage, the organism is capable in this stage not only of metabolic activity and growth, but also of reproduction. In this respect, this group shows also a close relationship to algae. All three types of nutrition, carried on separately or in com- bination, are to be found among the members of Mastigophora. In holophytic forms, the chlorophyll is contained in the chromato- phores which are of various forms among different species and which differ in colors, from green to red. The difference in color appears to be due to the pigments which envelop the chlorophyll body (p. 79). Many forms adapt their mode of nutrition to changed environmental conditions, for instance, from holophytic to saprozoic in the absence of the sunlight. Holozoic, saprozoic and holophytic nutrition are said to be combined in such a form as Ochromonas. In association with chromatophores, there occurs a refractile granule or body, the pyrenoid, which is connected 171 172 PROTOZOOLOGY with starch-formation. Reserve food substances are starch, oil, etc. (p. 94-95). In less complicated forms, the body is naked except for a slight cortical differentiation of the ectoplasm to delimit the body surface and is capable of forming pseudo])odia. In others, there occurs a thin plastic pellicle produced by the cytoplasm, which covers the body surface closely. In still others, the body form is constant, being encased in a shell, test, or lorica, which is composed of chitin, pseudochitin, or cellulose. Not infrequently a gelatinous secretion envelops the body. In three families of Protomonadina there is a collar-like structure located at the anterior end, through which the flagellum protrudes. The great majority of Mastigophora possess a single nucleus, and only a few are multinucleated. The nucleus is vesicular and contains a conspicuous endosome. Contractile vacuoles are always present in the forms inhabiting fresh water. In simple forms, the contents of the vacuoles are discharged directly through the body surface to the exterior; in others there are several contractile vacuoles arranged around a reservoir which opens to the exterior through the so-called cytopharynx. In the Dinoflagellata, there are apparently no contractile vacuoles, but non-contractile pusules (p. 217) occur in some forms. In chromatophore-bearing forms, there occurs usually a stigma which is located near the base of the flagellum and seems to be the center of phototactic activity of the organism which possesses it (p. 79). Asexual reproduction is, as a rule, by longitudinal fission, but in some forms multiple fission also takes place under certain circumstances, and in others budding may take place. Colony- formation (p. 140), due to incomplete separation of daughter in- dividuals, is widely found among this group. Sexual reproduction has been reported in a number of species. The Mastigophora are free-living or parasitic. The free-living forms are found in fresh and salt waters of every description; many are free-swimming, others creep over the surface of sub- merged objects, and still others are sessile. Together with algae, the Mastigophora compose a major portion of plankton hfe which makes the foundation for the existence of all higher aquatic organisms. The parasitic forms are ecto- or endo-parasitic, and the latter inhabit either the digestive tract or the circulatory system of the host animal. Trypanosoma, a representative genus MASTIGOPHORA, CHRYSOMONADINA 173 of the latter group, includes important disease-causing parasites of man and of domestic animals. The Mastigophora are divided into two subclasses as follows: With chromatophores Subclass 1 Phytomastigina Without chromatophores Subclass 2 Zoomastigina (p. 235) Subclass 1 Phytomastigina Doflein The Phytomastigina possess the chromatophores and their usual method of nutrition is holophytic, though some are holozoic, saprozoic or mixotrophic; the majority are conspicuously colored; some that lack chromatophores are included in this group, since their structure and development resemble closely those of typical Phytomastigina. 1-4 flagella, either directed anteriorly or trailing Chromatophores yellow, brown or orange Anabolic products fat, leucosin Order 1 Chrysomonadina Anabolic products starch or similar carbohydrates Order 2 Cryptomonadina (p. 184) Chromatophores green Simple contractile vacuole, anabolic products starch and oil. . Order 3 Phytomonadina (p. 188) Contractile vacuole complex Anabolic products paramylon. . Order 4 Euglenoidina (p. 203) Anabolic products oil Order 5 Chloromonadina (p. 213) 2 flagella, one of which transverse. . . .Order 6 Dinoflagellata (p. 216) Order 1 Chrysomonadina Stein The chrysomonads are minute organisms and are plastic, since the majority lack a definite cell-wall. Chromatophores are yellow to brown (rarely green or bluish) and usually discoid, though sometimes reticulated, in form. Metabolic products are refractile bodies, known collectively as leucosin (probably carbo- hydrates) and fats. Starches have not been found in them. 1-2 flagella are inserted at or near the anterior end of body where a stigma is present. Many chrysomonads are able to form pseudopodia for obtain- ing food materials which vary among different species. Nutrition, though chiefly holophytic, is sometimes holozoic or saprozoic. Contractile vacuoles are invariably found in freshwater forms, and are ordinarily of simple structure, although a complicated system seems to be found in some. 174 PROTOZOOLOGY Under conditions not fully understood, the Chrysomonadina lose their flagella and undergo division with development of mucilaginous envelope and thus transform themselves often into large bodies known as the palmella phase and undertake meta- bolic activities as well as multiplication (Fig. 84). Asexual re- production is, as a rule, by longitudinal division during either the motile or the palmella stage. Incomplete separation of the daughter individuals followed by repeated fission, results in numerous colonial forms mentioned elsewhere (p. 141). Some Fig. 84. The life-cycle of Chromulina, X about 200 (Kiihn). a, en- cystment; b, fission; c, colony-formation; d, palmella-formation. resemble higher algae very closely. Sexual reproduction is en- tirely unknown in this group. Encystment occurs commonly; in this the flagellum is lost and the cyst is enveloped by a sihcious wall possessing an opening with a plug. The chrysomonads inhabit both fresh and salt waters, often occurring abundantly in plankton. Motile stage dominant Suborder 1 Euchrysomonadina Palmella stage dominant Sarcodina-like; flagellate stage unknown Suborder 2 Rhizochrysidina (p. 181) Palmella phase dominant Suborder 3 Chrysocapsina (p. 182) Suborder 1 Euchrysomonadina Pascher With or without simple shell One flagellum Family 1 Chromulinidae (p. 175) 2 flagella Flagella equally long Family 2 Syncryptidae (p. 177) Flagella unequally long Family 3 Ochromonadidae (p. 179) MASTIGOPHORA, CHRYSOMONADINA 175 With calcareous or silicious shell Bearing calcareous discs and rods. .Family 4 Coccolithidae (p. 181) Bearing silicious skeleton Family 5 Silicoflagellidae (p. 181) Family 1 Chromulinidae Engler Minute forms, naked or with sculptured shell; with a single flagellum; often with rhizopodia; a few colonial; free-swimming or attached. Genus Chromulina Cienkowski. Oval; round in cross-section; amoeboid; 1-2 chromatophores; palmella stage often large; in fresh water. Numerous species. The presence of a large number of these organisms gives a golden-brown color to the surface of the water. C. pascheri Hofeneder (Fig. 85, a, h). 15-20/i in diameter. Genus Chrysapsis Pascher. Solitary; plastic or rigid; chromato- phore diffused or branching; with stigma; amoeboid movement; holophytic, holozoic; fresh water. C. sagene P. (Fig. 85, c). Anterior region actively plastic; stigma small; 8-14/i long; flagellum about 30/u long. Genus Chrysococcus Klebs. Shell spheroidal or ovoidal, smooth or sculptured and often brown-colored; through an opening a flagellum protrudes; 1-2 chromatophores; one of the daughter individuals formed by binary fission leaves the parent shell and forms a new one; fresh water. C. ornatus Pascher (Fig. 85, d). 14-16m by T-lO/x. Genus Mallomonas Perty {Pseudomallomonas Chodat). Body elongated; with silicious scales and often spines; 2 chromato- phores, rod-shaped; fresh water. Numerous species. M. litomosa Stokes (Fig. 85, e). Scales very dehcate, needle-like projections at both ends; flagellum as long as body; 24-3 2/i by 8m. Genus Pyramidochrysis Pascher. Body form constant; pyri- form with 3 longitudinal ridges; flagellate end drawn out; a single chromatophore; 2 contractile vacuoles; fresh water. P. modesta P. (Fig. 85,/). 11-13/x long. Genus Sphaleromantis Pascher. Triangular or heart-shaped; highly flattened; shghtly plastic; 2 chromatophores; 2 contractile vacuoles; stigma large; long flagellum; fresh water. S. ochracea P. (Fig. 85, g). 6-13/x long. Genus Kephyrion Pascher. With oval or fusiform lorica; body fills posterior half of lorica; one chromatophore; a single short flagellum; small; fresh water. 176 PROTOZOOLOGY Fig. 85. a, b, Chronmlina pascheri, X670 (Hofeneder) ; c, Chrysapsis sagene, XlOOO (Pascher); d, Chrysococcus ornatus, X600 (Pascher); e, Mallomonas litomosa, X400 (Stokes); f, Pyramidochrysis modesta, X670 (Pascher); g, Sphaleromantis ochracea, X600 (Pascher); h, Kephyrion ovum, X1600 (Pascher); i, Chrysopyxis cyathus, X600 (Pascher); j, Cyrtophora pedicellata, X400 (Pascher); k, Palatinella cyrtophora, X400 (Lauterborn); 1, Chrysosphaerella longispina, X600 (Lauterborn). MASTIGOPHORA, CHRYSOMONADINA 177 K. ovum P. (Fig. 85, h). Lorica up to 7n by 4/x. Genus Chrysopyxis Stein. With lorica of various forms, more or less flattened; 1-2 chromatophores; a flagellum; attached to algae in fresh water. C. cyathus Pascher (Fig. 85, i). One chromatophore; flagellum twice body length; lorica 20-25^ by 12-15/^. Genus Cyrtophora Pascher. Body inverted pyramid with 6-8 tentacles and a single flagellum; with a contractile stalk; a single chromatophore; a contractile vacuole; fresh water. C. pedicellata P. (Fig. 85, j). Body 18-22^ long; tentacles 40- 60)u long; stalk 50-80^ long. Genus Palatinella Lauterborn. Lorica tubular; body heart- shaped; anterior border with 16-20 tentacles; a single flagellum; a chromatophore; several contractile vacuoles; fresh water. P. cyrtopJiora L. (Fig. 85, k). Lorica 80-1 50m long; body 20-25iu by 18-25m; tentacles 50/x long. Genus Chrysosphaerella Lauterborn. In spherical colony, in- dividual cell, oval or pyriform, with 2 chromatophores; imbedded in gelatinous mass; fresh water. C. longispina L. (Fig. 85, I). Individuals up to 15/x by 9/x; colony up to 250/x in diameter; in standing water rich in vegeta- tion. Family 2 Syncryptidae Poche Solitary or colonial chrysomonads with 2 equal fiagella; with or without pellicle (when present, often sculptured) ; some possess stalk. Genus Syncrypta Ehrenberg. Spherical colonies; individuals with 2 lateral chromatophores, embedded in a gelatinous mass; 2 contractile vacuoles; without stigma; cysts unknown; fresh water. S. volvox E. (Fig. 86, a). 8-Ufx by 7-12ai; colony 20-70m in diameter; in standing water. Genus Synura Ehrenberg. Spherical or ellipsoidal colony com- posed of 2-50 ovoid individuals arranged radially; body usually covered by short bristles; 2 chromatophores lateral; no stigma; asexual reproduction of individuals is by longitudinal division; that of colony by bipartition; cysts spherical; fresh water. >S. uvella E. (Fig. 86, b). Cells oval; bristles conspicuous; 20- 40m by 8-1 7m; colony 100-400m in diameter; if present in large numbers, the organism is said to be responsible for an odor of the water resembhng that of ripe cucumber (Moore). 178 PKOTOZOOLOGY S. adamsi Smith (Fig. 86, c). Spherical colony with individuals radiating; individuals long spindle, 42-4 7)u by 6.5-7/x; 2 flagella up to 17ju long; in fresh water pond. Genus Hymenomonas Stein. Solitary; ellipsoid to cylindrical; membrane brownish, often sculptured; 2 chromatophores ; with- out stigma; a contractile vacuole anterior; fresh water. H. roseola S. (Fig. 86, d). 17-50^ by 10-20^. Genus Derepyxis Stokes. With cellulose lorica, with or without short stalk; body ellipsoid to spherical with 1-2 chromatophores; 2 equal flagella; fresh water. Fig. 86. a, Syncrypta volvox, X430 (Stein); b, Synura uvella, X500 (Stein); c, *S. adamsi, X280 (Smith); d, Hymenomonas roseola, X400 (Klebs); e, Derepyxis amphora, X540 (Stokes); f, D. ollula, X600 (Stokes); g, Stylochrysallis parasitica, X430 (Stein). D. amphora S. (Fig. 86, e). Lorica 25-30^ by 9-18/x; on algae in standing water. D. ollula S. (Fig. 86,/). Lorica 20-25^ by 15^. Genus Stylochrysallis Stein. Body fusiform; with a gelatinous stalk attached to Volvocidae; 2 equal flagella; 2 chromatophores; without stigma; fresh water. S. parasita S. (Fig. 86, g). Body 9-1 l/x long; stalk about 15//. long; on phytomonads. MASTIGOPHORA, CHRYSOMONADINA 179 Family 3 Ochromonadidae Pascher With 2 unequal flagella; body has no pellicle and is plastic; contractile vacuoles simple; with or without dehcate test; solitary or colonial; free-swimming or attached. Genus Ochromonas Wyssotzki. Solitary or colonial; body surface delicate; posterior end often drawn out for attachment; 1-2 chromatophores; usually with a stigma; encystment; fresh water. 0. mutdbilis Klebs (Fig. 87, a). Ovoid to spherical; plastic; 15-30m by 8-22/x. 0. ludihunda Pascher (Fig. 87, 6). Not plastic; 12-17ai by 6-1 2m. Genus Uroglena Ehrenberg. Spherical or ovoidal colony, com- posed of ovoid or ellipsoidal individuals arranged on periphery of a gelatinous mass; all individuals connected with one another by gelatinous processes running inward and meeting in a point; with a stigma and a plate-like chromatophore; asexual reproduc- tion of individuals by longitudinal fission, that of colony by bipartition; cysts spherical with spinous projections, and a long tubular process; fresh water. One species. U. volvox E. (Fig. 87, c). Cells 12-20^ by S-IBm; colony 40- 400/z in diameter; in standing water. Genus Uroglenopsis Lemmermann. Similar to Uroglena, but individuals without inner connecting processes. U. americana (Calkins) (Fig. 87, d). Each cell with one chro- matophore; 5-8jU long; flagellum up to 32/i long; colony up to 300/i in diameter; when present in abundance, the organism gives an offensive odor to the water (Calkins). U'. europaea Pascher. Similar to the last-named species; but chromatophores 2; cells up to 7^ long; colony 150-300/^ in diameter. Genus Cyclonexis Stokes. Wheel-like colony, composed of 10- 20 wedge-shaped individuals; young colony funnel-shaped; chro- matophores 2, lateral; no stigma; reproduction and encystment unknown; fresh water. C. annularis S. (Fig. 87, e). Cells ll-14;u long; colony 25-30/i in diameter; in marshy water with sphagnum. Genus Dinobryon Ehrenberg. Sohtary or colonial; individuals with vase-like, hyahne, but sometimes, yellowish cellulose test, drawn out at its base; elongated and attached to the base of test 180 PROTOZOOLOGY with its attenuated posterior ti]); 1-2 lateral chroniatophores; usually with a stigma; asexual r(>production by binary fission; one of the daughter individuals leaving test as a swarmer, to form a new one; in colonial forms daughter individuals remain attached Fig. 87. a, Ochromonas mutabilis, X670 (Senn); b, 0. ludibunda, X540 (Pascher); c, Uroglena volvox, x430 (Stein); d, Uroglenopsis americana, X470 (Lemmermann) ; e, Cyclonexis annularis, X540 (Stokes); f, ■Dinohryon sertularia, X670 (Scherffel) ; g, Hyalobryon ramosu7n, X540 (Lauterborn); h, Stylopyxis mucicola, X470 (Bol- ochonzew). to the inner margin of aperture of parent tests and there secrete new tests; encystment common; the spherical cysts possess a short process; Ahlstrom (1937) studied variability of North American species and found the organisms occur more com- monly in alkaline regions than elsewhere; fresh water. Numerous species. MASTIGOPHORA, CHRYSOMONADINA 181 D. sertularia E. (Fig. 87,/). 30-44^ by 10-14^. D. divergens Imhof. 31-53^ long; great variation in different localities (Ahlstrom). Genus Hyalobryon Laiiterborn. Solitary or colonial; individual body structure similar to that of Dinobryon; lorica in some cases tubular, and those of young individuals are attached to the ex- terior of parent tests; fresh water. H. ramosum L. (Fig. 87, g). Lorica 50-70/i long by 5-9/x in diameter; body up to 30^ by 5m; on vegetation in standing fresh water. Genus Stylopyxis Bolochonzew. Solitary; body located at bot- tom of a delicate stalked lorica with a wide aperture; 2 lateral chromatoi)hores; fresh water. S. mucicola B. (Fig. 87, h). Lorica 17-18/x long; stalk about 33m long; body 9-11m long; fresh water. Family 4 Coccolithidae Lohmann The members of this family, with a few exceptions, occur in salt water only; with perforate (tremahth) or imperforate (discohth) discs, composed of calcium carbonate; 1-2 flagella; 2 yellowish chromatophores; a single nucleus; oil drops and leuco- sin ; holophytic. Examples : Pontos'phaera haeckeli Lohmann (Fig. 88, a). Discosphaera tuhijer Murray et Blackman (Fig. 88, 6). Family 5 Silicoflagellidae Borgert Exclusively marine planktons; with siliceous skeleton which envelops the body. Example: Distephanus speculum (Mliller) (Fig. 88, c). Suborder 2 Rhizochrysidina Pascher No flagellate stage is knoMai to occur; the organism possesses pseudopodia; highly provisional group, based wholly upon the absence of flagella; naked or with test; various forms; in some species chromatophores are entirely lacking, so that the organisms resemble some members of the Sarcodina. Several genera. Genus Rhizochrysis Pascher. Body naked and amoeboid; with 1-2 chromatophores; fresh water. R. scherffeli P. (Fig. 88, d). 10-14^ in diameter; 1-2 chromato- phores; branching rhizopods; fresh water. Genus Chrysidiastrum Lauterborn. Naked; spherical; often 182 PROTOZOOLOGY Fig. 88. a, Pontosphaera haeckeli, X1070 (Kiihn); b, Discosphaera tubifer, X670 (Kiihn); c, Distephanus speculum, X530 (Kiihn); d, Rhizochrysis scherffeli, X670 (Doflein); e-g, Hydrurus foetidus (e, en- tire colony; f, portion; g, cyst), e (Bertholcl), f, X330, g, X800 (Klebs); h, i, Chrysocapsa paludosa, X530 (West); j, k, Phaeosphaera gelatinosa (j, part of a mass, X70; k, three cells, x330) (West). several in linear association by pseudopodia; one yellow-brown chromatophore; fresh water. C. catenatum L. Cells 12-14yu in diameter. Suborder 3 Chrysocapsina Pascher Palmella stage prominent; flagellate forms transient; colonial; individuals enclosed in a gelatinous mass; 1-2 flagella, one chro- matophore, and a contractile vacuole; one group of relatively minute forms and the other of large organisms. Genus Hydrurus Agardh. In a large (1-30 cm. long) branching gelatinous cylindrical mass; cells yellowish brown; spherical to eUipsoidal; with a chromatophore; individuals arranged loosely MASTIGOPHORA, CHRYSOMONADINA 183 in gelatinous matrix; apical growth resembles much higher algae; multiphcation of individuals results in formation of pyramidal forms with a flagellum, a chromatophore, and a leucosin mass; cyst may show a wing-like rim; cold freshwater streams. H. foetidus Kirschner (Figs. 31, d-f; 88, e-g). OHve-green, feathery tufts, 1-30 cm. long, develops an offensive odor; sticky to touch; occasionally encrusted with calcium carbonate; in running fresh water. Genus Chrysocapsa Pascher. In a spherical to ellipsoidal gelatinous mass; cells spherical to ellipsoid; 1-2 chromatophores; with or without stigma; freshwater. C. paludosa P. (Fig. 88, h, i). Spherical or elhpsoidal wdth cells distributed without order; \\\i\i a stigma; 2 chromatophores; swarmer pyriform with 2 flagella; cells l\^x long; colony up to 100/i in diameter. Genus Phaeosphaera West et West. In a simple or branching cylindrical gelatinous mass; cells spherical with a single chroma- tophore; fresh water. P. gelatinosa W. et W. (Fig. 88, j, k). Cells 14-17.5)u in diameter. References BtJTSCHLi, 0. 1883-1887 Mastigophora. In: 'Bromi' s Klassen und Ordnungen des Thierreichs. Vol. 1, part 2. Calkins, G. N. 1926 The biology of the Protozoa. Philadelphia. DoFLEiN, F. and E. Reichenow. 1929 Lehrbuch der Protozoen- kunde. Jena. Kent, S. 1880-1882 A Manual of Infusoria. London. Pascher, A. 1914 Flagellatae; Allgemeiner Teil. In: Die Silss- wasserfiora Deutschlands. Part 1, Stein, F. 1878, 1883 Der Organismus der Infusionsthiere. 3 Abt. Der Organismus der Flagellate oder Geisselinfusorien. Parts 1 and 2. Leipzig. Ahlstrom, E. H. 1937 Studies on variabiUty in the genus Dino- bryon (Mastigophora). Trans. Amer. Micr. Soc, Vol. 56. Fritsch, F. E. 1935 The structure and re-production of the algae. Cambridge. Pascher, A. 1913 Chrysomonadinae. In: Die Siisswasserflora Deutschlands. Part 2. Smith, G. M. 1933 The freshwater algae 'of the United States. New York. West, G. S. and F. E. Fritsch. 1927 A treatise on the British freshwater algae. Cambridge. Chapter 8 Order 2 Cryptomonadina Stein THE cryptomonads differ from the chrysomonads in having a constant body form. Pseudopodia are very rarely formed, as the body is covered by a pelhcle. The majority show dorso- ventral differentiation, with an oblique longitudinal furrow. 1-2 unequal flagella arise from the furrow or from the cytopharynx. In case 2 flagella are present, both may be directed anteriorly or one posteriorly. These organisms are free-swimming or creeping. 1-2 chromatophores are usually present. They are discoid or band-form. The color of chromatophores vary from common brown, red, olive-green up to blue-green. The nature of the pig- ment is not well understood, but it is said to be similar to that which is found in the Dinofiagellata (Pascher). One or more spherical pyrenoids which are enclosed within a starch envelope appear to occur outside the chromatophores. Nutrition is mostly holophytic; a few saprozoic or holozoic. Assimilation products are solid discoid carbohydrates which stain blue with iodine in Cryptomonas or which stain reddish violet by iodine as in Crypto- chrysis; fat and starch are produced in holozoic forms which feed upon bacteria and small Protozoa. The stigma is usually as- sociated with the insertion point of the flagella. Contractile vacuoles, one to several, are simple and are situated near the cytopharynx. A single vesicular nucleus is ordinarily located near the middle of the body. Asexual reproduction, by longitudinal fission, takes place in either the active or the non-motile stage. Sexual reproduction is unknown. Some cryptomonads form palmella stage and others gelatinous aggregates. In the suborder Phaeocapsina, the pal- mella stage is permanent. Cysts are spherical, and the cyst wall is composed of cellulose. The Cryptomonadina occur in fresh or sea water, living also often as symbionts in marine organisms. Flagellate forms predominant Suborder 1 Eucryptomonadina (p. 185) Palmella stage permanent Suborder 2 Phaeocapsina (p. 187) 184 CRYPTOMONADINA 185 Suborder 1 Eucryptomonadina Pascher Truncate anteriorly; 2 anterior flagella; with an oblique furrow near anterior end Familj^ 1 Cryptomonadidae Reniform; with 2 lateral flagella; furrow equatorial Family 2 Nephroselmidae (p. 186) Family 1 Cryptomonadidae Stein Genus Cryptomonas Ehrenberg. Body elliptical with a firm pellicle; anterior end truncate; dorsal side convex, ventral side slightly so or flat; nucleus posterior; longitudinal furrow; tubular cavity extending to the middle of body, through which equally long flagella arise; 2 lateral chromatophores vary in color from green to blue-green, brown or rarely red; holophytic; with small starch-like bodies which stain blue in iodine; 1-3 contractile vacuoles anterior; fresh water. Several species. C. ovata E. (Fig. 89, a). 20-30/i long; among vegetation. Genus Chilomonas Ehrenberg. Similar to Cryptomonas in general body form and structure, but colorless because of the absence of chromatophores; without pyrenoid; cytopharynx deep, lower half marked by "rudimentary trichocysts" ; 1-2 contractile vacuoles, anterior; nucleus in posterior half; endoplasm often filled with polygonal starch grains; fresh water. C. Paramecium E. (Fig. 89, h). Posterior end narrowed, slightly bent "dorsally"; 20-40^1 long; saprozoic; widely distributed in stagnant water and hay infusion. C. ohlonga Pascher. Oblong; posterior end broadly rounded; 20-50m long. Genus Chrysidella Pascher. Somewhat similar to Cryptomonas^ but much smaller; yellow chromatophores much shorter; those occurring in Foraminifera or Radiolaria as symbionts are known as Zooxanthellae. Several species. C. schaudinni (Winter) (Fig. 89, c, d). Body less than lO/i long; in the foraminiferan Peneroplis pertusus. Genus Cyathomonas Fromentel. Body small, somewhat oval; without chromatophores; much flattened; anterior end obliquely truncate; with 2 equal or subequal anterior flagella; colorless; nucleus central; anabolic products, stained red or reddish violet by iodine; contractile vacuole usually anterior; a row of refractile granules, protrichocysts (p. 65), close and parallel to anterior margin of body; asexual reproduction by longitudinal fission; holozoic; in stagnant water and infusion. One species. 186 PROTOZOOLOGY C. tnmcata Ehreiiborg (Fig. 89, e). 15-30/^ long. Genus Cryptochrysis Pascher. Furrow indistinctly granulated; 2 or more chromatoi)hores brownish, olive-green, or dark green, rarely red; pyrenoid central; 2 equal flagella; some lose flagella and may assume amoeboid form; fresh water. C. commutata P. (Fig. 89,/). Bean-shaped; 2 chromatophores; 19m by 10/1. Fig. 89. a, Cryptomonas ovata, XSOO (Pascher); b, Chilomonas Para- mecium., X1330 (Btitschli); c, d, Chrysidella schaudinni, X1330 (Win- ter); e, Cyathomonas truncata, X670 (Ulehla); f, Cryptochrysis com- ynutata, X670 (Pascher); g, RJiodomonas lens, X1330 (Ruttner); h, Nephroselmis olvacea, X670 (Pascher) ;i, Protochrysis phaeophycearum, XSOO (Pascher); j, k, Phaeothamnion confervicolum, X600 (Klihn). Genus Rhodomonas Karsten. Furrow granulated; chromato- phore one, red (upon degeneration the coloring matter becomes dissolved in water) ; pyrenoid central ; fresh water. R. lens Pascher et Ruttner (Fig. 89, g). Spindle-form; about IQn long; in fresh water. Family 2 Nephroselmidae Pascher Body reniform; with lateral equatorial furrow; 2 flagella arising from furrow, one directed anteriorly and the other posteriorly. Genus Nephroselmis Stein. Reniform; flattened; furrow and cytopharynx distinct; no stigma; 1-2 chromatophores, discoid. CRYPTOMONADINA 187 brownish green; nucleus dorsal; a central pyrenoid; 2 contractile vacuoles; with reddish globules; fresh water. N. olvacea S. (Fig. 89, h). 20-25// by 15^. Genus Protochrysis Pascher. Reniform; not flattened; with a distinct furrow, but without cytopharynx; a stigma at base of flagella; 1-2 chromatophores, brownish yellow; pyrenoid central; 2 contractile vacuoles; fission seems to take place during the rest- ing stage; fresh water. P. phaeophycearum P. (Fig. 89, i). 15-17/1 by 7-9iu. Suborder 2 Phaeocapsina Pascher Palmella stage predominant; perhaps border-line forms be- tween brown algae and cryptomonads. Example: Phaeothamnion confervicolum Lagerheim (Fig. 89, j, k) which is less than lOfx long. References Fritsch, F. E. 1935 The structure and reproduction of the algae. Cambridge. Pascher, A. 1913 Cryptomonadinae. Siisswasserflora Deutsch- lands, etc. part 2. Jena. West, G. S. and F. E. Fritsch. 1927 A treatise on the British freshwater algae. Cambridge. Chapter 9 Order 3 Phytomonadina Blochmann THE phytomonads are small, more or less rounded, green flagellates with a close resemblance to the algae. They show a definite body form, and most of them possess a cellulose mem- brane, which is thick in some and thin in others. There is a defi- nite opening in the membrane at the anterior end, through which 1-2 (or seldom 4 or more) flagella protrude. The majority possess numerous grass-green chromatophores, each of which contains one or more pyrenoids. The method of nutrition is mostly holo- phytic or mixotrophic; some colorless forms are, however, sapro- zoic. The metabolic products are usually starch and oils. Some phytomonads are stained red, owing to the presence of haemato- chrome. The contractile vacuoles may be located in the anterior part or scattered throughout the body. The nucleus is ordinarily centrally located, and its division seems to be mitotic, chromo- somes having been definitely noted in several species. Asexual reproduction is by longitudinal fission, and the daughter individuals remain within the parent membrane for some time. Sexual reproduction seems to occur widely. Colony formation also occurs, especially in the family Volvocidae. Encystment and formation of the palmella stage are common among many forms. The phytomonads have a much wider distribution in fresh than in salt water. Solitary Membrane a single piece; rarely indistinct 2 flagella Family 1 Chlamydomonadidae 3 flagella Family 2 Trichlorididae (p. 193) 4 flagella Family 3 Carteriidae (p. 194) 5 flagella Family 4 Chlorasteridae (p. 196) 6 or more flagella Family 5 Polyblepharididae (p. 196) Membrane bivalve Family 6 Phacotidae (p. 196) Colonial, of 4 or more individuals; 2 (1 or 4) flagella Family 7 Volvocidae (p. 197) Family 1 Chlamydomonadidae Biitschli Solitary; spheroid, oval, or ellipsoid; with a cellulose mem- brane; 2 flagella; chromatophores, stigma, and pyrenoids usually present. 188 PHYTOMONADINA 189 Genus Chlamydomonas Ehrenberg. Spherical, ovoid or elon- gated; sometimes flattened; 2 flagella; membrane often thickened at anterior end; a large chromatophore, containing one or more pyrenoids; stigma; a single nucleus; 2 contractile vacuoles an- terior; asexual reproduction and palmella formation known; sexual reproduction isogamy or anisogamy; fresh water. Numer- ous species. C. monadina Stein (Fig. 90, a-c). 15-30/i long; fresh water; Landacre noted that the organisms obstructed the sand filters used in connection with a septic tank, together with the diatom Navicula. C. angulosa Dill. About 20^ by 12-15/i; fresh water. C. epiphytica Smith (Fig. 90, d). 8-9 fj, by I-Sjjl; in freshwater lakes. C. glohosa Snow (Fig. 90, e). Spheroid or ellipsoid; 5-7/^ in diameter; in freshwater lakes. C. gracilis Snow (Fig. 90,/). 10-13^ by 5-7m; fresh water. Genus Haematococcus Agardh {Sphaerella Sommerfeldt). Spheroidal or ovoid with a gelatinous envelope; chromatophores peripheral and reticulate, with 2-8 scattered pyrenoids; several contractile vacuoles; haematochrome frequently abundant in both motile and encysted stages; asexual reproduction in motile form; sexual reproduction isogamy; fresh water. H. pluvialis (Flotow) (Figs. 38; 90, g). Oval or elhpsoid; wdth numerous radial cytoplasmic processes; chromatophores thick- walled; body up to 60ju by SO^u; stigma about 13yu long; fresh water; according to Reichenow (1909), the haematochrome dis- appears under experimental condition if the culture medium is rich in nitrogen and phosphorus. Genus Sphaerellopsis Korschikoff (Chlamydococcus Stein). With gelatinous envelope which is usually ellipsoid with rounded ends; body elongate fusiform or pyriform, no protoplasmic processes to envelope; 2 equally long flagella; chromatophore large; a pyrenoid; with or without stigma; nucleus in anterior half; 2 contractile vacuoles; fresh water. ,S. fluviatilis (Stein) (Fig. 90, h). 14-30^ by 10-20^; fresh water. Genus Brachiomonas Bohlin. Lobate; with horn-like processes, all directed posteriorly; contractile vacuoles; ill-defined chro- matophore; pyrenoids; with or without stigma; sexual and asexual reproduction; fresh, brackish or salt water. 190 PROTOZOOLOGY Fig. 90. a-c, Chlamijdomonas monadina, X470 (Goroschankin); d, C. epiphytica, X1030 (Smith); e, C. globosa, X2000 (Snow); f, C. gracilis, X770 (Snow); g, Haemalococcus pluvialis, X500 (Reichenow); h. Sphaerellopsis fluviatilis, X490 (Korschikoff) ; i, Brachivionas ivedi- ana X960 (West); j, Lohomonas rostrata, X1335 (Hazen); k, Diplo- stauron pentagonium, XlllO (Hazen); 1, Gigantochloris permaxima, X370 (Pascher); m, Gloeomonas ovalis, X330 (Pascher); n, Scourjieldia complanata, X1540 (West); o, Thorakomonas sabulosa, X670 (Kor- schikoff). B. westiana Pascher (Fig. 90, i). 15-24/i by 13-23^; brackish water. Genus Lobomonas Dangeard. Ovoid or irregularly angular; PHYTOMONADINA 191 chromatophore cup-shaped; pyrenoid; stigma; a contractile vacuole; fresh water. L. rostrata Hazen (Fig. 90, j). 5-12^1 by 4-8/x. Genus Diplostauron Korschikoff. Rectangular with raised corners; 2 equally long flagella; chromatophore; one pyrenoid; stigma; 2 contractile vacuoles anterior; fresh water. D. pentagonium (Hazen) (Fig. 90, k). 10-13/z by 9-10;u. Genus Gigantochloris Pascher. Unusually large form, equalling in size a colony of Eudorina; flattened; oval in front view; elongate ellipsoid in profile; membrane radially striated; 2 flagella widely apart, less than body length; chromatophore in network; numerous pyrenoids; often without stigma; in wood- land pools. G. permaxima P. (Fig. 90, 0- 70-150^ by 40-80^ by 25-50^. Genus Gloeomonas Klebs. Broadly ovoid, nearly subspherical; with a delicate membrane and a thin gelatinous envelope; 2 flagella widely apart; chromatophores numerous, circular or oval discs; pyrenoid (?); stigma; 2 contractile vacuoles, anterior; fresh water. G. ovalis K. (Fig. 90, m). 38-42iU by 23-33/x; gelatinous envelope over 2yu thick. Genus Scourfieldia West. Whole body flattened; ovoid in front view; membrane delicate; 2 flagella 2-5 times body length; a chromatophore; wdthout pyrenoid or stigma; contractile vacuole anterior; nucleus central; fresh water. S. complanata W. (Fig. 90, n). 5.2-5.7 /j. by 4.4-4. 6^; fresh water. Genus Thorakomonas Korschikoff. Flattened; somewhat ir- regularly shaped or ellipsoid in front view; membrane thick, enclustered with iron-bearing material, deep brown to black in color; protoplasmic body similar to that of Chlamydomonas; a chromatophore with a pyrenoid; 2 contractile vacuoles; standing fresh water. T. sahulosa K. (Fig. 90, o). Up to 16^ by 14/x. Genus Coccomonas Stein. Shell smooth; globular; body not filling intracapsular space; stigma; contractile vacuole; asexual reproduction into 4 individuals; fresh water. C. orbicularis S. (Fig. 91, a). 18-25/^ in diameter; fresh water. Genus Chlorogonium Ehrenberg. Fusiform; membrane thin and adheres closely to protoplasmic body; plate-like chromato- phores usually present, sometimes ill-contoured; one or more 192 PROTOZOOLOGY Fig. 91. a, Coccomonas orbicularis, x500 (Stein); b, Chlorogonium euchlorum, X430 (Jacobsen); c, Phyllomonas phacoides, X200 (Kor- schikoff); d, Sphaenochloris printzi, X600 (Printz); e, Korschikoffia guttula, X1670 (Pascher); f, Furcillalobosa, X670 (Stokes); g, Hyalo- gonium klebsi, X470 (Klebs); h, Polytoma iwella, X670 (Dangeard); i, Parapolytorna satura, Xl600 (Jameson); j, Trichloris paradoxa, X990 (Pascher). pyrenoids; numerous scattered contractile vacuoles; usually a stigma; a central nucleus; asexual reproduction by 2 successive transverse fissions during motile phase; isogamy reported; fresh water. C. euchlorum E. (Fig. 91, 6). 25-70yu by 4-15^1; in stagnant water. Genus Phyllomonas Korschikoff. Extremely flattened; mem- brane delicate; 2 flagella; chromatophore often faded or indistinct; numerous pyrenoids; with or without stigma; many contractile vacuoles; fresh water. P. phacoides K. (Fig. 91, c). Leaf-like; rotation movement; up to 100/x long; in standing fresh water. Genus Sphenochloris Pascher. Body truncate or concave at flagellate end in front view; sharply pointed in profile; 2 flagella widely apart; chromatophore large; pyrenoid; stigma; contractile vacuole anterior; fresh water. S. printzi P. (Fig. 91, d). Up to 18m by 9/^. PHYTOMONADINA 193 Genus Korschikoffia Pascher. Elongate pyriform with an undulating outline; anterior end narrow, posterior end more bluntly rounded; plastic; chromatophores in posterior half; stigma absent; contractile vacuole anterior; 2 equally long flagella; nucleus nearly central; salt water. K. guttula P. (Fig. 91, e). 6-10^ by 5^; brackish water. Genus Furcilla Stokes. U-shape, with 2 posterior processes; in side view somewhat flattened; anterior end with a papilla; 2 flagella equally long; 1-2 contractile vacuoles anterior; oil droplets; fresh water. F. lohosa S. (Fig. 91,/). ll-14/i long; fresh water. Genus Hyalogonium Pascher. Elongate spindle-form; anterior end bluntly rounded; posterior end more pointed; 2 flagella; protoplasm colorless; with starch granules; a stigma; asexual reproduction results in up to 8 daughter cells; fresh water. H. klebsi P. (Fig. 91, g). 30-80^ by up to lOfx; stagnant water. Genus Polytoma Ehrenberg (Chlamydohlepharis France; Tus- setia Pascher). Ovoid; no chromatophores; membrane yellowish to brown; pyrenoid unobserved; 2 contractile vacuoles; 2 flagella about body length; stigma if present, red or pale-colored; many starch bodies and oil droplets in posterior half of body; asexual reproduction in motile stage; isogamy; saprozoic; in stagnant fresh water. P. uvella E. (Figs. 8, e; 91, h). Oval to pyriform; stigma may be absent; 15-30^ by 9-20m. Genus Parapolytoma Jameson. Anterior margin obliquely truncate, resembling a cryj^tomonad, but without chromato- phores; without stigma and starch; division into 4 individuals within envelope; fresh water. P. satura J. (Fig. 91, i). About 15/i by lO/z; fresh water. Family 2 Trichlorididae With three flagella. Genus Trichloris Scherffel et Pascher. Bean-shape; flagellate side flattened or concave; opposite side convex; chromatophore large, covering convex side; 2 pyrenoids surrounded by starch granules; a stigma near posterior end of chromatophore; nucleus central; numerous contractile vacuoles scattered; 3 flagella near anterior end. T. paradoxa S. et P. (Fig. 91, j). 12-15/^ broad by 10-12^ high; flagella up to 30/^ long. 194 PROTOZOOLOGY Family 3 Carteriidae With four flagella arising from anterior pole. Genus Carteria Dicsing (Corhierea, Pithiscus, Dangeard; Teiramastix Korschikoff). Ovoid, chromatophore cup-shaped; pyrenoid; stigma; 2 contractile vacuoles; fresh water. Numerous species. C. cordiformis (Carter) (Fig. 92, a). Heart-shaped in front view; ovoid in profile; chromatophore large; 18-23^1 by 16-20;u. C. elUpsoidalis Bold. Ellipsoid; chromatophore; a small stigma; division into 2, 4, or 8 individuals in encysted stage; 6-24ju long; fresh water, Maryland (Bold, 1938). Genus Pyramimonas Schmarda (Pyramidomonas Stein). Small pyramidal or heart-shaped body; with bluntly drawn-out posterior end; usually 4 ridges in anterior region; 4 flagella; green chromatophores cup-shaped; with or without stigma; with a large pyrenoid in the posterior part; contractile vacuoles in the anterior portion; fresh water. Several species. P. tetrarhnnchus S. (Fig. 92, h). 20-28/^ by 12-18yu; fresh water; Wisconsin (Smith, 1933). P. montana Geitler. Bluntly conical; anterior end 4-lobed or truncate; posterior end narrowly rounded; plastic; pyriform nu- cleus anterior, closely associated with 4 flagella; stigma; 2 con- tractile vacuoles anterior; chromatophore cup-shaped, granular, with scattered starch grains and oil droplets; a pyrenoid with a ring of small starch grains; 17-22.5^ long (Geitler); 12-20/i by 8-16^1 (Bold), flagella about body length; fresh water, Maryland (Bold, 1938). Genus Polytomella Aragao. ElHpsoid, or oval, with a small papilla at anterior end, where 4 equally long flagella arise; with stigma; starch; fresh water. P. agilis A. (Fig. 92, c, d). Numerous starch grains; 8-18/1 by 5-9/i; flagella 12-17/* long; fresh water; hay infusion. Genus Medusochloris Pascher. Hollowed hemisphere with 4 processes, each bearing a flagellum at its lower edge; a lobed plate-shaped chromatophore; without pyrenoid below convex surface. One species. M. phiale P. In salt water pools with decaying algae in the Baltic. Genus Spirogonium Pascher. Body spindle-form; membrane delicate; flagella a little longer than body; chromatophore con- PHYTOMONADINA 195 Fig. 92. a, Carteria cordiformis, X600 (Dill); b, Pyramimonas tetra- rhynchus, X400 (Dill); c, d, Polytomella agilis, XlOOO (Doflein); e, Spirogonium chlorogonioides, X670 (Pascher); f, Tetrablepharis multifile, X670 (Pascher); g, Spermatozopsis exultans, X1630 (Pascher) ; h, Chloraster gyrans, X670 (Stein) ; i, Polyblepharides singu- laris, XS70 (Dangeard); j, k, Pocillomonas flos aquae, X920 (Stein- ecke);l, m, Phacotus lenticularis, X430 (Stein); n, Pteromonas angu- losa, X670 (West); o, p, Dysmorphococcus variabilis, XlOOO (Bold). spicuous; a pyrenoid; stigma anterior; 2 contractile vacuoles; fresh water. One species. *S. chlorogonioides (P.) (Fig. 92, e). Body up to 25^1 by 15ju. Genus Tetrablepharis Senn. Ellipsoid to ovoid; pyrenoid pres- ent; other characters are those of Polytoma; fresh water. T. multifilis (Klebs) (Fig. 92, /). 12-20^ by 8-1 5At; stagnant water. Genus Spermatozopsis Korschikoff. Sickle-form; bent easily, occasionally plastic; chromatophore mostly on convex side; a distinct stigma at more rounded anterior end; flagella equally long; 2 contractile vacuoles anterior; fresh water infusion. S. exultans K. (Fig. 92, g). 7-9/^ long; also biflagellate; in fresh water with algae, leaves, etc. 196 PROTOZOOLOGY Family 4 Chlorasteridae With 5 flajj;('lla arising from anterior pole. Genus Chloraster Ehrenbcrg. Similar to Pyramvnonas, but anterior half with a conical envelope drawn out at four corners; with 5 flagella; fresh or salt water. C. gyrans E. (Fig. 92, h). Up to 18At long; standing water; also reported from salt water. Family 5 Polyblepharididae Dangeard With 6 or more flagella arising from anterior end. Genus Polyblepharides Dangeard. Ellipsoid or ovoid; flagella 6-8, shorter than body length; chromatophore; a pyrenoid; a central nucleus; 2 contractile vacuoles anterior; cysts; a question- able genus ; fresh water. P. singularis D. (Fig. 92, i). 10-14^ by 8-9^. Genus Pocillomonas Steinecke. Ovoid with broadly concave anterior end; covered wdth gelatinous substance with numerous small projections; 6 flagella; chromatophores disc-shaped; 2 contractile vacuoles anterior; nucleus central; starch bodies; without pyrenoid. P. flos aquae S. (Fig. 92, j, k). 13/x by IO/jl; fresh water pools. Family 6 Phacotidae Poche The shell typically composed of 2 valves; 2 flagella protrude from anterior end; with stigma and chromatophores; asexual re- production within the shell; valves may become separated from each other owing to an increase in gelatinous contents. Genus Phacotus Perty. Oval to circular in front view; lentic- ular in profile; protoplasmic body does not fill dark-colored shell completely; flagella protrude through a foramina; asexual repro- duction into 2 to 8 individuals; fresh water. P. lenticularis (Ehrenberg) (Fig. 92, I, m). 13-20^ in diameter; in stagnant water. Genus Pteromonas Sehgo. Body broadly winged in plane of suture of 2 valves; protoplasmic body fills shell; chromatophore cup-shaped; one or more pyrenoids; stigma; 2 contractile vacuoles; asexual reproduction into 2-4 individuals; sexual re- production by isogamy; zygotes usually brown; fresh water. Several species. P. angulosa (Lemmermann) (Fig. 92, n). With a rounded wing PHYTOMONADINA 197 and 4 protoplasmic projections in profile; 13-17^1 by 9-20^1; fresh water. Genus Dysmorphococcus Takeda. Circular in front view; an- terior region narrowed; posterior end broad; shell distinctly flattened posteriorly, ornamented by numerous pores; sutural ridge without pores; 2 fiagella; 2 contractile vacuoles; stigma, pyrenoid, cup-shaped chromatophore; nucleus; multiplication by binary fission; fresh water. D. variahilis Takeda (Fig. 92, o, p). Shell 14-19^ by 13-17/z; older shells dark brown; fresh water; Maryland (Bold, 1938). Family 7 Volvo cidae Ehrenberg An interesting group of colonial flagellates; individual similar to Chlamydomonadidae, with 2 equally long flagella (one in Mastigophaera; 4 in Spondylomorum), green chromatophores, pyrenoids, stigma, and contractile vacuoles; body covered by a cellulose membrane and not plastic; colony or coenobium is dis- coid or spherical; exclusively freshwater inhabitants. Genus Volvox Linnaeus. Often large spherical or subspherical colonies, consisting of a large number of cells which are dif- ferentiated into somatic and reproductive cells; somatic cells numerous, embedded in gelatinous matrix, and contains a chro- matophore, one or more pyrenoids, a stigma and several contrac- tile vacuoles; in some species protoplasmic connections occur between adjacent cells; generative cells few and large. Both mono- and bi-sexual reproduction occurs; monosexual gametes usually fewer and larger in size than bisexual ones, each producing a young colony by repeated division; bisexual reproduction aniso- gamy; zygotes usually brownish red in color, with smooth, un- dulating, or spinous envelopes; fresh water. V. glohator L. (Fig. 93, a). Elhpsoid colony, composed of 5000- 20,000 cells which are 3-5yu high by up to 8/x wide; monoecious; up to SOOyu in diameter; in European waters. V. yerglohaior Powers. Colony up to 1.5 mm. in diameter; cells resemble those of V. glohator; in American waters. V. aureus Ehrenberg (Figs. 71; 93, h). Dioecious; cytoplasmic threads relatively thin and long; cells pyriform, 5-9/^ in diameter; colony 500-800/i in diameter. V. spermatosphaera Powers. Monoecious; cells number 1000- 3000; without any cytoplasmic connections; colony 150-1000/x in diameter. 198 PROTOZOOLOGY Fig. 93. a, Volvox globator, X200 (Janet); b, V. aureus, XllO (Klein); c, Gonium sociale, X270 (Chordat); d, G. pedorale, X670 (Hartmann); e, G. fortnosum, X600 (Pascher). PHYTOMONADINA 199 V. tertius Meyer. Dioecious; without cytoplasmic connections in mature state; individuals about 7-8ai in diameter. Genus Gonium M tiller. 4 or 16 individuals arranged in one plane; cell ovoid or slightly polygonal; with 2 flagella arranged in the plane of coenobium; with or without a gelatinous envelope; protoplasmic connections among individuals occur occasionally; asexual reproduction through simultaneous divisions of com- ponent cells; sexual reproduction isogamy; zygotes reddish; fresh water. G. sociale (Dujardin) (Fig. 93, c). 4 individuals form a discoid colony; cells 10-22/^ by 6-16ai wide; in open waters of ponds and lakes. G. pectorale M. (Fig. 93, d). 16 (rarely 4 or 8) individuals form a colony; 4 cells in center; 12 peripheral, closely arranged; cells 5-14^t by 10;u; colony up to 90/i in diameter; fresh water. G. formosiim Pascher (Fig. 93, e). 16 cells in a colony further apart; peripheral gelatinous envelope reduced; cells similar in size to those of G. sociale but colony somewhat larger: freshwater lakes. Genus Stephanoon Schewiakoff . Spherical or ellipsoidal colony, surrounded by gelatinous envelope, and composed of 8 or 16 biflagellate cells, arranged in 2 alternating rows on equatorial plane; fresh water. S. askenasii S. (Fig. 94, a). 16 individuals in ellipsoidal colony; cells dfx in diameter; flagella up to 30yu long; colony 78^ by 60/i. Genus Platydorina Kofoid. 32 cells arranged in a slightly twisted plane; flagella directed alternately to both sides; fresh water. P. caudata K. (Fig. 94, h). Individual cells lO-lSju long; colony up to 165^1 long, 145yu wide, and 25^ thick; rivers and lakes. Genus Spondylomorum Ehrenberg. 16 cells in a compact group in 4 transverse rings; each with 4 flagella; asexual reproduction by simultaneous division of component cells; fresh water. One species. S. quaternarium E. (Fig. 94, c). Cells 12-26^1 by 8-15At; colony up to 60/i long. Genus Chlamydobotrys Korschikoff. Colony composed of 8 or 16 individuals; cells with 2 flagella; chromatophore; stigma; no pyrenoid; fresh water. C. stellata K. (Fig. 94, d). Colony composed of 8 individuals 200 PROTOZOOLOGY Fig. 94. a, Stephanoon askenasii, X4'40 (Schewiakoff) ; b, Platij- dorina caudata, X280 (Kofoid); c, Sipondylomorum quaternnrium, X330 (Stein); d, Chlamydobotrys stellata, X430 (Korschikoff); e, Ste- phanosphaera pluvialis, x250 (Hieronymus); f, Pandorina morum, XBOO (Smith); g, Mastigosphaera gobii, X520 (Schewiakoff); h, Eu- dorina elegans, X310 (Goebel); i, Pleodorina illinoisensis, X200 (Ko- foid). arranged in 2 rings; individuals 14-15/i long; colony 30-40// in diameter; Maryland (Bold, 1933). Genus Stephanosphaera Cohn. Spherical or subspherical colony, with 8 (rarely 4 or 16) cells arranged in a ring; cells pyri- form, but with several processes; 2 flagella on one face; asexual reproduction and isogamy (p. 146); fresh water. S. pluvialis C. (Figs. 70; 94, e). Cells 7-13yu long; colony 30-60m in diameter. PHYTOMONADINA 201 Genus Pandorina Bory. Spherical or subspherical colony of usually 16 (sometimes 8 or 32) biflagellate individuals, closely packed within a gelatinous, but firm and thick matrix; individuals often angular; with stigma and chromatophores ; asexual repro- duction through simultaneous division of component indi\dduals; anisogamy preceded by division of each cell into 16 to 32 gametes; zygotes colored and covered by a smooth wall; fresh water. One species. P. morum (Mtiller) (Figs. 72; 94,/). Cells 8-17ju long; colony 20-40m, up to 250^1 in diameter; ponds and ditches. Genus Mastigosphaera Schewiakoff . Similar to Pandorina; but individual with a single flagellum which is 3.5 times the body length; fresh water. M. gobii S. (Fig. 94, g). Individual 9m long; colony 30-33m. Genus Eudorina Ehrenberg. Spherical or ellipsoidal colony of usually 32 or sometimes 16 spherical cells; asexual reproduction similar to that of Pandorina; isogamy with 32-64 spherical green macrogametes and numerous clustered microgametes ; reddish zygote with a smooth wall; fresh water. E. elegans E. (Figs. 73; 94, h). Cells 10-24/^ in diameter; colony 40-150ai in diameter; in ponds, ditches and lakes. Genus Pleodorina Shaw. Somewhat similar to Eudorina, being composed of 32, 64, or 128 ovoid or spherical cells of 2 types: small somatic and large generative, and are located within a gelatinous matrix; fresh water. P. illinoisensis Kofoid (Figs. 31, 6, c; 94, i). 32 cells in ellipsoid colony, 4 vegetative and 28 reproductive individuals; arranged in 5 circles, 4 in each polar circle, 8 at equator and 8 on either side of equator; 4 small vegetative cells at anterior pole; vegeta- tive cells 10-1 6m in diameter; reproductive cells 19-25m in diame- ter; colony up to 160yu by 130^. P. calif ornica S. Spherical colony with 64 or 128 cells, of which 1/2-2/3 are reproductive cells; vegetative cells 13-15/x; reproduc- tive cells up to 27/x; colony up to 450/x, both in diameter. References Bold, H. C. 1938 Notes on Maryland Algae. Bull. Torrey Bot. Club., Vol. 65. Crow, W. B. 1918 The classification of some colonial chlamy- domonads. New Phytologist, Vol. 17. Dangeard, p. 1900 Observations sur la structure et le develop- pement du Pandorina morum. Le Botaniste, Vol. 7. 202 PROTOZOOLOGY Entz, G. Jr. 1913 Cytologische Beobachtungen an Polytoma uvella. Verb. Dcutscb. Zool. Ges. Ver., Vol. 23. Fritsch, F. E. 1935 The structure and reproduction of the algae. Cambridge. Janet, C. 1912, 1922, 1923 Le Volvox. I, II, and III Memoires. Paris. KoFOiD, C. A. 1900 Plankton Studies, Nos. 2 and 3. Ann. Mag. Nat. Hist., Ser. 7, Vol. 6. Mast, S. O. 1928 Structure and function of the eye-spot in uni- cellular and colonial organisms. Arch. f. Protistenk., Vol. 60. Pascher, a. 1927 Vovocales — Phytomonadinae. In: Die Suss- wasserflora Deutschlands, Part 4. Shaw, W. R. 1894 Pleodorina, a new genus of the Volvocideae. Bot. Gaz., Vol. 19. Smith, G. M. 1933 The freshwater algae of the United States. New York. West, G. S. and F. E. Fritsch 1927 A treatise on the British freshwater algae. Cambridge. Chapter 10 Order 4 Euglenoidina Blochmann THE body is as a rule elongated; some are plastic, others have a definite body form with a well-developed, striated or variously sculptured pellicle. At the anterior end, there is an opening shrough which a flagellum protrudes. In holophytic forms the to-called cytostome and cytopharynx, if present, are apparently not concerned with the food-taking, but seem to give a passage- way for the flagellum and also to excrete the waste fluid matters which become collected in one or more contractile vacuoles located around the reservoir. In holozoic forms, a well-developed cytostome and cytopharynx are present. Ordinarily there is only one flagellum, but some possess two or three. Chromatophores are present only in the majority of the Euglenidae and absent in the other two families. They are green, vary in shape, such as spheroidal, band-form, cup-form, discoidal, or fusiform, and usually possess pyrenoids. Some forms may contain haemato- chrome. A small but conspicuous stigma is invariably present near the anterior end of the body in chromatophore-bearing forms. Reserve food material is the paramylon body, fat, and oil, the presence of which depends naturally on the metabolic condi- tion of the organism. The paramylon body assumes diverse forms in different species, but is, as a rule, constant in each species, and this facilitates specific identification to a certain extent. Nutrition is holophytic in chromatophore-possessing forms, which, however, may be saprozoic, depending on the amount of light and organic substances present in the water. The holozoic forms feed upon bacteria, algae, and smaller Protozoa. The nucleus, as a rule, is large and distinct and contains almost always a large endosome. Asexual reproduction is by longitudinal fission; sexual reproduction has been observed in a few species. Encystment is common. The majority inhabit fresh water, but some live in brackish or salt water, and a few are parasitic in animals. 203 204 I'ROTOZOOLOGY With stigma Family 1 Euglenidae Without stigma With 1 flageUum Family 2 Astasiidae (p. 209) With 2 flagella Family 3 Anisonemidae (p. 212) Family 1 Euglenidae Stein Body plastic ("euglonoid"), but, as a rule, more or less spindle- shaped during movement; the majority possess a single anterior flagellum (with the exception of Eutreptia and Euglenamorpha) ; green (sometimes red) chromatophores (except one genus) and stigma occur, though in some cases absent; metabolic products oil and paramylon; asexual reproduction by longitudinal fission in either active or resting stage; mostly freshwater inhabitants. Genus Euglena Ehrenberg. Short or elongated spindle, cylin- drical, or band-form; pelhcle usually marked by longitudinal or spiral striae; some highly plastic with a thin pellicle; others regu- larly spirally twisted; stigma usually anterior; chromatophores numerous and discoid, band-form, or fusiform; pyrenoids may or may not be surrounded by starch envelope; metabolic products paramylon bodies which may be two in number; one being located on either side of nucleus, and rod-like to ovoid in shape or numerous and scattered throughout; contractile vacuoles small, near reservoir; asexual reproduction by longitudinal fission; sexual reproduction reported in Euglena sanguinea; common in stagnant water, especially where algae occur; when present in large numbers, the active organisms may form a green film on the surface of water and resting or encysted stages may produce conspicuous green spots on the bottom of pond or pool; in fresh water. Numerous species. E. pisciformis Klebs (Fig. 95, a). 25-30^ by 7-1 0^; spindle- form, with bluntly pointed anterior and sharply attenuated posterior end; slightly plastic; highly active; paramylon indis- tinct; chromatophores lateral and discoidal; 2 pyrenoids; flagellum fairly long. E. viridis Ehrenberg (Fig. 95, 6). 50-60/i by 14-18/x; anterior end rounded, posterior end pointed; spindle-shaped during motion, highly plastic when stationary; pellicle obhquely striated; chromatophores more or less band-form, arranged in a stellate form; nucleus posterior; nutrition holophytic, but also able to carry on saprozoic nutrition, during which period chromato- phores degenerate. EUGLENOIDINA 205 E. acus E. (Figs. 24, h; 95, c). 100-200^ long; long spindle-form; posterior end sharply pointed; flagelliim short; spiral striation on pellicle very delicate; paramylon bodies rod-form; nucleus cen- tral; stigma distinct; numerous disc-like chromatophores; with- out pyrenoids; sluggish. Fig. 95. a, Euglena pisciformis, X270 (Klebs); b, E. viridis, X370 (Lemmermann); c, E. acus, X270 (Klebs); d, E. spirogyra, X430 (Stein); e, E. oxyuris, X430 (Stein); f, E. sanguinea, Xl30 (Klebs); g, E. deses, X230 (Lemmermann); h, E. gracilis, X270 (Klebs). E. spirogyra E. (Figs. 24, c; 95, d). 80-125^ by 10-20m; cylin- drical; with spiral striae, consisting of small knobs; numerous disc-like chromatophores; without pyrenoids; 2 ovoidal paramy- lon bodies, one on either side of centrally located nucleus; flagellum short; stigma prominent; sluggish. E. oxyuris Schmarda (Fig. 95, e), 375-500^ by 30-45^; almost 206 PROTOZOOLOGY always spirally twisted, somewhat flattened; pellicle with spirally arranged striae; numerous chromatophores; without pyrenoids; 2 ovoid paramylon bodies conspicuously observable, one on either side of nucleus; flagellum short. E. sanguinea E. (Figs. 37, e-h; 95, /). 55-120^ by 28-33^; with haematochrome; often found in crust on surface or half-dry bed of a pool; considered by some workers as a variety of E. viridis. E. deses E. (Figs. 24, a; 95, g). 85-155m by 15-22^; elongate, highly plastic; body striation faintly visible; stigma distinct; nucleus central, numerous chromatophores hemi-lenticular; several small rod-shaped paramylon bodies scattered; flagellum short. E. gracilis Klebs (Figs. 37, a-d; 95, h). 37-45/x by 6-23m; cylindrical to elongated oval; highly plastic; flagellum less than body length; chromatophores numerous, discoid; nucleus central; pyrenoids. Genus Khawkinea Jahn et McKibben. Similar to Genus Euglena, but without chromatophores and thus permanently colorless; fresh water. K. halli J. et Mc. 40-45^ (30-65^) by 12-14^; fusiform; pellicle spirally striated; plastic; flagellum slightly longer than body; stigma 2-3/i in diameter, yellow-orange to reddish-orange, com- posed of numerous granules; numerous (25-100) paramylon bodies elliptical or polyhedral; cysts 20-30ju in diameter; putrid leaf infusion; saprozoic. K. ocellata (Khawkine). Similar to above; flagellum 1.5-2 times body length; fresh water. Genus Phacus Nitzsch. Highly flattened; asymmetrical; body- form constant; pellicle often with prominent longitudinal or oblique striae; a flagellum and a stigma; nucleus posterior; a short "cytopharynx"; green chromatophores rounded discoid; with or without paramylon bodies around a pyrenoid; in fresh water. Numerous species. P. pleuronectes (Miiller) (Fig. 96, a). 4:5-50^i by 30-33/1 ; short posterior prolongation slightly curved ; a prominent fold on con- vex side, extending to middle of body; longitudinally striated; one or more circular paramylon bodies ; colorless forms sometimes appear; flagellum as long as body. P. longicaudus (Ehrenberg) (Fig. 96, 6). 85-115/1 by 45-70/t; EUGLENOIDINA 207 usually twisted with a long caudal prolongation; stigma promi- nent; discoidal paramylon body central; pellicle longitudinally striated. P. pyrum (E.) (Fig. 96, c). About 40^1 long; pyriform, with a straight caudal prolongation; pellicle obliquely striated. P. triqueter (E.) (Fig. 96, d). 50-55/x by 30-35^; ovate; with a longitudinal ridge; posterior end acuminate; oblique striation distinct; 1-2 paramylon bodies. P. anacoelus Stokes (Fig. 96, e). About 42/^ long; oval or round; with flagellum as long as body. P. acuminata S. (Fig. 96, /). About 25m long; nearly circular in outline; longitudinally striated; fold long; flagellum as long as body; 2 small paramylon bodies. Genus Crumenula Dujardin (LepocincUs Perty). Body more or less ovo-cylindrical; rigid with spirally striated pellicle; often with a short posterior spinous projection; stigma sometimes present; numerous discoidal chromatophores marginal; paramy- lon bodies usually large and ring-shaped, laterally disposed; without pyrenoids; fresh water. Several species. C. ova (Ehrenberg) (Fig. 96, g). 20-40^ long; in fresh water with Euglena. Genus Trachelomonas Ehrenberg. With a lorica which often possesses numerous spinous projections; sometimes yellowish to dark brown; a single flagellum protrudes from anterior aperture, the rim of which is frequently thickened to form a collar; chroma- tophores either 2 curved plates or numerous discs; paramylon bodies small grains; stigma and pyrenoids; multiplication by longitudinal fission; one daughter individual retains lorica and flagellum, while the other escapes through flagellar aperture, forms a new flagellum and secretes a lorica; cysts common; specific differentiation is based upon the lorica; fresh water. Numerous species. T. hispida (Perty) (Figs. 31, a; 96, h). Lorica oval, with numer- ous minute spines; brownish; 8-10 chromatophores; 20-42/i by 15-26/x; many varieties. T. urceolata Stokes (Fig. 96, i). Lorica vasiform, smooth with a short neck; about 45^ long. T. piscatoris (Fisher) (Fig. 96, j). Lorica cylindrical with a short neck and with numerous short, conical spines; 25-40/z long; flagellum 1-2 times body length. 208 PROTOZOOLOGY Fig. 96. a, Phacus pleuronectes, X670 (Lemmermann); b, P. longi- caudus, X430 (Stein); c, P. pyrum, X400 (Lemmermann); d, P. triqueter, x430 (Stein); e, P. anacoelus, X330 (Stokes); f, P. acuminata X560 (Stokes); g, Crumenula ova, X430 (Stein); h, Trachelomonas hispida, X430 (Stein); i, T. urceolata, x430 (Stokes); j, T. piscatoris, X520 (Fischer); k, T. verrucosa, X550 (Stokes); 1, T. vermiculosa, X800 (Palmer); m, Cryptoglena pigra, X430 (Stein); n, Ascoglena vaginicola, X390 (Stein); o, Colacmm vesiculosuvi, X390 (Stein); p, Eutreptia viridis, X270 (Klebs); q, E. marina, X670 (da Cunha); r, Euglenamorpha hegneri, X730 (Wenrich). T. verrucosa Stokes (Fig. 96, h). Lorica spherical, with numer- ous knob-like attachments; no neck; 24-25ai in diameter. T. vermiculosa Palmer (Fig. 96, I). Lorica spherical; with sausage-form markings; 23^t in diameter. Genus Cryptoglena Ehrenberg. Body rigid, flattened; 2 band- EUGLENOIDINA 209 form chromatophores lateral; a single flagelliim; nucleus pos- terior; among freshwater algae. One species. C. pigra E. (Fig. 96, ?n). Ovoid, pointed posteriorly; fiagelUim short; stigma prominent; 10-15/^ by 6-10^; standing water. Genus Ascoglena Stein. Encased in a flexible, colorless to brown lorica, attached with its base to foreign object; solitary, without stalk; body ovoidal, plastic; attached to test with its posterior end; a single fiagellum; a stigma; numerous chromato- phores discoid; with or without pyrenoids; reproduction as in Trachelomonas (p. 207); fresh water. A. vaginicola S. (Fig. 96, n). Lorica about ^2>^x by 15^. Genus Colacium Ehrenberg. Stalked individuals form colony; frequently attached to animals such as copepods, rotifers, etc.; stalk mucilaginous; individual cells pyriform, ellipsoidal or cylindrical; without fiagellum; a single fiagellum only in free- swimming stage; discoidal chromatophores numerous; with pyrenoids; multiplication by longitudinal fission; also by swarmers, possessing a fiagellum and a stigma; fresh water. Several species. C. vesiculosum E. (Fig. 96, o). Colony of 2-8 cells; also solitary; 20-30/i by 9-18^; attached to freshwater copepods. Genus Eutreptia Perty (Eutreptiella Cunha). With 2 flagella at anterior end; pellicle distinctly striated; plastic; spindle- shaped during movement; stigma; numerous discoid chromato- phores; pyrenoids absent; paramylon bodies spherical or sub- cylindrical; multiplication as in Euglena; cyst with a thick stratified wall; fresh or salt water. E. viridis P. (Fig. 96, p). 50-70^ by 5-13/x; in fresh water; a variety was reported from brackish water ponds. E. marina (da Cunha) (Fig. 96, q). Flagella unequal in length; longer one as long as body, shorter one 1/3; body 40-50At by 8-1 0/x; salt water. Genus Euglenamorpha Wenrich. Body form and structure similar to those of Euglena, but with 3 flagella; in gut of frog tadpoles. One species. E. hegneri W. (Fig. 96, r). 40-50/x long. Family 2 Astasiidae Biitschli Similar to Euglenidae in body form and general structure, but without chromatophores; body is plastic, although it assumes 210 PROTOZOOLOGY I FiG. 97. a, Astasia klebsi, X500 (Klebs); b, Urceolus cyclostomus, X430 (Stein); c, U. sabulosiis, X430 (Stokes); d, Peranema triclio- 'phorum, X530 (Kudo); e, Petalmonas mediocanellata, XlOOO (Klebs); f, Menoidium incurvum, X1400 (Hall); g, Scytomonas pusilla, x430 (Stein); h, Anisonema acinus, X400 (Klebs); i, A. truncatum, X430 (Stein); j, ^. emerginalum, X530 (Stokes); k, Heteronema acus, X430 (Stein); 1, H. mutabile, Xl20 (Stokes); m, TropidoscAjphus odocostatus, X290 (Lemmermann); n, Distigma proteus, x430 (Stein); o, Enlosi- phon sulcatum, x430 (Stein); p, Notosolenus apocamptus, X1200 (Stokes); q, N. sinatus, x600(Stokes); r, Marsupiogaster striata, X590 (Schewiakoff) ; s, M. picta (Faria, da Cunha and Pinto). EUGLENOIDINA 211 usually an elongated form; there is a cytopharynx and cytostome, the former being connected with the reservoir of contractile vacuoles; without stigma; flagellum usually straight and its free end vibrates in a characteristic manner; asexual reproduction by longitudinal fission. Genus Astasia Dujardin. Body plastic, although ordinarily elongate; fresh water or endoparasitic (?) in Cyclops, etc. Several species. A. klehsi Lemmermann (Fig. 97, a). Spindle-form; posterior portion drawn out; flagellum as long as body; plastic; paramylon bodies oval; 50-60ai by 13-20^; stagnant water. Genus Urceolus Mereschkowsky {Phialonema Stein). Body colorless; plastic; flask-shaped; striated; a funnel-like neck; posterior region stout; a single flagellum protrudes from funnel and reaches inward the posterior third of body; fresh or salt water. U. cyclostomus (Stein) (Figs. 8, /; 97, h). 25-50yu long; fresh water. U. sahulosus (Stokes) (Fig. 97, c). Spindle-form ; surf ace covered with minute sand-grains; about 58^ long; fresh water. Genus Peranema Dujardin. Elongate with a broad, rounded or truncate posterior end during locomotion; highly plastic when stationary; delicate pellicle shows a fine striation; flagellum long, tapers toward free end and vibrates; nucleus central; con- tractile vacuoles; saprozoic and holozoic; in stagnant water; often in hay infusion. P. trichophorum (Ehrenberg) (Figs. 26; 97, d). 20-70/i long; very common. P. granulijera Penard. Much smaller, 8-15/x long; spherical or elongate; pellicle granulated; standing water. Genus Petalomonas Stein. Colorless; constant in form; pellicle often with longitudinal keels on one side; a single flagellum; holozoic or saprozoic; cytostome at anterior end; cytopharynx fairly deep; in fresh water, rich in vegetable matter. Many species. P. mediocanellata S. (Fig. 97, e). Ovoid with longitudinal fur- row; flagellum about as long as body; 22-23ai long. Genus Menoidium Perty. Rigid body, more or less curved; peUicle striated; a single flagellum; fresh water. M. incurvum (Fresenius) (Figs. 24, d; 64; 97,/). Crescentic cyl- 212 • PHOTOZOOLOCY indcr; flagellum as long as body; nuclous central or terminal; 15- 25m by 7-8m; in standing fresh water. Hall (1923) made a careful cytological study of the organism. M. tortuosum Stokes. S-form; posterior end drawn out to a sharp point; elongate paramylon bodies; 42-78^ long; in infusion. Genus Scytomonas Stein. Oval or pyriform, with a delicate pel- licle; a single flagellum; a contractile vacuole with a reservoir; holozoic on bacteria; longitudinal fission in motile stage; stag- nant water and coprozoic. S. pusilla S. (Fig. 97, g). About 15m long. Genus Copromonas Dobell. Elongate ovoid; with a single fla- gellum; a small cytostome at anterior end; holozoic on bacteria; permanent fusion followed by encystment (p. 145); coprozoic in faecal matters of frog, toad, and man; several authors hold that this genus is probably identical with Scytomonas which was in- completely described by Stein. C. siibtilis D. (Fig. 69). 7-20^ long. Family 3 Anisonemidae Schewiakofif Colorless body plastic or rigid with a variously marked pellicle; 2 flagella, one directed anteriorly and the other usually posteri- orly; contractile vacuoles and reservoir; stigma absent; paramy- lon bodies as a rule present; free-swimming or creeping. Genus Anisonema Dujardin. Generally ovoid; more or less flattened; asymmetrical; plastic or rigid; a slit-like ventral fur- row; flagella at anterior end; cytopharynx long; contractile vacu- ole anterior; nucleus posterior; in fresh water. Several species. A. acinus D. (Fig. 97, h). Rigid; oval; somewhat flattened; pel- licle slightly striated; 25-40/x by 16-22^. A. truncatum Stein (Fig. 97, i). Rigid; ovoid; 60/i by 20/^. A. emarginatum Stokes (Fig. 97, j). Rigid; 14/x long; flagella long. Genus Heteronema Dujardin. Plastic; rounded or elongate; flagella arise from anterior end, one directed forward and the other trailing; cytostome near base of flagella; holozoic; fresh water. Several species. H. acus (Ehrenberg) (Fig. 97, k). Extended body tapers to- wards both ends ; anterior flagellum as long as body, trailing one about 1/2; contractile vacuole anterior; nucleus central; 45-50^ long; freshwater. EUGLENOIDINA 213 H. mutabile (Stokes) (Fig. 97, I). Elongate; highly ])Iastic; longitudinally striated; about 254/i long; in cypress swamp. Genus Tropidoscyphus Stein. Slightly plastic; pellicle with 8 longitudinal ridges; 2 unequal flagella at anterior pole; holozoic or saprozoic; fresh or salt water. T. octocostatus S. (Fig. 97, m). 35-63/^ long; fresh water, rich in vegetation. Genus Distigma Ehrenberg. Plastic; elongate when extended; body surface without any marking; 2 flagella unequal in length, directed forward; cytostome and cytopharynx located at anterior end; endoplasm transparent; holozoic. One species. D. proteus E. (Fig. 97, n). 50-1 10/z long when extended; nu- cleus central; stagnant water; infusion. Genus Entosiphon Stein. Oval, flattened; more or less rigid; flagella arise from a cytostome, one flagellum trailing; protrusible cytopharynx a long conical tubule almost reaching posterior end ; nucleus centro-lateral; fre>sh water. E. sulcatum (Dujardin) (Fig. 97, o). About 20// long. E. ovatum Stokes. Anterior end rounded; 10-12 longitudinal striae; about 25-28/z long. Genus Notosolenus Stokes. Free-swimming; rigid oval; ventral surface convex, dorsal surface with a broad longitudinal groove; flagella anterior; one long, directed anteriorly and vibratile; the other shorter and traihng; fresh water with vegetation. N. apocamptus S. (Fig. 97, p). Oval with broad posterior end; 6-1 1m long. N. sinuatus S. (Fig. 97, q). Posterior end truncate or concave; about 22/x long. Genus Marsupiogaster Schewiakoff. Oval; flattened; asym- metrical; cytostome occupies entire anterior end; cytopharynx conspicuous, 1/2 body length; body longitudinally striated; 2 flagella, one directed anteriorly, the other posteriorly; spherical nucleus; contractile vacuole anterior; fresh or salt water. M. striata Schewiakoff (Fig. 97, r). About 27 fi by IS/x; fresh water; Hawaii. M. picta Faria, da Cunha et Pinto (Fig. 97, s). In salt water; Rio de Janeiro. Order 5 Chloromonadina Klebs The chloromonads are of rare occurrence and consequently not 214 rilOTOZOOLOGY b c Fig. 98. a, Gonyostomum semen, X540 (Stein); b, Vacuolaria vires- cens, X460 (Senn); c, Trentonia flagellata, X330 (Stokes); d, Thau- matomastrix setifera, X830 (Lauterborn), well known. The majority possess small discoidal grass-green chromatophores with a large amount of xanthophyll which on addition of an acid become blue-green. No pyrenoids occur. The metabolic products are fatty oil. Starch or allied carbohydrates are absent. Stigma is also not present. Genus Gonyostomum Diesing (Rhaphidomonas Stein). With grass-green chromatophores; highly refractile trichocyst-like structures in cytoplasm; in fresh water. A few species. G. semen D. (Fig. 98, a). Sluggish animal; about 45-60ju long; in marshy water among decaying vegetation. Genus Vacuolaria Cienkowski. Highly plastic; without tricho- cyst-like structures; anterior end narrow; with 2 flagella; cysts with a gelatinous envelope. One species. V. virescens C. (Fig. 98, h). About 50-150m long; fresh water. Genus Trentonia Stokes. Bi-flagellate as in the last genus; but flattened; anterior margin slightly bilobed. One species. T. flagellata S. (Fig. 98, c). Slow-moving organism; encystment followed by binary fission; about 60^1 long; fresh water. Genus Thaumatomastix Lauterborn. Colorless; pseudopodia formed; 2 flagella, one extended anteriorly, the other trailing; holozoic; perhaps a transitional form between the Mastigophora and the Sarcodina. One species. T. setifera L. (Fig. 98, d). About 20-35^ by 15-28m; fresh water. EUGLENOIDINA, CHLOROMONADINA 215 References Dangeard, p. 1901 Recherches sur les Eugleniens. Le Bota- niste. P. 97. Fritsch, F. E. 1935 The structure and reproduction of the algae. Vol. 1. Cambridge. Hall, R. P. 1923 Morphology and binary fission of Menoidium incurvuni (Fres.) Klebs. Uni. Cal. Publ. Zool., Vol. 20. Lemmermann, E. 1913 Eugleninae. In: Susswasserfl. Deutsch- lands, Part 2. Pascher, a. 1913 Chlorojnonadinae. Ibid. Part 2. Smith, G. M. 1933 The freshwater algae of the United States. New York, Wenrich, D. H. 1924 Studies on Euglenamorpha hegneri n. g., n. sp., a euglenoid flagellate found in tadpoles, Biol. Bull,, Vol. 47. West, G. S. and F. E. Fritsch. 1927 A treatise on the British freshwater algae. Cambridge. '''' k: Chapter 11 Order 6 Dinoflagellata Butschli THE dinoflagellates make one of the most distinct groups of the Mastigophora, inhabiting mostly marine water, and to a lesser extent fresh water. In the general appearance, the arrangement of the two flagella, the characteristic furrows, and the possession of brown chromatophores, they are closely related to the Crypto- monadina. The body is covered by an envelope composed of cellulose which may be a simple smooth piece, or may be composed of two valves or of numerous plates, that are variously sculptured and possess manifold projections. Differences in the position and course of the furrows and in the projections of the envelope pro- duce numerous asymmetrical forms. The furrows, or grooves, are a transverse annulus and a longitudinal sulcus. The annulus is a girdle around the middle or toward one end of the body. It may be a complete or incomplete ring or sometimes spiral. While the ma- jority show a single transverse furrow, a few may possess several. The part of the shell anterior to the annulus is called the epitheca and that posterior to the annulus the hypotheca. In case the en- velope is not developed, the terms epicene and hypocone are used (Fig. 99). The sulcus may run from end to end or from one end to the annulus. The two flagella arise ty]jically from the annulus, one being transverse and the other longitudinal. -Anterior flagellar pore x,^ y — "n. Epicene , Transverse flagelluni Annulus or girdle _ -Sulcus Hypocone Longitudinal flagellum [ "^Posterior flagellar pore Fig. 99. Diagram of a typical naked dinoflagellate (Lebour). The transverse flagellum which is often band-form, encircles the body and undergoes undulating movements, which in former 216 DINOFLAGELLATA 217 years were looked upon as ciliary movements (hence the name CiHoflagellata). In the suborder Adinida, this fiagellum vibrates freely in a circle near the anterior end. The longitudinal fiagellum often projects beyond the body and vibrates. Combination of the movements of these flagella produces whirling movements char- acteristic of the organisms. The majority of dinoflagellates possess a single somewhat mas- sive nucleus with evenly scattered chromatin, and usually several endosomes. There are two kinds of vacuoles. One is often sur- rounded by a ring of smaller vacuoles, while the other is large contains pink-colored fluid and connected with the exterior by a canal opening in a flagellar pore. The latter is known as the pusule which functions as a digestive organella (Kofoid and Swezy). In many freshwater forms a stigma is present, and in Pouchetiidae there is an ocellus composed of an amyloid lens and a dark pig- ment-ball. The majority of planktonic forms possess a large num- ber of small chromatophores which are usually dark yellow, brown or sometimes slightly greenish and are located in the pe- riphery of the body, while bottom-dwelling and parasitic forms are, as a rule, colorless, because of the absence of chromatophores. A few forms contain haematochrome. The method of nutrition is holophytic, holozoic, saprozoic, or mixotrophic. In holophytic forms, anabolic products are starch, oil, or fats. Asexual reproduction is by binary or multiple fission or bud- ding in either the active or the resting stage and differs among different goups. Encystment is of common occurrence. In some forms the cyst wall is formed within the test. The cysts remain alive for many years ; for example, Ceratium cysts were found to retain their vitahty in one instance for six and one-half years. Conjugation and sexual fusion have been reported in certain forms, but definite knowledge on sexual reproduction awaits further investigation. The dinoflagellates are abundant in the plankton of the sea and play an important part in the economy of marine life as a whole. A number of parasitic forms are also known. Their hosts include various diatoms, copepods and several pelagic animals. Bivalve shell without furrows Suborder 1 Prorocentrinea (p. 218) Nakes or with shell showing furrows Suborder 2 Peridiniinea (p. 219) Naked; without furrows; no transverse fiagellum Suborder 3 Cystoflagellata (p. 233) 218 a PROTOZOOLOGY c d Fig. 100. a, Prorocentnim micans, X420 (Schiitt); b, c, Exuviaella marina, X420 (Schiitt); d, e, Cystodinium steini, X370 (Klebs); f, Glenodinium cinctuni, X590 (Schilling); g, G. j)ulvisculum, x420 (Schilling); h, G. uliginosum, X590 (Schilling); i, G. edax, X490 (Schilling); j, G. negleduni, X650 (Schilling). Suborder 1 Prorocentrinea Poche Test bivalve; without any groove; with yellow chromato- phores; 2 flagella anterior, one directed anteriorly, the other vi- brates in a circle; fresh or salt water. Family Prorocentridae Kofoid Genus Prorocentrum Ehrenberg. Elongate oval; anterior one bluntly pointed, with a spinous projection at pole; chromato- phores small, yellowish brown; salt water. P. micans E. (Fig. 100, a). 36-52/x long; a cause of "red water." P. triangulatum Martin. Triangular with rounded posterior end; shell-valves flattened; one valve with a delicate tooth; sur- face covered with minute poroids; margin striated; chromato- phores yellow-brown, irregular, broken up in small masses; 17- 22/i (excluding tooth); Martin found it extremely abundant in brackish water in New Jersey. DINOFLAGELLATA 219 Genus Exuviaella Cienkowski. Subspherical or oval; no anterior projection, except 2 flagella; 2 lateral chromatophores, large, brown, each with a pyrenoid and a starch body; nucleus posterior; salt water. Several species. E. marina C. (Fig. 100, h, c). 36-50/i long. E. apora Schiller. Compressed, oval; striae on margin of valves; chromatophores numerous yellow-brown irregular in form; 30- 32m by 21-26m (Schiller); 17-22/z by 14-19; (Lebour; Martin); common in brackish water. New Jersey. Suborder 2 Peridiniinea Poche Typical dinofiagellates with one to many transverse annuli and a sulcus; 2 flagella, one of which undergoes a typical undulating movement, while the other usually directed posteriorly. Accord- ing to Kofoid and Swezy, this suborder is divided into two tribes. Body naked or covered by a thin shell . . . Tribe 1 Gymnodinioidae Body covered by a thick shell Tribe 2 Peridinioidae (p. 229) Tribe 1 Gymnodinioidae Poche Naked or covered by a single piece cellulose membrane with annulus and sulcus, and 2 flagella; chromatophores abundant, yellow or greenish platelets or bands; stigma sometimes present; asexual reproduction binary or multiple division; holophytic, holozoic, or saprozoic; the majority are deep-sea forms; a few coastal or fresh water forms also occur. With a cellulose membrane Family 1 Cystodiniidae Without shell Furrows rudimentary Family 2 Pronoctilucidae (p. 220) Annulus and sulcus distinct Solitary With ocellus Family 3 Pouchetiidae (p. 220) Without ocellus With tentacles Family 4 Noctilucidae (p. 222) Without tentacles Free-living Family 5 Gymnodiniidae (p. 223) Parasitic Family 6 Blastodiniidae (p. 227) Permanently colonial Family 7 Polykrikidae (p. 228) Family 1 Cystodiniidae Kofoid et Swezy Genus Cystodinium Klebs. In swimming phase, oval, with ex- 220 PROTOZOOLOGY tremely delicate envelope; annulus somewhat acyclic; cyst-mem- brane drawn out into 2 horns. C. steini K. (Fig. 100, d, e). Stigma beneath sulcus; chromato- phores brown; swarmer about 45ju long; freshwater ponds. Genus Glenodinium Ehrenberg. (Glenodiniopsis, Stasziecella, Woloszynska). Spherical; ellipsoidal or reniform in end-view; an- nulus a circle; several discoidal, yellow to brown chromatophores; horseshoe- or rod-shaped stigma in some; often with gelatinous envelope; fresh water. Many species. G. cinctum E. (Fig. 100, /). Spherical to ovoid; annulus equa- torial; stigma horseshoe-shaped; 43/x by 40/i. G. pulvisad'um Stein (Fig. 100, g). No stigma; 38At by 30^:. G. uliginosum Schilling (Fig. 100, h). 36-48/z by 30/i. G. edax S. (Fig. 100, i). Mix by 33^^. G. neglectum S. (Fig. 100, j). 30-32^ by 29/x. Family 2 Pronoctilucidae Lebour Genus Pronoctiluca Fabre-Domergue. Body with an antero- ventral tentacle and sulcus; annulus poorly marked; salt water. P. tentaculatum (Kofoid et Swezy) (Fig. 101, a). About 54/i long; off California. Genus Oxyrrhis Dujardin. Subovoidal, asymmetrical posterior- ly; annulus incomplete; salt water. 0. marina D. (Fig. 101, 6). 10-37^ long. Family 3 Pouchetiidae Kofoid et Swezy Ocellus consists of lens and melanosome (pigment mass) ; sulcus and annulus somewhat twisted; pusules usually present; cyto- plasm colored; salt water (pelagic). Genus Pouchetia Schiitt. Nucleus anterior to ocellus; ocellus with red or black pigment mass with a red, brown, yellow, or colorless central core; lens hyahne; body surface usually smooth; holozoic; encystment common; salt water. Many species. P.fusus S. (Fig. 101, c). About 94ju by 41^; ocellus 27m long. P. maxima Kofoid et Swezy (Fig. 101, d). 145^ by 92/x; ocellus 20iu; off California. Genus Protopsis Kofoid et Swezy. Annulus and sulcus similar to those of Gymnodinium or Gyrodinium ; with a simple or com- pound ocellus, no tentacles; body not twisted; salt water. A few species. DINOFLAGELLATA 221 Fig. 101. a, Pronodiluca tenia culat inn, x730 (Kofoid and Swezy); b, Oxyrrhis marina, XSIO (Senn); c, Pouchetia fusus, X340 (Schiitt); d, P. maxima, X330 (Kofoid and Swezy); e, Protopsis ochrea, X340 (Wright); f, Nematodinium partitum, X560 (Kofoid and Swezy); g, Proterythropsis crassicaudata, x740 (Kofoid and Swezy); h, Ery- thropsis cornuta, X340 (Kofoid and Swezy); i, j, Noctihica scintillans (i. side view; j, budding process), Xl40 (Robin). 222 PROTOZOOLOGY P. ochrea (Wright) (Fig. 101, e). 55/x by 45)u; ocellus 22/i long; Nova Scotia. Genus Nematodinium Kofoid et Swezy. With nematocysts; girdle more than 1 turn; ocellus distributed or concentrated, pos- terior; holozoic; salt water. N. partitum K. et S. (Fig. 101,/). Ol^i long; off CaHfornia. Genus Proterythropsis Kofoid et Swezy. Annulus median; ocel- lus posterior; a stout rudimentary tentacle or prod-like antapical process; salt water. One species. P. crassicaudata K. et S. (Fig. 101, g). 70/u long; off California. Genus Erythropsis Hertwig. Epicone flattened, less than 1/4 hypocone; ocellus very large, composed of one or several hyaline lenses attached to or imbedded in a red, brownish or black pig- ment body with a red, brown or yellow core, located at left of sulcus; sulcus expands posteriorly into ventro-posterior tentacle; salt water. Several species. E. cornuta (Schlitt) (Fig. 101, h). 104^ long; off California (Ko- foid and Swezy). Family 4 Noctilucidae Kent Tentacle somewhat contractile, arises from sulcal area and ex- tends posteriorly; this group had formerly been included in the Cystoflagellata; studies by recent investigators, particularly by Kofoid, show their affinities with the present suborder; holozoic; salt water. Genus Noctiluca Suriray. Spherical, bilaterally symmetrical; peristome marks median line of body; a cytostome at bottom of peristome; with a conspicuous tentacle; cytoplasm much vacuo- lated, and cytoplasmic strands connect central mass with periph- ery; peripheral granules phosphorescent (p. 95); cytoplasm colorless or blue-green; sometimes tinged with yellow coloration in center; swarmers formed by budding, and each possesses one flagellum, annulus, and tentacle; widely distributed in salt water; holozoic. One species. N. scintillans (Macartney) {N. miliaris S.) (Fig. 101, i, j). Usually 500-1000;u in diameter, with extremes of 200/i and 2 mm. Gross (1934) observed that complete fusion of two swarmers (isogametes) results in cyst formation from which trophozoites develop. Acid contents of the body fluid is said to be about pH 3. Genus Pavillardia Kofoid et Swezy. Annulus and sulcus similar to those of Gymnodinium; longitudinal flagellum absent; stout DINOFLAGELLATA 223 finger-like mobile tentacle directed posteriorly; salt water. One species. P. tentacuUfera K. et S. 58// by 27m; pale yellow; off California. Family 5 Gymnodiniidae Kofoid Naked forms with simple but distinct 1/2-4 turns of annulus; with or without chromatophores; fresh or salt water. Genus Gymnodinium Stein. Pellicle delicate; subcircular; bi- laterally symmetrical; numerous discoid chromatophores vari- colored (yellow to deep brown, green, or blue) or sometimes ab- sent; stigma present in few; many with mucilaginous envelope; salt, brackish, or fresh water. Numerous species. G. aeruginosum S. (Fig. 102, a). Chromatophores green; 33-35/x by 22m ; ponds and lakes. G. rotundatum Klebs (Fig. 102, 6). 32-35m by 22-25^; fresh wa- ter. G. palustre Schilling (Fig. 102, c). 45m by 38m; fresh water. G. agile Kofoid et Swezy (Fig. 102, d). About 28m long; in sandy beaches. Genus Hemidinium Stein. Asymmetrical; oval; annulus about half a turn, only on left half. One species. H. nasutu7n S. (Fig. 102, e). Sulcus on hjrpocone; chromato- phores yellow to brown; with a reddish brown oil drop; nucleus posterior; transverse fission; 24-28m by 16-1 7m; fresh water. Genus Amphidinium Claparede et Lachmann. Form variable; epicone small; annulus anterior; sulcus straight on hypocone or also on part of epicone; with or without chromatophores; mainly holophytic, some holozoic; coastal or fresh water. Numerous spe- cies. A. lacustre Stein (Fig. 102, /). 30m by 18m; in fresh and salt (?) water. A. scissum Kofoid et Swezy (Fig. 102, g). 50-60m long; in sandy beaches. A.fusiforme Martin. Fusiform, twice as long as broad; circular in cross-section; epicone rounded conical; annulus anterior; hypo- cone 2-2.5 times as long as epicone; sulcus obscure; body filled with yellowish green chromatophores except at posterior end; stigma dull orange, below girdle; nucleus ellipsoid, posterior to annulus; pellicle delicate; 17-22m by 8-1 1m in diameter. Martin (1929) found that it was extremely abundant in parts of Delaware 224 PROTOZOOLOGY Fig. 102. a, Gymnodinium aeruginosum, X500 (Schilling); b, G. ro- timdatum, X360 (Klebs); c, G. palustre, X360 (Schilling); d, G. agile, X740 (Kofoid and Swezy); e, Hemidinium nasutum, X670 (Stein); f, Amphidinium lacustre, X440 (Stein); g, A. scissum, X8S0 (Kofoid and Swezy); h, Gyrodiniwn hiconicum, X340 (Kofoid and Swezy); i, G. hyalinum, X670 (Kofoid and Swezy); j, Cochlodinimn atromacu- latum, X340 (Kofoid and Swezy); k, Torodinium robustum, X670 (Kofoid and Swezy); 1, Massartia nie^tportensis, X670 (Conrad); m, Chilodinium cruciatum, X900 (Conrad); n, o, Trochodiniitm pris- maticurn, X1270 (Conrad); p, Ceratodinium asymetricuvi, X670 (Con- rad). DINOFLAGELLATA 225 Bay and gave rise to red coloration of the water ("Red water"). Genus Gyrodinium Kofoid et Swezy. Annuliis descending left spiral; sulcus extending from end to end; nucleus central; pusules; surface smooth or striated; chromatophores rarely present; cyto- plasm colored; holozoic; salt or fresh water. Many species. G. hiconicum K. et S. (Fig. 102, h). 68m long; salt water; off Cali- fornia. G. hyalinum (Schilling) (Fig. 102, i). About 24^ long; fresh- water. Genus Cochlodinium Schiitt. Twisted at least 1.5 turns; annu- lus descending left spiral; pusules; cytoplasm colorless to highly colored; chromatophores rarely present; holozoic; surface smooth or striate; salt water. Numerous species. C. atromaculatum Kofoid et Swezy (Fig. 102, j). 183-185m by 72m; longitudinal flagellum 45ju long; ofif California. Genus Torodinium Kofoid et Swezy. Elongate; epicone several times longer than hypocone; annulus and hypocone form augur- shaped cone; sulcus long; nucleus greatly elongate; salt water. 2 species. T. robustum K. et S. (Fig. 102, h). Ql-7^ti long; off California. Genus Massartia Conrad. Cylindrical; epicone larger (9-10 times longer and 3 times wider) than hypocone; no sulcus; with or without yellowish discoid chromatophore. M. nieu'portensis C. (Fig. 102, I). 28-37m long; brackish water. Genus Chilodinium Conrad. Ellipsoid; posterior end broadly rounded, anterior end narrowed and drawn out into a digitform process closely adhering to body; sulcus, apex to 1/5 from pos- terior end; annulus oblique, in anterior 1/3. C. crvciatum C. (Fig. 103, m). 40-50/x by 30-40^; with trich- ocysts; brackish water. Genus Trochodinium Conrad. Somewhat similar to Amphidin- ium; epicone small, button-hke; hypocone with 4 longitudinal rounded ridges; stigma; without chromatophores. T. prismaticum C. (Fig. 102, n, o). 18-22^ by 9-12^; epicone 5-7m in diameter; brackish water. Genus Ceratodinium Conrad. Cuneiform; asymmetrical, color- less, more or less flattened; annulus complete, oblique; sulcus on half of epicone and full length of hypocone; stigma. C. asymetricum C. (Fig. 102, p). 68-80^ by about 10^; brackish water. 226 PROTOZOOLOGY Fig. 103. a, Blastodinium spinulosum, X240 (Chatton); b, c, Oodi- nium poucheti (c, a swarmer) (Chatton); d, e, Apodinium mycetoides (d, swarmer-formation, X450; e, a younger stage, X640) (Chatton); f, Chytriodinium parasilicum in a copepod egg (Dogiel); g, Trypano- dinium ovicola, X1070 (Chatton); h, Duboscquella tintinnicola (Du- boscq and Collin); i, j, Haplozoon clymenellae (i, mature colony, X300; j, a swarmer, Xl340) (Shumway); k, Syndinium turbo, X1340 (Chat- ton); 1, Paradinium poucheti, XSOO (Chatton); m, Ellobiopsis chattoni on Calanus finmarchicus (Caullery); n, Paraellobiopsis coutieri (Collin). DINOFLAGELLATA 227 Family 6 Blastodiniidae Kofoid et Swezy All parasitic in or on plants and animals; in colony forming genera, there occur trophoc5rte (Chatton) by which organism is attached to host and more or less numerous gonocytes (Chatton). Genus Blastodinium Chatton. In gut of copepods; spindle- shaped, arched, ends attenuate)!; envelope (not cellulose) often with 2 spiral rows of bristles; young forms binucleate; when pres- ent, chromatophores in yellowdsh brown network; swarmers sim- ilar to those of Gymnodinium; in salt water. Many species. B. spinulosum C. (Fig. 103, a). About 235^ by 33-39/^; swarm- ers 5-lOyu; in Palacalanus parvus, Clausocalanus arcuicornis and C. furcatus. Genus Oodinium Chatton. Spherical or pyriform ; with a short stalk; nucleus large; often with yellowish pigment; on Salpa, An- nelida, Siphonophora, etc. 0. poucheti (Lemmermann) (Fig. 103, 6, c). Fully grown indi- viduals up to 170 fji long; bright yellow ochre; mature forms be- come detached and float, dividing into numerous gymnodinium- like swarmers; on the tunicate, Oikopleura dioica. Genus Apodinium Chatton. Young individuals elongate, spher- ical or pyriform; binucleate; adult colorless; formation of numer- ous swarmers in adult stage is peculiar in that lower of the 2 individuals formed at each division secretes a new envelope, and delays its further division until the upper divides for the second time, leaving several open cups; on tunicates. A. mycetoides C. (Fig. 103, d, e). On gill-slits of Fritillaria pel- lucida. Genus Chytriodinium Chatton. In eggs of planktonic copepods; young individuals grow at the expense of host egg and when fully formed, body divides into many parts, each producing 4 swarm- ers. Several species. C. parasiticum (Dogiel) (Fig. 103,/). In copepod eggs; Naples. Genus Trypanodinium Chatton. In copepod eggs; swarmer- stage only known. T. ovicola C. (Fig. 103, g). Swarmers biflagellate ; about 15)u long. Genus Duboscquella Chatton. Rounded cell with a large nu- cleus; parasitic in Tintinnidiidae. One species. D. tintinnicola (Lohmann) (Fig. 103, h). Intracellular stage oval, about lOO^t in diameter with a large nucleus; swarmers bi- flagellate. 228 PROTOZOOLOGY Genus Haplozoon Dogiel. In gut of polychaetes; mature forms composed of variable number of cells arranged in line or in pyra- mid; salt water. Many species. H. clymenellae (Calkins) (Fig. 103, i, j). In Clymenella tor- quata; colonial forms consist of 250 or more cells; Woods Hole. Genus Syndinium Chatton. In gut and body cavity of marine copepods; multinucleate round cysts in gut considered as young forms; multinucleate body in host body cavity with numerous needle-like inclusions. S. turho C. (Fig. 103, h). In Paracalanus parvus, Corycaeus venustus, Calanus finmarchicus ; swarmers about 15m long. Genus Paradinium Chatton. In body-cavity of copepods; mul- tinucleate body without inclusions; swarmers formed outside the host body. P. poucheti C. (Fig. 103, I). In the copepod, Acartia clausi; swarmers about 25^1 long, amoeboid. Genus Ellobiopsis Caullery. Pyriform ; with stalk ; often a sep- tum near stalked end; attached to anterior appendages of marine copepods. E. chattoni C. (Fig. 103, m). Up to TOO^i long; on antennae and oral appendages of Calanus finmarchicus, Pseudocalanus elongatus and Acartia clausi. Genus Paraellobiopsis Collin. Young forms stalkless; spherical; mature individuals in chain-form; on Malacostraca. P. coutieri C. (Fig. 103, n). On appendages of Nebalia hipes. Family 7 Polykrikidae Kofoid et Swezy 2, 4, 8, or 16 individuals permanently joined in chain; individu- als similar to Gymnodinium; sulcus however extending entire body length; with nematocysts (Fig. 104, 6); greenish to pink; nuclei about 1/2 the number of individuals; holozoic; salt water. Genus Polykrikos Biitschli. With the above-mentioned char- acters; salt or brackish water. P. kofoidi (Chatton) (Fig. 104, a, h). Greenish grey to rose; composed of 2, 4, 8, or IG individuals; with nematocysts; each nematocyst possesses presumably a hollow thread, and discharged under suitable stimulation; a binucleate colony composed of 4 in- dividuals about llO/i long; off California. P. harnegatensis Martin. Ovate, nearly circular in cross-section, slightly concave ventrally; composed of 2 individuals; constric- DINOFLAGELLATA 229 tion slight; beaded nucleus in center; annuli descending left spiral, displaced twice their width; sulcus ends near anterior end; cyto- plasm colorless, with numerous oval, yellow-brown chromato- phores; nematocysts absent; 46ju by 31.5ju; in brackish water of Barnegat Bay. Tribe 2 Peridinioidae Poche The shell composed of epitheca, annulus and hypotheca, which may be divided into numerous plates; body form variable. With annulus and sulcus Shell composed of plates; but no suture Family 1 Peridiniidae Breast plate divided by sagittal suture Family 2 Dinophysidae (p. 233) Without annulus or sulcus Family 3 Phj^todiniidae (p. 233) Family 1 Peridiniidae Kent Shell composed of numerous plates; annulus usually at equator, covered by a plate known as cingulum ; variously sculptured and finely perforated plates vary in shape and number among differ- ent species; in many species certain plates drawn out into various processes, varying greatly in different seasons and localities even among one and the same species; these processes seem to retard descending movement of organisms from upper to lower level in water when flagellar activity ceases; chromatophores numerous, small platelets, yellow or green; some deep-sea forms without chromatophores; chain formation in some forms; mostly surface and pelagic inhabitants in fresh or salt water. Genus Peridinium Ehrenberg. Subspherical to ovoid; reniform in cross-section; annulus slightly spiral with projecting rims; hy- potheca often with short horns and epitheca drawn out ; colorless, green, or brown; stigma usually present; cysts spherical; salt or fresh water. Numerous species. P. tahulatum Claparede et Lachmann (Fig. 104, c). 48m by 44^; fresh water. P. divergens (E.) (Fig. 104, d). About 45m in diameter; yellow- ish, salt water. Genus Ceratium Schrank. Body flattened; with one anterior and 1-4 posterior horn-like processes; often large; chromato- phores yellow, brown, or greenish; color variation conspicuous; fission is said to take place at night and in the early morning; 230 PROTOZOOLOGY Fig. 104. a, b, Polykrikos kofoidi (a, colony of four individuals, X340; b, a nematocyst, X1040) (Kofoid and Swezy); c, Peridinium tabulatum, X460 (Schilling); d, P. diver gens, X340 (Calkins); e, Cera- tium hirundinella, x540 (Stein); f, C. longipes, XlOO (Wailes); g, C. tripos, Xl40 (Wailes); h, C. fiisus, XlOO (Wailes); i, Heterodinium scrippsi, X570 (Kofoid and Adamson). fresh or salt water. Numerous species; specific identification is difficult due to a great variation (p. 162). DINOFLAGELLATA 231 C. hirundineUa (Muller) (Figs. 80; 104, e). 1 apical and 2-3 antapical horns; seasonal and geographical variations; chain-for- mation frequent; 95-700yu long; fresh and salt water. Numerous varieties. C. longpipes (Bailey) (Fig. 104,/). About 210^ by 51-57^; salt water. C. tripos (Miiller) (Fig. 104, g). About 225)U by 75/x; salt water. Wailes (1928) observed var. atlantica in British Columbia; Mar- tin (1929) in Barnegat Inlet, New Jersey. C. fusus (Ehrenberg) (Fig. 104, h). 300-600^ by 15-30^; salt water; widely distributed; British Columbia (Wailes), New Jer- sey (Martin), etc. Genus Heterodinium Kofoid. Flattened or spheroidal; 2 large antapical horns; annulus submedian; with post-cingular ridge; sulcus short, narrow; shell hyahne, reticulate, porulate; salt wa- ter. Numerous species. H. scippsi K. (Fig. 104, i). 130-155/x long; Pacific and Atlantic (tropical). Genus Dolichodinium Kofoid et Adamson. Subcorneal, elon- gate; wdthout apical or antapical horns; sulcus not indenting epi- theca; plate porulate; salt water. D. lineatum (Kofoid et Michener) (Fig. 105, a). 58/x long; east- ern tropical Pacific. Genus Goniodoma Stein. Polyhedral with a deep annulus; epi- theca and hypotheca slightly unequal in size, composed of regu- larly arranged armored plates; chromatophores small brown platelets ; fresh or salt water. G. acuminata (Ehrenberg) (Fig. 105, h). About 50^ long; S'alt water. Genus Gonyaulax Diesing. Spherical, polyhedral, fusiform, or elongated with stout apical and antapical prolongations, or dorso- ventrally flattened; apex never sharply attenuated; annulus equa- torial; sulcus from apex to antapex, broadened posteriorly; plates 1-6 apical, 0-3 anterior intercalaries, 6 precingulars, 6 annular plates, 6 postincingulars, 1 posterior intercalary and 1 antapical; porulate; chromatophores yellow to dark brown, often dense; without stigma; fresh, brackish or salt water. Numerous species. G. polyedra Stein (Fig. 105, c). Angular, polyhedral; ridges along sutures, annulus displaced 1-2 annulus widths, regularly pitted; salt water. "Very abundant in the San Diego region in the 232 PROTOZOOLOGY Fig. 105. a, Dolichodinium lineatum, X670 (Kofoid and Adamson); b, Goniodoma acuminata, x340 (Stein); c, Gorumlax polyedra, X670 (Kofoid); d, G. apiculata, x670 (Lindemann); e, Spiraulax jolliffei, right side of theca, X340 (Kofoid) ; f, Dinophysis aciita, X580 (Schiitt); g, h, Oxyphysis oxytoxoides, X780 (Kofoid); i, Phytodinium simplex, X340 (Klebs); j, k, Dissodinium lunula: j, primary cyst (Dogiel); k, secondary cyst with 4 s warmers (Wailes), X220. summer plankton, July-September, when it causes local out- breaks of 'red water,' which extend along the coast of southern and lower California" (Kofoid, 1911). G. apiculata (Penard) (Fig. 105, d). Ovate, chromatophores yellowish brown; 30-60^ long; fresh water. Genus Spiraulax Kofoid. Bi conical; apices pointed; sulcus not reaching apex; no ventral pore; surface heavily pitted; salt water. DINOFLAGELLATA 233 S. jolliffei (Murray et Whitting) (Fig. 105, e). 132^ by 92m; California. Family 2 Dinophysidae Kofoid Genus Dinophysis Ehrenberg. Highly compressed; annulus widened, funnel-like, surrounding small epitheca; chromato- phores yellow; salt water. Several species. D. acuta E. (Fig. 105,/). Oval; attenuated posteriorly; 54-94/i long; widely distributed; British Columbia (Wailes). Genus Oxyphysis Kofoid. Epitheca developed; sulcus short; sulcal lists feebly developed; sagittal suture conspicuous; annulus impressed; salt water. 0. oxytoxoides K. (Fig. 105, g, h). 63-68ai by 15/^; off Alaska. Family 3 Phytodiniidae Klebs Genus Phytodinium Klebs. Spherical or ellipsoidal; without furrows; chromatophores discoidal, yellowish brown. P. simplex K. (Fig. 105, i). Spherical or oval; 42-50/i by 30- 45m; fresh water. Genus Dissodinium Klebs (Pyrocystis Paulsen). Primary cyst, spherical, uninucleate; contents divide into 8-16 crescentic sec- ondary cysts which become set free; in them are formed 2, 4, 6, or 8 Gymnodinium-like swarmers ; salt water. D. lunula (Schlitt) (Fig. 105, j, k). Primary cysts 80-155m in diameter; secondary cysts 104-130/1 long; swarmers 22^ long; widely distributed; British Columbia (Wailes). Fig. 106. a, Leptodiscus medusoides, X50 (Hertwig); b, Craspedotella pileolus, XllO (Kofoid). Suborder 3 Cystoflagellata Haeckel Since Noctiluca which had for many years been placed in this suborder has been removed, according to Kofoid, to the second suborder, the Cystoflagellata becomes a highly ill-defined group and includes two peculiar marine forms: Leptodiscus medusoides Hertwig (Fig. 106, a), and Craspedotella pileolus Kofoid (Fig. 106, b), both of which are medusoid in general body form. 234 PROTOZOOLOGY References Chatton, E. 1920 Les Peridieniens parasites; morphologie, reproduction, ethologie, Arch. zool. exp. et gen. Vol. 59. Fritsch, F, E. 1935 The structure and reproduction of the algae. Vol. 1. Cambridge. Gross, F. 1934 Zur Biologic und Entwicklungsgeschichte von Noctiluca miliaris. Archiv. f. Protistenk., Vol. 83. KoFOiD, C. A. 1906 On the significance of the symmetry of the Dinoflagellata. Uni. Calif. Piibl. Zool., Vol. 3. 1920 A new morphological interpretation of Noctiluca and its bearing on the status of Cystoflagellata. Ibid., Vol. 19. and A. M. Adamson 1933 The Dinoflagellata: The family Heterodiniidae of the Peridinioidae. Mem. Mus. Comp. Zool., Harvard, Vol. 54. and Olive Swezy. 1921 The free-living unarmored Dinoflagellata. Mem. Uni. Calif., Vol. 5. Lebour, Marie V. 1925 The dinoflagellates of northern seas. London. Martin, G. W. 1929 Dinoflagellates from marine and brackish waters of New Jersey. Uni. Iowa Studies in Nat. Hist., Vol. 12. Reichenow, E. 1930 Parasitische Peridinea. Grimpe's Die Tier- welt der Nord- und Ostsee. Part 19. Schilling, A. 1913 Dinoflagellatae (Peridineae). Siisswasser- fiora Deutschlands, etc. H. 3. Wailes, G. H. 1928 Dinoflagellates and Protozoa from British Columbia. Vancouver Mus. Notes. Vol. 3. Chapter 12 Subclass 2 Zoomastigina Doflein THE Zoomastigina lack chromatophores and their body or- ganizations vary greatly from a single to a very complex type. The majority possess a single nucleus which is, as a rule, vesicular in structure. A characteristic organella, the parabasal body (p. 66) is present in numerous forms and myonemes are found in some species. Nutrition is holozoic or saprozoic (parasitic). Asex- ual reproduction is by longitudinal fission; sexual reproduction is unknown. Encystment occurs commonly. The Zoomastigina are free-living or parasitic in various animals. With pseudopodia besides flagella Order 1 Rhizomastigina With flagella only With 1-2 flagella Order 2 Protomonadina (p. 239) With 3-8 flagella Order 3 Polyraastigina (p. 260) With more than 8 flagella Order 4 Hypermastigina (p. 277) Order 1 Rhizomastigina Biitschli A number of borderline forms between the Sarcodina and the Mastigophora are placed here. Flagella vary in number from one to several and pseudopods also vary greatly in number and in ap- pearance. With many flagella Family 1 Multiciliidae With 1-3 rarely 4 flagella Family 2 Mastigamoebidae (p. 236) Family 1 Multiciliidae Poche Genus Multicilia Cienkowski. Generally spheroidal, but amoe- boid; with 40-50 flagella, long and evenly distributed; one or more nuclei; holozoic; food obtained by means of pseudopodia; contractile vacuoles numerous ; multiplication by fission ; fresh or salt water. M. marina C. (Fig. 107, a). 20-30/x in diameter; uninucleate; salt water. M. lacustris Lauterborn (Fig. 107, h). Multinucleate; 30-40m in diameter ; fresh water. 235 236 PROTOZOOLOGY Fig. 107. a, Multicilia marina, x400 (Cienkowski) ; b, M. lacustris, X400 (Lauterborn); c, Mastigamoeba aspera, X200 (Schulze); d, M. longifilum, X340 (Stokes); e, M. setosa, x370 (Goldschmidt) ; f, Masti- gella vitrea, x370 (Goldschmidt). Family 2 Mastigamoebidae With 1-3 or rarely 4 flagella and axopodia or lobopodia; uni- nucleate; flagellum arises from a basal granule which is often con- nected with the nucleus by a rhizoplast; binary fission in both trophic and encysted stages; sexual reproduction has been re- ported in one species; holozoic or saprozoic; the majority are free-living, though a few parasitic. Genus Mastigamoeba Schulze (Mastigina Frenzel). Mono- mastigote, uninucleate, with finger-like pseudopodia; flagellum long and connected with nucleus; fresh water, soil or endocom- mensal. ZOOMASTIGINA, RHIZOMASTIGINA 237 M. aspera S. (Fig. 107, c). Subspherical or oval; during locomo- tion elongate and narrowed anteriorly, while posterior end rounded or lobed; numerous pseudopods slender, straight; nu- cleus near flagellate end; 2 contractile vacuoles; 150-200^1 by about 50/x; in ooze of pond. M. longifilum Stokes (Fig. 107, d). Elongate, transparent flagel- lum twice body length; pseudopods few, short; contractile vacu- ole anterior; body 28^^ long when extended, contracted about 10/x stagnant water. M. setosa (Goldschmidt) (Fig. 107, e). Up to 140/x long. Fig. 108. a, Mastigamoeba hylae, X690 (Becker); b, Adinomonas mirabilis, X1140 (Griessmann); c, Dimoryha mutans, X940 (Bloch- mann); d, Pteridomonas pulex, X540 (Penard); e, Histomonas melea- gris, X940 (Tyzzer); f, Rhizomastix gracilis, X1340 (Mackinnon). M. hylae (Frenzel) (Fig. 108, a). In hind gut of frogs and tad- poles; 80-100/x by 20ju; flagellum about IOjjl long. Genus Mastigella Frenzl. Flagellum apparently not connected with nucleus; pseudopods numerous, digitate; body form changes actively and continuously; contractile vacuole. M. vitrea Goldschmidt (Fig. 107,/). I50fx long; sexual reproduc- tion (Goldschmidt). Genus Actinomonas Kent. Generally spheroidal, with a single 238 PHUTOZUOl.OC.Y flagellum and radiating pseudopods; ordinarily attached to for- eign object with a cytoplasmic process, but swims freely by with- drawing it; nucleus central; several contractile vacuoles; holo- zoic. A. mirabilis K. (Fig. 108, h). Numerous simple filopodia; about lO^i in diameter; flagellum 20yu long; fresh water. Genus Dimorpha Gruber. Ovoid or subspherical ; with 2 flagella and radiating axopodia, all arising from an eccentric centriole ; nucleus eccentric; pseudopods sometimes withdrawn; fresh water. D. mutans G. (Fig. 108, c). 15-20yu in diameter; flagella about 20-30m long. Genus Pteridomonas Penard. Small, heart-shaped; usually at- tached with a long cytoplasmic process ; from opposite pole there arises a single flagellum, around which occurs a ring of extremely fine filopods; nucleus central; a contractile vacuole; holozoic; fresh water. P. pulex P. (Fig. 108, d). 6-12^ broad. Genus Histomonas Tyzzer. Parasitic in domestic fowls; body amoeboid with a blepharoplast; indication of flagellum (?); with- out cytostome. H. meleagris (Smith) (Fig. 108, e). 12-21/i long; rounded re- sistant phase up to ll^t by 9;u; the cause of the "blackhead" dis- ease of young turkeys, chicken, grouse, and quail (Tyzzer) in which the intestinal mucosa as well as liver tissues become in- fected by this organism. Genus Rhizomastix Alexeieff. Body amoeboid; nucleus central: blepharoplast located between nucleus and posterior end; a long fiber (rhizostyle) runs from it to anterior end and continues into the flagellum; without contractile vacuole; division in spherical cyst. R. gracilis A. (Fig. 108, /). 8-14/i long; flagellum l^ifx long; in intestine of axolotles and tipulid larvae. References Becker, E. R. 1925 The morphology of Mastigina hylae (Fren- zel) from the intestine of the tadpole. Jour. Paras., Vol. 11. Lemmermann, E. 1914 Pantostomatinae. Siisswasser flora Deutschlands, etc. H. 1. Tyzzer, E. E. 1919 Developmental phases of the Protozoon of "blackhead" in Turkeys. Jour. Med. Res., Vol. 40. Chapter 13 Order 2 Protomonadina Blochmann THE protomonads possess one or two flagella and are com- posed of a heterogeneous lot of Protozoa, mostly parasitic, whose affinities to one another are very incompletely known. The body is in many cases plastic, having no definite pelhcle, and in some cases amoeboid. The method of nutrition is holozoic, or saprozoic (parasitic). Reproduction is, as a rule, by longitudinal fission, although budding or multiple fission has also been known to occur, while sexual reproduction, though reported in some forms, has not been confirmed. With 1 flagellum With cytoplasmic collar Collar enclosed in jelly Family 1 Phalansteriidae Collar not enlosed in jelly Without lorica Family 2 Codosigidae With lorica Family 3 Bicosoecidae (p. 241) Without cytoplasmic collar Free-living Family 4 Oikomonadidae (p. 243) Parasitic Family 5 Trypanosomatidae (p. 244) With 2 flagella With undulating membrane Family 6 Cryptobiidae (p. 252) Without undulating membrane Flagella equally long Family 7 Amphimonadidae (p. 253) Flagella unequallj' long no traiUng flagellum Family 8 Monadidae (p. 255) one flagellum trailing Family 9 Bodonidae (p. 256) Family 1 Phalansteriidae Kent Genus Phalansterium Cienkowski. Small, ovoid; one flagellum and a narrow collar; numerous individuals are embedded in gelat- inous substance which presents a dendritic form, with protruding flagella; fresh water. P. digitatum Stein (Fig. 109, a). Cells about 17/i long; oval; colony dendritic; fresh water among vegetation. Family 2 Codosigidae Kent Small flagellates, sometimes with second flagellum which serves for fixation of body; delicate collar surrounds flagellum; ordina- 239 240 PROTOZOOLOGY rily sedentary forms; if temporarily freed, organisms swim with fiagellum directed backward; holozoic on bacteria or saprozoic; often colonial; free-living in fresh water. Genus Codosiga Kent (Codonocladium Stein; Astrosiga Kent). Individuals clustered at end of a simple or branching stalk; fresh water. C. utriculus Stokes (Fig. 109, b). About ll/j. long; attached to freshwater plants. C. disjuncta (Fromentel) (Fig. 109, c). In stellate clusters; cells about 15/x long; fresh water. Genus Monosiga Kent. Solitary; with or without stalk; occa- sionally with short pseudopodia; attached to freshwater plants. Several species. Fig. 109. a, Phalansterium digitatum, X540 (Stein); b, Codosiga utriculus, X1340 (Stokes); c, C. disjuncta, X400 (Kent); d, Monosiga ovata, X800 (Kent); e, M. rohusta, x770 (Stokes); f, Desmarella nioniliformis, X800 (Kent) ; g, Proiospon^ia haeckeli, X400 (Lemmer- mann); h, Sphaeroeca volvox, X890 (Lenimermann) ; i, Diplosiga francei, X400 (Lemmermann) ; j, D. socialis, X670 (Franc6). PROTOMONADINA 241 M. ovata K. (Fig. 109, d). 5-1 5m long; with a short stalk. M. robusta Stokes (Fig. 109, e). 13/i long; stalk very long. Genus Desmarella Kent. Cells united laterally to one another; fresh water. D. moniliformis K. (Fig. 109, /). Cells about 6/u long; cluster composed of 2-12 individuals; standing fresh water. D. irregularis Stokes. Cluster of individuals irregularly branch- ing, composed of more than 50 cells; cells 7-1 l/x long; pond water. Genus Protospongia Kent. Stalkless individuals embedded ir- regularly in a jelly mass, collars protruding; fresh water. P. haeckeli K. (Fig. 109, g). Body oval; 8^ long; flagellum 24- 32/i long; 6-60 cells in a colony. Genus Sphaeroeca Lauterborn. Somewhat similar to the last genus; but individuals with stalks and radiating; gelatinous mass spheroidal; fresh water. S. volvox L. (Fig. 109, h). Cells ovoid, 8-12^ long; stalk about twice as long; flagellum long; contractile vacuole posterior; colony 82-200m in diameter; fresh water. Genus Diplosiga Frenzel (Codonosigopsis Senn). With 2 collars; without lorica; a contractile vacuole; solitary or clustered (up to 4) ; fresh water. D. francei Lemmermann (Fig. 109, i). With a short pedicle; 12m long; flagellum as long as body. D. socialis F. (Fig. 109, j). Body about 15yu long; usually 4 ■ clustered at one end of stalk (15^ long). Family 3 Bicosoecidae Poche Small monomastigote; with lorica; solitary or colonial, collar may be rudimentary; holozoic; fresh water. Genus Bicosoeca James-Clark. With vase-Hke lorica; body small, ovoid with rudimentary collar, a flagellum extending through it ; protoplasmic body anchored to base by a cytoplasmic filament (flagellum?); a nucleus and a contractile vacuole; at- tached or free-swimming. B. socialis Lauterborn (Fig. 110, a). Lorica cylindrical, 23m by 12m; body about 10m long; often in groups; free-swimming in fresh water. Genus Salpingoeca James-Clark. With a vase-like chitinous lorica to which stalked or stalkless organism is attached; fresh or salt water. Numerous species. 242 PROTOZOOLOGY S. fusiforynis Kent (Fig. 110, b). Lorica short vase-like, about 15-16m long; body filling lorica; flagellum as long as body; fresh water. Fig. 110. a, Bicosoeca socialis, X560 (Lauterborn); b, Salpingoeca Jusijormis, X400 (Lemmermann); c, Diplosigopsis affinis, X590 (Franc6); d, Histiona zachariasi, x440 (Lemmermann); e, Poterioden- dron petiolatum, X440 (Stein); f, Codonoeca inclinata, X540 (Kent); g, Lagenoeca ovata, x400 (Lemmermann). Genus Diplosigopsis France. Similar to Diplosiga (p. 241), but with lorica; solitary; fresh water on algae. D. affinis Lemmermann (Fig. 110, c). Chitinous lorica, spindle- form, about \bn long; body not filling lorica; fresh water. Genus Histiona Voigt. With lorica; but body without attaching filament; anterior end with lips and sail-like projection; fresh water. H. zachariasi V. (Fig. 110, d). Lorica cup-like; without stalk; about ISyu long; oval body 13/i long; flagellum long; standing fresh water. Genus Poteriodendron Stein. Similar to Bicosoeca; but colo- nial; lorica vase-shaped; with a prolonged stalk; fresh water. P. petiolatum (S.) (Fig. 110, e). Lorica 17-50/x high; body 21- 35m long; flagellum twice as long as body; contractile vacuole terminal ; standing fresh water. Genus Codonoeca James-Clark. With a stalked lorica; a single flagellum; 1-2 contractile vacuoles; fresh or salt water. C. inclinata Kent (Fig. 110,/). Lorica oval; aperture truncate; about 23ju long; stalk twice as long; body oval, about 17/x long; PROTOMONADINA 243 flagellum 1.5 times as long as body; contractile vacuole posterior; standing fresh water. Genus Lagenoeca Kent. Resembles somewhat Salpingoeca; with lorica; but without any pedicle between body and lorica; solitary; free-swimming; fresh water. L. ovata Lemmermann (Fig. 110, g). Lorica oval, IS/z long; body loosely filling lorica; flagellum 1.5 times body length; fresh water. Family 4 Oikomonadidae Hartog Genus Oikomonas Kent. A rounded monomastigote; uninu- cleate; encystment common; stagnant water, soil and exposed faecal matter. Fig. 111. a, Oikomonas termo, X1330 (Lemmermann); b, Thylaco- monas compressa, X640 (Lemmermann); c, Ancijromonas contorta, X2000 (Lemmermann); d, Platytheca microspora, X650 (Stein). 0. termo (Ehrenberg) (Fig. Ill, a). Spherical or oval; anterior end hp-like; flagellum about twice body length; a contractile vacuole; 5-9/i in diameter; stagnant water. Genus Thylacomonas Schewiakoff. Pellicle distinct; cytostome anterior; one flagellum; contractile vacuole anterior; rare. T. compressa S. (Fig. Ill, fe). 22^t by IS/x; flagellum body length; fresh water. Genus Ancyromonas Kent. Ovate to triangular; free-swimming or adherent ; flagellum trailing, adhesive or anchorate at its distal end, vibratile throughout remainder of its length; nucleus central; a contractile vacuole; fresh or salt water. A. contorta (Klebs) (Fig. Ill, c). Triangular, flattened, poste- rior end pointed; 6-7/x by 5-6^; flagellum short; a contractile vac- uole ; standing fresh water. Genus Platytheca Stein. With a flattened pyriform lorica, with a small aperture; 1 or more contractile vacuoles; fresh water. 244 PKOTOZOOLOCY P. microspora S. (Fig. Ill, d). Lorica yellowish brown, with a small aperture; 12-18// long; flagcllum short; among roots of Lemna. Family 5 Trypanosomatidae Doflein Body characteristically leaf-like, although changeable to a cer- tain extent; a single nucleus and a blepharoplast; a flagellum originates in a basal granule w^hich may be independent from, or united with, the blepharoplast (Figs. 9, 112); basal portion of flagellum forms outer margin of undulating membrane which ex- tends along one side of body; exclusively parasitic; a number of important parasitic Protozoa which are responsible for serious diseases of man and domestic animals in various parts of the world are included in it. In vertebrate host In invertebrate host In vertebrate host Trypanosoma Trypanosoma Crithidia Leptomonas Leishmania Leishmania Leptomonas and Phytomonas (in plant) Leishmania Crithidia Herpetomonas Trypanosoma Fig. 112. Diagram illustrating the structural differences among the genera of Trypanosomatidae (Wenyon). Genus Trypanosoma Gruby. Parasitic in the circulatory system of vertebrates; highly flattened, pointed at flagellate end, and bluntly rounded, or pointed, at other; polymorphism due to dif- ferences in development common; nucleus central; near bluntly rounded end, there is a blepharoplast and usually a basal granule from which the flagellum arises and runs toward^opposite end, marking the outer boundary of the undulating membrane; in PROTOMONADINA 245 most cases flagelliim extends freely beyond body; many with myonemes; multiplication by binary or multiple fission. The or- ganism is carried from host to host by blood-sucking invertebrates and undergoes a series of changes in the digestive system of the latter (Fig. 113). A number of forms are pathogenic to their hosts and the diseased condition is termed trypanosomiasis in general. T. gambiense Button (Fig. 114, a). Parasitic in blood and lymph of man in certain regions of Africa; transmitted by the tsetse fly, Glossi7ia palpalis; reservoir hosts are domestic and wild animals. Body 15-30m long; mature forms slender and long, w^th a long flagellum; individuals formed by longitudinal fission short and broad with no projecting flagellum; half-grown forms intermediate in size and structure; the cause of the "sleeping sickness" of man in Africa. T. (Schizotrypanum) cruzi (Chagas) (Fig. 114, h). Parasitic in children in South America (Brazil, Peru, Venezuela, etc.). A small curved form about 20/i long; nucleus central; blepharoplast large, located close to sharply pointed non-flagellate end; multi- plication takes place in the cells of nearly every organ of the host body; upon entering a host cell, the trypanosome loses its flagel- lum and undulating membrane, and assumes a leishmania form which measures 2 to 5yu in diameter; this form undergoes repeated binary fission, and a large number of daughter individuals are produced; they develop sooner or later into trypanosomes which, through rupture of host cell, become liberated into blood stream ; transmitted by the reduviid bug, Triatoma megista and allied species; the diseased condition is known as "Chagas' disease." Apparently the organism occurs in wood rats (Neotoma) and meadow mice (Microtus) in south-western United States, trans- mitted from host to host by the cone-nose or kissing bug, Triatoma protracta (Kofoid and others). T. hrucei PHmmer et Bradford (Figs. 9, a; 114 c). Polymorphic; 15-30/i long (average 20^); transmitted by various species of tsetse flies, Glossina; the most virulent of all trypanosomes; the cause of the fatal disease known as "nagana" among mules, don- keys, horses, camels, cattle, swine, dogs, etc., which terminates in the death of the host animal in from two weeks to a few months ; wild animals are equally susceptible; the disease occurs, of course, only in the region in Africa where the tsetse flies live. 246 PROTOZOOJ.OGY Fig. 113. The life-cycle of Trypanosoma lewisi in the flea, Cera- tophyllus fasciattis (Minchin and Thomson, modified), a, trypanosome from rat's blood; b. individual after being in flea's stomach for a few hours; c-1, stages in intracellular schizogony in stomach epithelium; m-r, two ways in which rectal phase may arise from stomach forms in rectum; s, rectal phase, showing various types; t, secondary infection of pylorus of hind-gut, showing forms similar to those of rectum. PROTOMONADINA 247 Fig. 114. a, Trypanosoma gambiense (5 individuals) and a human erythrocyte; b, T. cruzi; c, T. hrucei; d, T. theileri; e, T. vielophagimn ; f, T. evansi; g, T. equinum; h, T. equiperdum, all X 1000 (various in- vestigators) . T. theileri Layeran (Fig. 114, d). Non-pathogenic large tryp- anosome which occurs in blood of cattle; sharply pointed at both ends; 60-70/x long; myonemes are well developed. T. americanum Crawley. In American cattle; probably iden- tical with T. theileri; transmitted from cattle to cattle by tabanid flies. T. melophagium (Flu) (Fig. 114, e). Non-pathogenic trypano- some of the sheep; 50-60yu long with attenuated ends ; transmitted by Melophagus ovinus. T. evansi (Steel) (Fig. 114,/). In horses, mules, donkeys, cattle, dogs, camels, elephants, etc.; infection in horses seems to be usu- ally fatal and known as "surra"; about 25/ilong; monomorphic; transmitted by tabanid flies; widely distributed. T. equinum Vages (Fig. 114, g). In horses in South America, causing an acute disease known as "mal de Caderas"; other do- mestic animals do not suffer as much as do horses; 2()-25fx long; without blepharoplast. T. equiperdum Doflein (Fig. 114, h). In horses and donkeys; causes "dourine," a chronic disease; widely distributed; 25-30)u long; no intermediate host; transmission takes place directly from host to host during sexual act. T. lewisi (Kent) (Figs. 113; 114, i). In blood of various species of rat; widely distributed; non-pathogenic under ordinary condi- tions; about 25/x long; very active; slender; with a long flagellum; 248 PROTOZOOLOGY transmitted by the flea, Ceratophyllus fasciatus, in which the or- ganism undergoes changes (Fig. 113); when, a rat swallows freshly voided faecal matter containing the organisms, it becomes in- fected. T. duttoni Thiroux. In the mouse; similar to T. lewisi, but rats are not susceptible,, hence considered as a distinct species; trans- mission by fleas. T. peromysci Watson. Similar to T. lewisi; in Canadian deer mice, Peromyscus maniculatus and others. T. nahiasi Railliet. Similar to T. lewisi; in rabbits, Lepus domesticus and L. cuniculus. T. paddae Laveran et Mesnil. In Java sparrow, Munia oryzi- vora. T. noctuae (Schaudinn). In the little owl, Athene noctua. Several other species are known. Crocodiles, snakes, and turtles are hosts for Trypanosoma. Transmission by blood-sucking arthropods or leeches. T. rotatorium (Meyer) (Fig. 115, a). In tadpoles and adults of various species of frog ; between a slender form with a long pro- jecting flagellum measuring about 35m long and a very broad one without free portion of flagellum, various intermediate forms are to be noted in a single host; blood vessels of internal organs, such as kidneys, contain more individuals than the peripheral vessels; nucleus central, hard to stain; blepharoplast small; undulating membrane highly developed; myonemes prominent; multiplica- tion by longitudinal fission; the leech, Placohdella marginata, has been found to be the transmitter in some localities. T. inopinatum Sergent et Sergent (Fig. 115, b). In blood of various frogs; slender; 12-20^ long; larger forms 30-35/i long; blepharoplast comparatively large; transmitted by leeches. Numerous species of Trypanosoma have been reported from the frog, but specific identificsPtion is indistinct; it is better and safer to hold that they belong to one of the 2 species mentioned above until their development and transmission become known. T. diemyctyli Tobey (Fig. 115, c). In blood of the newt, Tri- turus viridescens ; a comparatively large form; slender; about 50^ by 2-5^; flagellum 20-25;u long; with well developed undulating membrane. Both fresh and salt water fish are hosts to different species of trypanosomes; what effects these parasites exercise upon the host PROTOMONADINA 249 Fig. 115. a, Tnj-panosovia rolatoriuvi, X750 (Kudo); b, T. ino'pina- tum, X1180 (Kudo); c, T. diemyctyli, X800 (Hegner); d, T. giganteum, X500 (Neumann); e, T. granulosum, XlOOO (Minchin); f, T. reniaki, X1650 (Kudo); g, T. percae, XlOOO (Minchin); h, T. danilewskyi, XlOOO (Laveran and Mesnil); i, T. rajae, X1600 (Kudo), fish are not understood; as a rule, only a few individuals are ob- served in the peripheral blood of the host. T. granulosum Laveran et Mesnil (Fig. 115, e). In the eel, An- guilla vulgaris; 70-80/^ long. T. giganteum Neumann (Fig. 115, d). In Raja oxyrhynchus; 125-130Mlong. T. remaki Laveran et Mesnil (Fig. 115,/). In Esox lucius, E. reticulatus and probably other species; dimorphic; 24-33/i long. T. percae Brumpt (Fig. 115, g). In Perca fluviatilis; 45-50)U long. T. danilewskyi Laveran et Mesnil (Fig. 115, h). In carp and goldfish; widely distributed; 40/x long. T. rajae Laveran et Mesnil (Fig. 115, i). In various species of Raja; 30-35/x long. Genus Crithidia Leger. Parasitic in arthropods and other in- vertebrates; blepharoplast located between central nucleus and fiagellum-bearing end (Fig. 112); undulating membrane not so well developed as in Trypanosoma; it may lose the flagellum and form a leptomonas or rounded leishmania stage which leaves host 250 PKOTOZOOLOGY intestine with faecal matter and becomes the source of infection in other host animals. C. gerridis Patton (Fig. 116, d). In intestine of water bugs, Ger- ris and Micro veha; 22-45)U long. C. hyalommae O'Farrell (Fig. 116, e, /). In body cavity of the cattle tick, Hyalomma aegyptmm in Egypt; the flagellate through its invasion of ova is said to be capable of infecting the offspring while it is still in the body of the parent tick. C. euryophthalmi McCulloch (Fig. 116, a-c). In gut of Eury- ophthalmus convivus; California coast. Fig. 116. a-c, Crithidia euryophthalmi (a, b, in mid-gut; c, in rec- tum), X880 (McCulloch); d, C. gerridis, X1070 (Becker); e, f, C. hyalommae, XlOOO (O'Farrell); g, h, Leptomonas denocephali, XlOOO (Wenyon); i, j, Phytomonas elmassiani (i, in milkweed, Asclepias sp.; j, in gut of a suspected transmitter, Oncopeltus fasciatus), X1500 (Holmes); k, Herpetomonas muscarum, X1070 (Becker); 1-n, H. drosophilae, XlOOO (Chatton and L^ger). Genus Leptomonas Kent. Exclusively parasitic in inverte- brates; blepharoplast very close to flagellate end; without un- dulating membrane (Fig. 112); non-flagellate phase resembles Leishmania. L. ctenocephali Fantham (Fig. 116, g, h). In hindgut of the dog flea, Ctenocephalus cants; widely distributed. Genus Phytomonas Donovan. Morphologically similar to Lep- tomonas (Fig. 112); in the latex of plants belonging to the fami- Hes: Euphorbiaceae, Asclepiadaceae, Apocynaceae, Sapotaceae and Utricaceae; transmitted by hemipterous insects; often found in enormous numbers in localized areas in host plant; infection PROTOMOXADINA 251 spreads from part to part; infected latex is a clear fluid, owing to the absence of starch grains and other particles, and this results in degeneration of the infected part of the plant. Several species. P. davidi (Lafront). 15-20/z by about 1.5)u; posterior portion of body often twisted two or three times; multiplication by longi- tudinal fission; widely distributed; in various species of Euphor- bia. P. elmassiani (Migone) (Fig. 116, i, j). In various species of milk- weeds; 9-20;u long; suspected transmitter, Oncopeltus fas- ciatus (Holmes) ; in South and North America. Genus Herpetomonas Kent. Ill-defined genus (Fig. 112); ex- clusively invertebrate parasites; Trypanosoma-, Crithidia-, Lep- tomonas-, and Leishmania-forms occur during development. Several species. H. muscaruyn (Leidy) {H. muscae-domesticae (Burnett)) (Fig. 116, k). In gut of flies, belonging to the genera Musca, CalHphora, Sarcophaga, Lucilia, Phormia, etc.; up to 30a£ by 2-3//. H. drosophilae (Chatton et Alilaire) (Fig. 116, l-n). In intestine of Drosophila confusa; large leptomonad forms 21-25^ long, flagellum body-length; forms attached to rectum 4-5^ long. Genus Leishmania Ross. Parasitic in vertebrate and inverte- brate hosts, the latter not having been actually demonstrated, but suspected; non-flagellate and flagellate forms occur (Fig. 112); very minute; in vertebrate host the organism not flagellated; spherical or ovoid, with a definite pellicle; nucleus eccentric; a blepharoplast ; 2-5;u in diameter; organism enters endothelial cells of blood capillaries and mucosae; spleen becomes highly en- larged; transmitting agent believed to be blood-sucking arthro- pods; in culture, the organism develops into leptomonad forms; 4 "species" in man, all of which are practically indistinguishable morphologically from one another, and 2 of which are considered as identical. L. donovani (Laveran et Mesnil) (L. infantum Nicolle) (Fig. 117, a-f). The organism attacks endothelial cells and macrophage of man, causing the disease known as "kala azar"; it occurs in India, China, west to southern Russia, and regions bordering the Mediterranean Sea. L. tropica (Wright) (Fig. 117, g, h). The organism invades ex- posed skin and sometimes mucous lining of mouth, pharynx, and nose of man; the disease is known as "Oriental sore"; distribution is similar to the above-mentioned species. 252 PROTOZOOLOGY ^ Q Q a Fig. 117. a-f, Leishmania donovani (a, three individuals from lymph smear of a kala azar patient; b, from a spleen smear; c-f, cultural forms), X2000 (Wenyon; Thomson and Robertson); g, h, L. tropica (g, from an Oriental sore; h, the organisms in a polymorphonuclear cell from a sore), X2000 (Wenyon; Thomson and Robertson); i, Cryptobia helicis, X2530 (Belaf); j, C. borreli, X870 (Mavor); k, C. cyprini, X890 (Plehn). L. hrasiliensis Vianna. The organism occurs in South and Cen- tral America; some authors consider this species as identical with L. tropica. Although morphologically identical, these species show specific serum reactions. Family 6 Cryptobiidae Poche Biflagellate trypanosome-like protomonads; 1 flagellum free, the other marks outer margin of undulating membrane; bleph- aroplast an elongated rod-like structure, often referred to as the parabasal body; all parasitic. Genus Cryptobia Leidy (Trypanoplasma Laveran et Mesnil). Parasitic in reproductive organs of molluscs and other inverte- brates and in blood and gut of fish. PROTOMONADINA 253 C. helicis L. (Fig. 117, i). In reproductive organs of various species of Helix in America and Europe; 6-20)U long; asexual re- production through binary fission. C. horreli (Laveran et Mesnil) (Fig. 117, j). In blood of various freshwater fish such as Catostomus, Cyprinus, etc. ; 20-25m long. C. cyprini (Plehn) (Fig. 117, k). In blood of carp and goldfish; lO-SOfx long; rare. C. grohbeni (Keysselitz). In coelenteric cavity of Siphonophora; about 65ju by 4/x. Family 7 Amphimonadidae Kent Body naked or with a gelatinous envelope; 2 equally long an- terior flagella; often colonial; 1-2 contractile vacuoles; free- swimming or attached; mainly fresh water. Genus Amphimonas Dujardin. Small oval or rounded amoe- boid; flagella at anterior end; free-swimming or attached by an elongated stalk-like posterior process; fresh or salt water. A. glohosa Kent (Fig. 118, a). Spherical; about 13yu in diameter; stalk long, delicate; fresh water. Genus Spongomonas Stein. Individuals in granulated gelati- nous masses; flagella with 2 basal granules; one contractile vacuole; colony often several centimeters high; with pointed pseudopodia in motile stage; fresh water. S. uvella S. (Fig. 118, h). Oval; 8-12yu long; flagella 2-3 times as long; colony about 50ju high; fresh water. Genus Cladomonas Stein. Individuals are embedded in dichot- omous dendritic gelatinous tubes which are united laterally; fresh water. C . fruticulosa S. (Fig. 118, c). Oval; about 8^ long; colony up to 85/i high. Genus Rhipidodendron Stein. Similar to Cladomonas, but tubes are fused lengthwise; fresh water. R. splendidurn S. (Fig. 118, d, e). Oval; about 13yu long; flagella about 2-3 times body length; fully grown colony 350^ high. Genus Spiromonas Perty. Elongate; without gelatinous cover- ing ; spirally twisted ; 2 flagella anterior ; solitary ; fresh water. S. augusta (Dujardin) (Fig. 118, /). Spindle-form; about lOfx long; stagnant water. Genus Diplomita Kent. With transparent lorica; body at- tached to bottom of lorica by a retractile filamentous process; a rudimentary stigma (?); fresh water. 254 PROTOZOOLOGY Fig. 118. a, AmpJmnonas globosa, X540 (Kent); b, Spongomonas uvella, X440 (Stein); c, Cladomonas fruticulosa, X440 (Stein); d, e, Rhipidodendron splendidum (d, a young colony, X440; e, a free- swimming individual, X770) (Stein); f, Spiromonas augusta, XlOOO (Kent); g, Diplomita socialis, XlOOO (Kent); h, Streptomonas cordata, X890 (Lemmermann); i, Dinomonas vorax, X800 (Kent). D. socialis K. (Fig. 118, g). Oval; flagellum about 2-3 times the body length; lorica yellowish or pale brown; broadly spindle in form; about 15/x long; pond water. Genus Streptomonas Klebs. Free-swimming; naked; distinctly keeled; fresh water. S. cordata (Perty) (Fig. 118, h). Heart-shaped; IS^t by 13/^; rota- tion movement. Genus Dinomonas Kent. Ovate or pyriform, plastic, free-swim- ming; 2 flagella, equal or sub-equal, inserted at anterior extrem- ity, where large oral aperture visible only at time of food inges- tion, is also located, feeding on other flagellates; vegetative infusions. D. vorax K. (Fig. 118, i). Ovoid, anterior end pointed; 15-16ju long; flagella longer than body; hay infusion and stagnant water. PROTOMONADINA Family 8 Monadidae Stein 255 2 unequal flagella; one primary and the other secondary; motile or attached; 1-2 contractile vacuoles; colony formation frequent; free-living. Genus Monas M tiller {Physomonas Kent). Plastic and actively motile ("dancing movement"); often attached to foreign objects; not longer than 20/^; known for a long time, but still very in- completely. Krijgsman (1925) studied the flagellar movements (p. 107). M. guttula Ehrenberg (Fig. 119, a). Spherical to ovoid; 14-16m long; free-swimming or attached; longer fiagellum about 1-2 times body length; cysts 12/x in diameter; stagnant water. M. elongata (Stokes) (Fig. 119, h). Elongate; about 11/x long; Fig. 119. a, Mo7ias guttula, X620 (Fisch); b, M. elongata, X670 (Stokes); c, M. socialis, X670 (Kent); d, M. vestita, X570 (Stokes); e, Stokesiella dissimilis, X500 (Stokes); f, S. leptostonia, x840 (Stokes); g, Stylohryon abbotti, X480 (Stokes); h, Dendromonas virgaria, a young colony of, X670 (Stein); i, Ce-phalothamniuni cyclopicm, X440 (Stein); j, k, Anthophysa vegetans (j, part of a colonv, X230; k, an individual, X770) (Stein). 256 PROTOZOOLOGY free-swimming or attached; anterior end obliquely truncate; fresh water. M. socialis (Kent) (Figs. 8, g; 119, c). Spherical; 5-10^ long; among decaying vegetation in fresh water. M. vestita (Stokes) (Fig. 119, d). Spherical; about 13.5/x in diameter; stalk about 40;u long; pond water. Reynolds (1934) made a careful study of the organism. Genus Stokesiella Lemmermann. Body attached by a fine cytoplasmic thread to a delicate and stalked vase-Hke lorica; 2 contractile vacuoles ; fresh water. S. dissimilis (Stokes) (Fig. 119, e). Solitary; lorica about 28yu long. S. leptostoma (S.) (Fig. 119,/). Lorica about 17yu long; often in groups; on vegetation. Genus Stylobryon Fromentel. Similar to Stokesiella; but colonial; on algae in fresh water. S. ahbotti Stokes (Fig. 119, g). Lorica campanulate; about 17^ long; main stalk about 100 fj. high; body oval or spheroidal; flagella short. Genus Dendromonas Stein. Colonial; individuals without lorica, located at end of branched stalks; fresh water among vegetation. D. virgaria (Weisse) (Fig. 119, h). About S/j, long; colony 200ijl high; pond water. Genus Cephalothamnium Stein. Colonial; without lorica, but individuals clustered at end of a stalk which is colorless and rigid; fresh water. C. cyclopum S. (Fig. 119, i). Ovoid; 5-10^ long; attached to body of Cyclops and also among plankton. Genus Anthophysa Bory. Colonial forms, somewhat similar to Cephalothamnium; stalks yellow or brownish and usually bent; detached individuals amoeboid with pointed pseudopodia. A. vegetans (Mliller) (Fig. 119, j, k). About 5-6/z long; common in stagnant water and infusion. Family 9 Bodonidae Btitschh With 2 flagella; one directed anteriorly and the other pos- teriorly and trailing; flagella arise from anterior end which is drawn out to a varying degree; one to several contractile vacuoles; asexual reproduction by binary fission; holozoic or saprozoic (parasitic). PROTOMONADINA 257 Genus Bodo Ehrenberg {Prowazekia Hartmann et Chagas). Small, ovoid, but plastic; cytostome anterior; nucleus central or anterior; flagella connected \^^th 2 blepharoplasts in some species; encystment common; in stagnant water and coprozoic. Numerous species. B. caudatus (Dujardin) (Fig. 120, a, 6). Highly flattened, usually tapering posteriorly; ll-22ju by 5-10)u; anterior flagellum about body length, trailing flagellum longer; blepharoplast ; cysts spherical; stagnant water. B. edax Klebs (Fig. 120, c). Oval with pointed anterior end; 11-15^1 by 5-7/x; stagnant water. Genus Pleuromonas Perty. Naked, somewhat amoeboid; usually attached with trailing flagellum; active cytoplasmic movement; fresh water. P. jaculans P. (Fig. 120, d). Body G-lO^t by about 5ai; flagellum 2-3 times body length; 4-8 young individuals are said to emerge from a spherical cyst ; stagnant water. Genus Rhynchomonas Klebs (Cruzella Faria, da Cunha et Pinto). Similar to Bodo, but there is an anterior extension of body, in which one of the flagella is embedded, while the other flagellum trails; a single nucleus; minute forms; fresh or salt water; also sometimes coprozoic. R. nasuta (Stokes) (Fig. 120, e). Oval, flattened; 5-6yLt by 2-3 fx; fresh water and coprozoic. R. marina (F., C. et P.). In salt water. Genus Proteromonas Kunstler (Prowazekella Alexeieff). Elon- gated pyriform; 2 flagella from anterior end, one directed an- teriorly and the other, posteriorly; nucleus anterior; encysted stage is remarkable in that it is capable of increasing in size to a marked degree; exclusively parasitic; in gut of various species of lizards. P. lacertae (Grassi) (Figs. 9, h; 120, /). Elongate, pyriform; 10-30/z long; gut of lizards belonging to the genera Lacerta, Tarentola, etc. Genus Retortamonas Grassi (Emhadomonas Mackinnon). Spindle-form or pyriform, drawn out posteriorly; ventral side usually more convex than dorsal side; large oval pouch on ventral side, about 1/3 as long as body; nucleus anterior; 2 flagella longer than body, anterior flagellum shorter than the posterior one which usually shows 2 or 3 undulations; cysts ovoid; parasitic in gut of various animals. PROTOZOOLOGY Fig. 120. a, b, Bodo caudatus, X1500 (Sinton); c, B. edax, X1400 (Kiihn); d, Pleuromonas jaculans, X650 (Lemmermann); e, Rhin- chomonas nasuta, X1800 (Parisi); f, Proteromonas lacertae, X2500 (Kiihn); g, Retortamonas gryllotalpae, X2000 (Wenrich); h, R. blattae, X2000 (Wenrich); i, R. intestinalis, X2000 (Wenrich); j, PhyUomitus undidans, XlOOO (Stein); k, Colj)onema loxodes, X650 (Stein); 1, Cer- comonas longicauda, X2000 (Wenyon); m, C. crassicauda, x2000 (Dobell). R. gnjllotalpae (G.) (Fig. 120, g). About 7-14/x (average lOyu) long; in intestine of the mole cricket, Gryllotalpa gryllotalpa. R. hlattae (Bishop) (Fig. 120, h). About Q-Q/j. long; in colon of cockroaches. R. intestinalis (Wenyon et O'Connor) (Fig. 120, i). 6-1 4/^ long; in human intestine. Genus PhyUomitus Stein. Oval; highly plastic; cytostome large and conspicuous; 2 unequal flagella, each originates in a basal granule; apparently no blepharoplast; fresh water or coprozoic. PROTOMONADINA 259 P. undulans S. (Fig. 120, j)- Ovoid; 21-27yu long; trailing flagel- lum much longer than anterior one; stagnant water. Genus Colponema Stein. Body small; rigid; ventral furrow conspicuous, wide at anterior end; one flagellum arises from anterior end and the other from middle of body; fresh water. C. loxodes S. (Fig. 120, A;). 18-30^1 by 14/^; cytoplasm with re- fractile globules. Genus Cercomonas Dujardin. Biflagellate, both flagella arising from anterior end of body; one directed anteriorly and the other runs backward over body surface, becoming a traihng flagellum; plastic; pyriform nucleus connected with the basal granules of flagella; spherical cysts uninucleate; fresh water or coprozoic. C. longicauda D. (Fig. 120, I). Pyriform or ovoid; posterior end drawn out; 18-36/x by 9-14/^; flagella as long as body; pseudo- podia; fresh water and coprozoic. C. crassicauda D. (Fig. 120, m). IO-ICai by 7-10/u; fresh water and coprozoic. References Holmes, F. O. 1925 The relation of Herpetomonas elmassiani (Migone) to its plant and insect hosts. Biol. Bull., Vol. 49. Laveran, a. and F. Mesnil. 1912 Trypanosomes et Trypano- somiases. Second edition. Paris. Lemmermann, E. 1914 Protomastiginae. Susswasserjl. Deutsch- lands, etc., H. 1. MiNCHiN, E. A. and J. D. Thomson. 1915 The rat trypanosome, Trypanosoma lewisi, in its relation to the rat flea, Cerato- phyllus fasciatus. Quart. Jour. Micr. Sci., Vol. 60. Nelson, R. 1922 The occurrence of Protozoa in plants affected with mosaic and related diseases. Michigan Agr. Coll. Bot. Stat., Tech. Bull. No. 58. Reynolds, B. D. 1934 Studies on monad flagellates. I, II. Arch. f. Protistenk., Vol. 81. Wenyon, C. M. 1926 Protozoology, Vol. 1. London. Chapter 14 Order 3 Polymastigina Blochmann THE Zoomastigina placed in this group possess 3-8 (in one family up to a dozen or more) flagella and generally speak- ing, are minute forms with varied characters and structures. Many possess a cytosome and one to many nuclei and the body is covered by a thin pellicle which allows the organism to change form, although each species shows a typical form. The cyto- plasm does not show any special cortical differentiation; in many, there is an axial structure known as axostyle or axostylar fila- ments (p. 61). In forms with an undulating membrane, there is usually a rod-like structure beneath the membrane which is known as costa (Kunstler). Parabasal body of various forms occur in many species. The majority of Polymastigina inhabit the di- gestive tract of animals and nutrition is holozoic or saprozoic (parasitic). Asexual reproduction is by longitudinal fission, some- times multiple. Encystment is common, and the cyst is responsi- ble for infection of new hosts through mouth. Sexual reproduction has not been definitely established. With 1 nucleus Suborder 1 Monomonadina With 2 nuclei Suborder 2 Diplomonadina (p. 272) With more than 2 nuclei Suborder 3 Polymonadina (p. 274) Suborder 1 Monomonadina Without axial organella With 3 flagella Family 1 Trimastigidae With 4 flagella None undulates on body surface. . T Family 2 Tetramitidae (p. 263) One undulates on body surface Family 3 Chilomastigidae (p. 264) With more than 4 flagella Family 4 Callimastigidae (p. 265) With axial organella Without undulating membrane. .Family 5 Polymastigidae (p. 265) With undulating membrane. .Family 6 Trichomonadidae (p. 269) Family 1 Trimastigidae Kent Free-swimming or attached; with 3 flagella; no cytostome; free- living in fresh or salt water, coprozoic or parasitic. 260 POLYMASTIGINA 261 Genus Trimastix Kent. Ovate or pyriform; naked; free- swimming; with a laterally produced membranous border; 3 flagella, 1 anterior flagellum vibrating, 2 trailing; salt water. 'M Fig. 121. a, Trimastix marina, X1250 (Kent); b, DaUingeria drys- dali, X2000 (Kent); c, Macromastix lapsa, XloOO (Stokes); d, En- teromonas hominis, X2000 (da Fonseca). T. marina K. (Fig. 121, a). About 18)U long; salt water. Genus DaUingeria Kent. Free-swimming or attached; with trailing flagella; body small; with drawn-out anterior end; fresh water with decomposed organic matter. D. drysdali K. (Fig. 121, b). Small; elongate oval; less than 6/x long; stagnant water. Genus Macromastix Stokes. Free-swimming, somewhat like DaUingeria, but anterior region not constricted; 3 flagella from anterior end; one contractile vacuole; fresh water. M. lapsa S. (Fig. 121, c). Ovoid; 5. 5m long; anterior flagellum 1/2 and trailing flagella 2-3 times body length; pond water. Genus Enteromonas da Fonseca. Body globular; 2 anterior flagella and one traihng flagellum. E. hominis d. F. (Fig. 121, d). Small, 5-6^ in diameter; in human faeces. Genus Mixotricha Sutherland. Large; elongate; anterior tip spirally twisted and motile; posterior end probably eversible; 262 PROTOZOOLOGY body surface with a coat of cilia in closely packed transverse bands (insertion and movement entirely different from those of Trichonytnpha) exce})t })()st(n'i()r end; 3 short flagella at anterior end; nucleus, 20// by 2/x, conn(^cted with blepharoplasts by pro- FiG. 122. Diagram illustrating the life-cycle of Tetramitus rostratus (Bunting), a, cyst; b, vegetative amoeba; c, division; d, after division; e, f, stages in transformation to flagellate form; g, fully formed flagel- late; h, flagellate prior to division; i, flagellate after division; j-1, trans- formation stages to amoeba. longed tube which encloses nucleus itself; cytoplasm with scat- tered wood chips; in termite gut. One species. Taxonomic position undetermined. M. paradoxa S. About 340)u long, 200/^ broad and 25/x thick; in gut of Mastotermes darwiniensis ; Australia. POLYMASTIGINA 263 Family 2 Tetramitidae Biitschli With 4 flagella, no one of which undulates on body surface. Genus Tetramitus Perty. Ellipsoidal or pyriform; free-swim- ming; cytostome at anterior end; 4 flagella unequal in length; a contractile vacuole; holozoic; fresh or salt water. T. rostratus P. (Fig. 123, a). Form variable; usually ovoid with narrow posterior region; 18-30/x by 8-1 1/z; stagnant water. Bunt- ing (1922, 1926) observed a very interesting life-cycle of an organism which she found in culture of caecal contents of rat and which she identified as T. rostratus (Fig. 122). Fig. 123. a, Tetramitus rostratus, X620 (Lemmermann); b, T. pyri- f or mis, X670 (Klebs); c, T. salinus, X1630 (Kirby); d, Collodictyon triciliatimi , X400 (Carter): e-j, Costia necatrix (e, f, X800 (Weltner); g-i, X1400 (Moroff); j, two individuals attached to host integument X500 (Kudo)); k, Tricercomonas intestinalis, X1730 (Wenyon and O'Connor); 1, Coproviastix prowazeki, X1070 (Aragao). T. pyriformis Klebs (Fig. 123, h). Pyriform, with pointed pos- terior end; 11-13^ by 10-12^i; stagnant water. T. salinus (Entz) (Fig. 123, c). 2 anterior flagella, 2 long trailing flagella; nucleus anterior; cytostome anterior to nucleus; a 264 PROTOZOOLOGY groove to posterior end; cytopharynx temporary and length variable; 20-30/x long (Entz); 15-19// long (Kirby). Kirby ob- served it in a pool with salinity of about 15 per cent at Marina, California. Genus Collodictyon Carter. Body highly plastic; with longi- tudinal furrows; posterior end bluntly narrowed or lobed; no apparent cytostome; 4 flagella; a contractile vacuole anterior; fresh water. C. triciliatum C. (Fig. 123, d). Spherical, ovoid or heart-shaped; 27-60/1 long; flagella as long as the body; pond water. Rhodes (1919) made a comprehensive cytological study of the organism. Genus Costia Leclerque. Ovoid in front view; pyriform in pro- file; toward right side, a funnel-like depression, at the posterior end of which are located cytostome (?) and 2 long and 2 short flagella; contractile vacuole in posterior half; longitudinal divi- sion; encystment; ectoparasitic in various freshwater fishes. C. necatrix (Henneguy) (Fig. 123, e-j). 10-20^ by 5-10//; compact nucleus central; a contractile vacuole; cyst uninucleate, spherical, 7-10/z in diameter; when present in large numbers, the epidermis of fish appears to be covered by a whitish coat. Genus Tricercomonas Wenyon et O'Connor. Body similar to that of Cercomonas (p. 259), but with 3 anterior flagella and a posterior flagellum; oblong cyst with 4 nuclei when mature; parasitic. T. intestinalis W. et O'C. (Fig. 123, k). 4-8/1 long; in human intestine. Genus Copromastix Aragao. 4 anterior flagella equally long; body triangular or pyramidal; coprozoic. C. prowazeki A. (Fig. 123, /). About I6-I8/1 long; in human and rate faeces. Family 3 Chilomastigidae Wenyon 4 flagella, one of which undulates on body surface. Genus Chilomastix Alexeieff . Pyriform ; with a large cytostomal cleft at anterior end; nucleus anterior; 3 anteriorly directed flagella; short fourth flagellum undulates within cleft; cysts common; in intestine of vertebrates. Several species. C. mesnili (Wenyon) (Fig. 124, a-c). 10-15/t long; cyst 5-10/t long; in human intestine; commensal, although often found in diarrhoeic stools. C. intestinalis Kuczynski. In guinea pigs. POLYMASTIGINA 265 C. hettencourti da Fonseca. In rats and mice. C. cuniculi da Fonseca. In rabbits. C. caprae da Fonseca. In goat. C. gallinarum Martin et Robertson. 11-20/i by S-Qfj.; in domes- tic fowls. Family 4 Callimastigidae da Fonseca Flagella 12 or more; in stomach of ruminants or in caecum and colon of horse. Genus Callimastix Weissenberg. Ovoid; compact nucleus central or anterior; 12-15 long flagella near anterior end, vibrate in unison. Weissenberg (1912) considered this genus to be related to Lophomonas (p. 280), but organism lacks axial organellae; in Cyclops and alimentary canal of ruminants and horse. C. cyclopis W. In body-cavity of Cyclops sp. C. frontalis Braune (Fig. 124, d). 12 flagella; about 12ju long; flagella 30// long; in cattle, sheep and goats. C. equi Hsiung (Fig. 124, e). 12-15 flagella; 12-18/z by 7-10^; nucleus central; in caecum and colon of horse. Family 5 Polymastigidae Biitschli With axial structures; without undulating membrane; flagella variable in number. Genus Polymastix BiitschH. Pyriform; 4 flagella arise from 2 blepharoplasts located at anterior end; cytostome and axostyle inconspicuously present; ectoplasm covered by longitudinal ridges; endocommensal in insects. P. melolonthae (Grassi) (Fig. 124,/). 5-22ju long; in hindgut of Melolontha, Oryctes, Cetonia, Rhizotrogus, Tipula, etc. Genus Eutrichomastix Kofoid et Swezy (Trichomastix Bloch- mann). Pyriform; anterior end rounded; cytostome and nucleus anterior; 3 flagella of equal length arise from anterior end, the fourth traihng; axostyle projects beyond posterior end of body; all endocommensal. E. serpentis (Dobell) (Fig. 124, g). About 10-25/^ long; in in- testine of snakes; Pituophis, Eutaenia, and Python. E. batrachorum (Dobell) (Fig. 124, h). Ovoid; 6-20// long; in intestine of Rana fusca. E. axostylis Kirby (Fig. 124, i). Elongate, ellipsoid, or pyri- form; axostyle projecting; 5-10. 5ju by 2-3. 5^; 3 anterior flagella 5-10m long; in gut of Nasutitermes kirhyi. 266 PKOTOZOOLOGY Fig. 124. a-c, Chilomastix mesnili, X1350 (Kudo); d, Callimastix frontalis, XlSOO (Braune); e, C. equi, XHOO (Hsiung); f, Polymastix melolonthae, X540 (Hamburger); g, Eutrichomastix serpentis, X1450 (Kofoid and Swezy) ; h, E. hatrachorum, X 1350 (Dobell) ; i, E. axostylis, X2000 (Kirby); j, Hexamastix termopsidis, X2670 (Kirby) ; k, H. hatrachorum, XlOOO (Alexeieff); 1, Protrichomonas legeri, XlOOO (Alexeieff); m, Parajoenia grassii, X890 (Kirby); n, Oxynionas pro- jector, X1260 (Kofoid and Swezv); o, Streblomastix strix, XlOBO (Kidder). Genus Hexamastix Alexeieff. Body similar to Eutrichomastix, but with 6 flagella, of which one trails; axostyle conspicuous; parabasal body prominent. H. termopsidis Kirby (Fig. 124, j). Ovoidal or pyriform; S-ll/i long; flagella 15-25ju long; in gut of Zootermopsis angusticollis and Z. nevadensis ; California. POLYMASTIGINA 267 H. hatrachorum Alexeieff (Fig. 124, k). Oval or spindle form; 8-14/i by 4-8/z; flagella about body length; in gut of Triton taeniatus. Genus Protrichomonas Alexeieff. 3 anterior flagella of equal length, arising from a blepharoplast located at anterior end; parasitic. P. legeri A. (Fig. 124, I). In oesophagus of the marine fish, Box boops. Genus Parajoenia Janicki. Medium large; ends rounded; 3 anterior flagella; 1 long trailing flagellum; axostyle stout; para- basal body in 2 parts; Kirby (1937) showed that this genus belongs to Polymastigina; in termite gut. P. grassii J. (Fig. 124, m). 29-59^ by 12-33/x; numerous spiro- chaetes, about 15-20/1 long, adherent to anterior and posterior parts of body; in Neotermes connexus; Hawaii. Genus Oxymonas Janicki. Oval or pyriform; extensible and retractile rostellum (proboscis) at anterior end; at its base 2 groups of 3 flagella; nucleus anterior; bundle of axial filaments; in termite gut. 0. projector Kofoid et Swezy (Fig. 124, n). 12-40^ long; in Kalotermes perparvus. 0. dimorpha Connell. Subovoid; delicate pellicle; axostyle pro- truding; a pair of long anterior flagella from 2 blepharoplasts connected by rhizoplast; nucleus anterior, Feulgen negative; when attached to intestine, rostellum elongate, flagella disappear; xylophilous; 165-195/x by 14-17^; in Neotermes simplicicornis ; California and Arizona. Genus Streblomastix Kofoid et Swezy. Elongate, spindle; 4 anterior flagella; nucleus elongate spindle; with spiral ridges; in termite gut. S. strix K. et S. (Fig. 124, o). 200-530m by 20-80m; in Zooter- mopsis angusticollis. Genus Devescovina Foa. Oblong; axostyle rigid, extends to posterior end; 3 anterior flagella and one long trailing flagellum; parabasal body; in termite gut. D. lemniscata Kirby (Fig. 125, a). 12-41ju by 7-15ju; in Crypto- termes hermsi. Genus Pseudodevescovina Sutherland. Relatively large and stout; a single anterior flagellum; without (Sutherland) or with one comparatively short trailing flagellum (Kirby); axostyle 268 PROTOZOOLOGY stout; parabasal body large; investment of short spirochaetes; in termite gut. P. unijlagdlata S. About 65/i by 40-45^ (Sutherland); 52-95^ by 26-60^1 (Kirby); in Kalotermes insularis. Genus Monocercomonas Grassi. Small; 4 flagella inserted in pairs in 2 places; 2 directed anteriorly and the other 2, posteriorly; axostyle filamentous; parasitic. Fig. 125. a, Devescovina lemniscata, XlOOO (Kirby); b, Monocer- comonas bufonis, X1670 (Alexeieff); c, Pyrsonympha vertens, X260 (Comes); d, Dinenyni'pha finihriala, X830 (Kirby); e, Metadevescovina debilis, X1130 (Light); i, Foaina nana, X1670 (Kirby); g, Saccino- bac'ulus ambloaxostylus, XSOO (Cleveland et al.). M. bufonis Dobell (Fig. 125, h). Spindle-form; 12-15// long; cysts spherical; in Axolotle, Triton, frogs and toads. Genus Pyrsonympha Leidy. Ovoid or ellipsoid; axostyle divided into 2 parts along its posterior portion and the whole POLYMASTIGINA 269 vibrates in life; 4-8 flagella adhering to the body; in termite gut. P. vertens L. (Fig. 125, c). 100-1 60ju long; in Reticulitermes flavipes. Genus Dinenympha Leidy. Elongate; 4-8 flagella spirally ad- hering to the body; axostyle conspicuous; in termite gut. D. gracilis L. In Reticulitermes flavipes and R. lucifugus; Duboscq and Grasse hold that this is an immature stage of Pyrsonympha vertens. D. fimbriata Kirby (Fig. 125, d). 52-78m by about 18^; in Reticulitermes hesperus. Genus Metadevescovina Light. Spindle to elongate oval; circular in cross-section; body surface smooth, but often with attached bacteria; nucleus anterior; axostyle not extending be- yond the posterior end of body; parabasal body a spiral rod around axostyle; one primary flagellum and 3 long secondary flagella; spirochaetes adhering to body surface (Ivirby) ; in termite gut. M. debilis L. (Figs. 23; 125, e). 30-70^ by 15-30/x; in Kalo- termes hubhardi. Genus Foaina Janicki (Paradevescovina Kirby). Ellipsoidal; rigid axostyle protrudes a little; flagella similar to those of Devescovina in number and appearance; parabasal body a long curved rod; in termite gut. F. nana Kirby (Fig. 125, /). 10/x by 7/^; in Cryptoterynes hermsi. Genus Saccinobaculus Cleveland. Elongate to spherical; 4 (8 or 12) flagella; axostyle large, paddle-like, deeply stained with Heidenhain, undulates, and serves for locomotion; parasitic. S. amhloaxostylus C. (Fig. 125, ^f). 65-1 10/x by 18-26^; in Cryptocercus punctulatus. Family 6 Trichomonadidae With both axial organellae and an undulating membrane. Genus Trichomonas Donne {Ditricho?7ionas Cutler). Pyriform; 4 anterior flagella; another flagellum along the margin of undu- lating membrane; costa along the base of the membrane; axostyle projects beyond the posterior end of body; cysts observed in forms inhabiting the animal intestines, but not in those living in man; parasitic in gut of vertebrates and invertebrates. Numerous species. T. hominis (Davaine) (Fig. 126, a). 5-18/i long; in human intestine. 270 PROTOZOOLOGY T. elongata Steinberg (T. hiiccalis Goodey et Wellings) (Fig. 126, h). About 10-20yLt long; in hunuin mouth. T. vaginalis Donne (Fig. 126, c). 10-25)U long; in human vagina. T. batrachonim (Perty). Ovoid; 14-18ai by 6-10/x; in frog gut. T. auguda Alexeieff. Spindle-form; 18-22yu by 8-14/x; in frog gut. T. linearis Kirby (Fig. 126, d). Elongate, spindle-form; 9-24/i by 3-8/jl; in gut of Orthognathotermes wheeleri; Panama. T. termitis (Cutler) (Fig. 126, e). 30-88m by 13-57^ (Imms); in gut of Archotermopsis wroughioni ; India. Genus Gigantomonas Dogiel (Myxomonas D.). Somewhat similar to Trichomonas, but much larger; 3 short flagella and a very long flagellum; axostyle large; undulating membrane well developed; parasitic. G. herculea D. (Fig. 126, /). 60-75ac by 30-35yu; in gut of Hodotermes mossambicus. Myxomonas polymorpha D. (g) reported from the same host appears to be a degenerating specimen. Genus Tritrichomonas Kofoid. Similar to Trichomonas in ap- pearance and structure; but 3 anterior flagella; parasitic. T. hrevicollis Kirby (Fig. 126, h). Ovoid; undulating membrane curved around end; 10-17yu by 4-8ai; in gut of Kalotermes hrevicollis; Panama. T. foetus (Riedmiiller). Pathogenic; in genitalia of cattle; simi- lar to Trichomonas vaginalis; but 3 anterior flagella; body about 15m by 5fx; transmission by sexual act, from cow to bull or bull to cow ; in infected cow conception temporarily or permanently sus- pended or death of foetus occurs. T.fecalis Cleveland. 5/i by 4^ to 12^ by 6m; average dimensions 8.5m by 5.7m; axostyle long, protruding 1/3-1/2 the body length from the posterior end; of 3 flagella, one is longer and less active than the other two; in the faeces of man. Its remarkable adapta- bihty observed by Cleveland was noted elsewhere (p. 28"). Genus Tricercomitus Kirby. Small; 3 anterior flagella; a long trailing flagellum, adhering to body; nucleus anterior, without endosome; blepharoplast large, with a parabasal body and an axial filament; parasitic. T. termopsidis K. (Fig. 126, i, j). 4-12m by 2-3m; anterior flagella 6-20m long; trailing flagellum 19-65m long; in gut of Zootermopsis angusticollis, Z. nevadensis and Z. laticeps; Cali- fornia and Arizona. POLYMASTIGINA 271 Fig. 126. a, Trichomonas hominis, X1070 (Kudo); b, T. elongata, X1070 (Kudo); c, T. vaginalis, X870 (Wenyon); d, T. linearis, X2000 (Kirby); e, T. termitis, X630 (Cutler); f, Gigantomonas herculea, X530 (Dogiel); g, a degenerating form, X400 (Dogiel); h, Tritrichomonas brevicollis, x2000 (Kirby); i, j, Tricercomitus termopsidis, X890 (Kirby); k, Pentatrichomonas scroa, X2000 (Kirby); 1, Pseudotryp- anosoma giganteum, X580 (Kirby). Genus Pentatrichomonas Chatterjee. 5 anterior flagella; axo- style very slightly developed; parabasal body fusiform; nucleus at some distance from anterior end; parasitic. 272 PROTOZOOLOGY P. scroa Kirby (Fig. 126, k). 18-45m by 6-1 5/x; in Kalotermes dudleyi and K. longicollis ; Panama. Gonus Pseudotrypanosoma Grassi. Large, elongate; 3 anterior flagella; undulating membrane; slender axostyle; costa conspicu- ous; band-like structure between blepharoplast and nucleus; striae near body surface; parabasal body long; parasitic. P. giganteum G. (Fig. 126, I). 55-1 ll/x long (Grassi); 145-205^ by 20-40/i; anterior flagella about 30)U long (Kirby); cytostome not observed; in gut of Porotermes adamsoni and P. grandis; Australia. Suborder 2 Diplomonadina The suborder consists of a number of binucleate flagellates possessing bilateral symmetry. Family Hexamitidae Kent Genus Hexamita Dujardin {Octomitus Prowazek). Pyriform; 2 nuclei at anterior pole, 6 anterior and 2 posterior flagella; 2 axostyles; 1-2 contractile vacuoles; cytostome obscure; endo- plasm with refractile granules; encystment; in stagnant water or parasitic. H. inflata D. (Fig. 127, a). Broadly oval; posterior end trun- cate; 13-25m by 9-15^; in stagnant water. H. iiitestinalis D. (Fig. 127, h, c). 10-16m long; in intestine of frogs, also in midgut of Trutta fario and in rectum of Motella tricirrata and M. mustela in European waters. H. salmonis (Moore) (Fig. 127, d). 10-12/i by 6-8/^; in intestine of various species of trout and salmon; schizogony in epithelium of pyloric caeca and intestine; cysts; pathogenic to young host fish (Davis, 1925). H. periplanetae (Belaf) In gut of cockroaches. H. cryptocerci Cleveland (Fig. 127, e). 8-13/i by 4-5. 5m; in Cryptocercus puntulatus. Genus Giardia Kunstler {Lamhlia Blanchard). Pyriform; bi- laterally symmetrical; dorsal side convex; ventral side with sucking disc at anterior region; 8 flagella; 4 from margin of suck- ing disc; 2 from middle part and 2 from posterior end of body; parasites in intestine of various vertebrates. Several species. G. intestinalis (Lambl) (Fig. 127, f-h). 10-20m by 6-10/x; com- mensal in human intestine. G. muris (Grassi). 7-13^ by 5-10ai; in intestine of mice and rats. POLYMASTIGINA 273 Fig. 127. a, Hexamita inflata, X690 (Klebs); b, c, H. intestinalis, X1600 (Alexeieff); d, H. salmonis, X2100 (Davis); e, H. cryptocerci, X1600 (Cleveland); f-h, Giardia inlestinalis, X1070 (Kofoid and Swezy); i, Treipomonas agilis, X1070 (Klebs); j, T. rotans, X710 (Lemmermann); k, Gyromonas amhulans, X530 (Seligo); 1, Trigo- noynonas compressa, X490 (Klebs); m, Urophagus rostratus, X800 (Klebs). Genus Trepomonas Dujardin. Free-swimming; flattened; more or less rounded; cytosomal grooves on posterior half, one on each side; 8 flagella (one long and 3 short flagella on each side) arise from anterior margin of groove; at anterior end there is a horse- 274 PROTOZOOLOGY shoe-like structure, in which two nuclei are located; fresh water, parasitic, or coprozoic. T. agilis D. (Fig. 127, i). More or less ovoid; 7-30^ long; 1 long and 3 short flagella on each side; rotation movement; stagnant water; also reported from intestine of Amphibians. T. rolans Klebs (Fig. 127, j). Broadly oval; posterior half highly flattened; 2 long and 2 short flagella on each of 2 cytostomes; stagnant water. Genus Gyromonas Seligo. Free-swimming; small; form con- stant, flattened; slightly spirally coiled; 4 flagella at anterior end; cytostome not observed; fresh water. G. ambulans S. (Fig. 127, k). Rounded; 8-15yu long; standing water. Genus Trigonomonas Klebs. Free-swimming; pyriform; plastic; cytostome on either side, from anterior margin of which arise 3 flagella; flagella 6 in all; 2 nuclei situated near anterior end; move- ment rotation; holozoic; fresh water. T. compressa K. (Fig. 127, I). 24-33^ by 10-16m; flagella of different length; standing water. Genus Urophagus Klebs. Somewhat similar to Hexamita; but a single cytostome; 2 moveable posterior processes; holozoic; stagnant water. U. rostratus (Stein) (Fig. 127, m). Spindle-form; 16-25iu by 6- 1 2m. Suborder 3 Polymonadina This group includes forms which inhabit the intestine of various species of termites, most probably as symbionts. The majority are multinucleate. Each nucleus gives rise to a basal body (from which flagella extend), a parabasal body, and an axial filament. Janicki called this complex a karyomastigont, and the other type of complex which does not contain a nucleus akaryomastigont. Genus Calonympha Foa. Body rounded; large; numerous long flagella arise from anterior region; nuclei arranged near insertion points of flagella; with karyomastigonts or akaryomastigonts; axial filaments form a bundle; in termite gut. C. grassi F. (Fig. 128, a). In Cryptotermes grassii; Q9-90fjL long. Genus Stephanonympha Janicki. Oval, but plastic; pellicle sculptured with foreign bodies ; numerous nuclei spirally arranged around anterior end; in termite gut. POLYMASTIGINA 275 S. nelumhium Kirby (Fig. 128, b). 45/i by 27^; in Cryptotermes hermsi. Genus Microrhopalodina Grassi et Foa (Prohoscidiella Kofoid et Swezy). One to many nuclei, each in a karyomastigont com- FiG. 128. a, Calonympha grassii, X900 (Janicki); b, Stephaiionympha nelumhium, X400 (Kirby); c, Microrhopalodina multinudeata, x440 (Kofoid and Swezy); d, Coronympha clevelandi, XlOOO (Kirby); e, Snyderella tabogae, X350 (Kirby). plex; a single extensible and retractile rostellum; binary fission; in termite gut. M. multinudeata (Kofoid et Swezy) (Fig. 128, c). 25-160a£ long; in Kalotermes nocens. M. occidentis (Lewis). 26-133^ by 11-80/^; average number of 276 PROTOZOOLOGY nucloi 5.5, about 23 per cent uninucleate; in Kalotermes Oc- cident if^. Geiuis Coronympha Kirby. Pyriform with 16 nuclei, arranged in a single circle in anterior region; each nucleus center of a karyo- mastigont; in termite gut. C. clevelandi K. (Fig. 128, d). 25-53^ by 18-46yu, in Kalotermes clevelandi; Panama. Genus Snyderella Kirby. Numerous nuclei scattered through cytoplasm; akaryomastigonts close together and extend through greater part of peripheral region; axial filaments in bundle; in termite gut. S. tahogae K. (Fig. 128, e). Pyriform; rounded posteriorly; bluntly conical anteriorly; 77-1 72ju by 53-97yu; in Kalotermes longicollis; Panama. References Cleveland, L. R. et al. 1934 The wood-feeding roach, Crypto- cercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts and Sci., Vol. 17. DoBELL, C. and F. W. O'Connor 1921 The intestinal Protozoa of man. London. Grasse, p. p. 1926 Contribution a I'etude des Flagelles para- sites. Arch. zool. exp., et gen.. Vol. 65. Kirby, H. 1930, 1931 Trichomonad flagellates from termites. I, IL Uni. Cal. Pub. Zool., Vols. 33, 36. KoFOiD, C. A. and Olive Swezy 1915 Mitosis and multiple fis- sion in trichomonad flagellates. Proc. Amer. Acad. Arts and Sci., Vol. 51. 1920 On the morphology and mitosis of Chilo- mastix mesnili (Wenyon), a common flagellate of the human intestine. Uni. Cal. Pub. Zool., Vol. 20. 1922 Mitosis and fission in the active and en- cysted phases of Giardia enterica etc. Ibid., Vol. 20. Rees, C. W. 1938 Observations on bovine venereal trichomoni- asis. Veterin. Med., Vol. 33. Sutherland, J. L. 1933 Protozoa from Australian termites. Quart. Jour. Micr. Sci., Vol. 76. Wenyon, C. M. 1926 Protozoology, Vol. 1. London. Chapter 15 Order 4 Hypermastigina Grassi ALL members of this order are inhabitants of the alimentary ,. canal of the termite or other insects. The cytoplasmic or- ganization is of high complexity, although there is only a single nucleus. Flagella are numerous and have their origin in blepharo- plasts located at the anterior region of the body. In some species, it has been established by Cleveland that there exists a true symbiotic relationship between the host insects and the proto- zoans (p. 24). Method of nutrition is either holozoic or saprozoic (parasitic). No cytostome has been detected and bits of wood, starch grains, and other food materials, are taken in by means of pseudopodia. Asexual reproduction is by longitudinal fission; multiple divi- sion has also been noted in some species under certain conditions, while sexual reproduction has not been observed. Encystment occurs in some genera of Lophomonadidae and certain species in- habiting wood-roaches, in which moulting of the host insect leads to encystment. Because of the peculiarity and complexity of their structures and also of their common occurrence in termites, the Hypermastigina have in recent years been frequently studied. Body without segmented appearance Flagella in spiral rows Family 1 Holomastigotidae Flagella not arranged in spiral rows Flagella in one or more anterior tufts 1 tuft of flagella Family 2 Lophomonadidae (p. 280) 2 tufts of flagella Family 3 Hoplonymphidae (p. 282) 4 tufts of flagella Family 4 Staurojoeninidae (p. 284) Several tufts (loriculae) Family 5 Kofoidiidae (p. 284) Flagella not arranged in tufts Posterior part without flagella Family 6 Trichonymphidae (p. 284) Flagella over entire body . Family 7 Eucomonymphidae (p. 286) Body with segmented appearance. Family 8 Teratonymphidae (p. 287) Family 1 Holomastigotidae Janicki Flagella are arranged in spiral rows; posterior region may be without flagella; the "anterior body" surrounds, or occurs near, 277 278 PROTOZOOLOGY the nucleus; reproduction by longitudinal division; inhabitants of termito gut. Genus Holomastigotes Grassi. Body small; spindle-shajjed; few spiral rows reach from anterior to i)osterior end; nucleus anterior, surrounded by a mass of dense cytoplasm; nutrition by absorp- tion of fluid material; in termite gut. H. elongatum G. (Fig. 129, a). In gut of Reticulitermes lucifugus, R. speratus, R. flaviceps, and Macrohodotermes mossambicus; up to 70ai by 24m (Grassi). Genus Holomastigotoides Grassi et Foa. Large; spindle-shaped; spiral rows of flagella as in the last genus, but more numerous (12-40 rows); a mass of dense cytoplasm surrounds ovoid nucleus; in termite gut. H. hartmanni Koidzumi (Fig. 129, 6). 50-140yu long; in Copto- termes formosanus. Genus Spirotrichonympha Grassi. Moderately large; elongate pyriform; flagella deeply embedded in cytoplasm in anterior region, arising from 1-several spiral bands; mass of dense cyto- plasm conical and its base indistinct; nucleus spherical ; in termite gut. S. leidyi Koidzumi (Fig. 129, c). In Coptotennes formosanus; 15-50m by 8-30^. ,S. pulchella Brown (Fig. 129, d). 36 42/x by 14-16^; in Reticu- litermes hageni. S. polygyira Cupp. (Fig. 58). In Kalotermes simplicicornis ; 63-1 12/x by 25-60/x; four flagellar bands. Genus Spirotrichonymphella Grassi. Small; without spiral ridges; flagella longer; not wood-feeding; in termite gut. S. pudihunda G. In Porotermes adamsoni; Australia. Multiple fusion (Sutherland). Genus Microspirotrichonympha Koidzumi {Spironympha Koid- zumi). Small, surface not ridged; spiral rows of flagella only on anterior half; a tubular structure between nucleus and anterior extremity; a mass of dense cytoplasm surrounds nucleus; with or without axial rod; in termite gut. M. porteri K. (Fig. 129, e). In Leucotermes flaviceps; 20-55^ by 20-40m. M. ovalis (Brown) (Fig. 129,/). 36-48^ by about 40^; in Reticu- litermes hesperus. Genus Spire tricho soma Sutherland. Pyriform or elongate; be- HYPERMASTIGINA 279 low operculum, two deeply staining rods from which flagella arise and which extends posteriorly into 2 spiral flagellar bands; with- out axostyle; nucleus anterior, median; wood chips ahvays present, but method of feeding unknown; in Stolotermes victoriensis ; Australia. 'f'f'il'll t-fM \ ^^^ vMJ 'H Fig. 129. a, Holomastigotes elongatum, x700 (Koidzumi); b, Holo- mastigotoides hartmanni, X250 (Koidzumi); c, Spirotrichonyinpha leidyi, X400 (Koidzumi); d, S. pidchella, X900 (Brown); e, Micro- spirotrichonympha porteri, X250 (Koidzumi); f, M. ovalis, X600 (Brown); g, Macrospironympha xylopletha, X300 (Cleveland et al.); h, Leptospironympha eupora, X1050 (Cleveland et al.). S. capitata S. 97/x by 38/i; flagellar bands closely spiral, reach posterior end. Genus Macrospironympha Cleveland. Broadly conical; flagella on 2 broad flagellar bands which make 10-12 spiral turns, 2 inner bands; axostyles 36-50 or more; during mitosis nucleus migrates posteriorly; encystment, in which only nucleus and centrioles are 280 PROTOZOOLOGY retained, takes place at each ecdysis of host; in Cryptocercus punctulatus. M. xijlopletha C. (Fig. 129, g). 112-154m by 72-127ax. Genus Leptospironympha Cleveland. Cylindrical; small; flagella on 2 bands winding spirally along body axis; axostyle single, hya- line; nucleus does not migrate posteriorly during division; en- cystment unknown; in Cryptocercus punctulatus. L. eupora C. (Fig. 129, h). 30-38^ by 18-21m. Family 2 Lophomonadidae Kent Numerous flagella arise from anterior end in a tuft; each flagellum originates in a blepharoplast from which extends in- ward an axostylar filament; nucleus anterior, surrounded by a funnel-shaped space formed by filaments; no cytostome; para- basal body; nutrition holozoic or parasitic; reproduction by bi- nary or multiple fission; encystment common; sexual reproduc- tion unknown; in cockroaches and termite guts. Genus Lophomonas Stein. Ovoid or elongate; small; a vesicular nucleus anterior; cysts common; in colon of cockroaches. L. hlattarum S. (Figs. 23; 59; 66; 130, a-e). Small, pyriform, but plastic; bundle of axostylar filaments may project beyond pos- terior margin; active swimming movements; binary or multiple fission; 25-30^ long; holozoic in colon of cockroaches; widely distributed. L. striata Biitschli (Fig. 130, f-h). Elongate spindle; surface with obliquely arranged needle-like structures which some in- vestigators believe to be a protophytan (to which Grasse gave the name, Fusiformis lopho7nonadis) ; bundle of axial filaments short, never protruding; movement sluggish; cyst spherical with needle-like structures; in same habitat as the last species. Genus Eulophomonas Grassi et Foa. Similar to Lophomonas, but flagella vary from 5-15 or a little more in number; in termite gut. E. kalotermitis Grassi. In Kalotermes flavicollis; this flagellate has not been observed by other workers. Genus Prolophomonas Cleveland. Similar to Eulophomonas; established since Eulophomonas had not been seen by recent workers; would become synonym "if Eulophomonas can be found in K. flavicollis" (Cleveland). P. tocopola C. (Fig. 130, i). 14-19m by 12-15^; in Cnjptocercus punctulatus. HYPERMASTIGINA 281 Genus Joenia Grassi. Ellipsoidal; anterior portion capable of forming pseudopodia; flagellar tufts in part directed posteriorly; surface covered by numerous immobile short filamentous process- es, which some hold to be symbiotic bacteria; nucleus spherical Fig. 130. a-e, Lopho7nonas blattarum (a, b, in life, X320; c, d, stained specimens; e, cyst, X1150) (Kudo); f-h, L. striata (f, in life, X320; g, h, stained individuals, X1150) (Kudo); i, Prolophomonas tocopola, X1200 (Cleveland et al.); j, Joenia annectens (Grassi and Foa) ; k, Mi- crojoenia pyriformis, X 920 (Brown); 1, Torquenympha octoplus, x920 (Brown). anterior; posterior to it a conspicuous axostyle composed of numerous axial filaments, a parabasal apparatus surrounding it; bits of wood used as food; in termite gut. J. annectens G. (Fig. 130, j)- Ii^ Kalotermes flavicollis. Genus Joenina Grassi. More complex in structure than that of 282 PROTOZOOl.OCJY Joenia; flagclla inserted at anterior end in a semi-circle; j^ara- basal bodies 2 elongated curved rods; feeding on wood fragments. J. pidchcUa G. In Porotermes adam„som. Genus Joenopsis Cutler. Oval; large; a horseshoe-shaped pillar at anterior end, flagella arising from it; some directed an- teriorly, others posteriorly; parabasal bodies long rods; a strong axostyle; feeding on bits of wood; in termite gut. J. polytricha C. In Archotermopsis wroughtoni ; 95-129)u long. Genus Microjoenia Grassi. Small, pyriform; anterior end flat- tened; flagella arranged in longitudinal rows; axostyle; parabasal body simple; in termite gut. M. pyriformis Brown (Fig. 130, k). 44-52/x by 24-30)li; in Reticulitermes hageni. Genus Mesojoenia Grassi. Large; flagellar tuft spread over a wide area; distinct axostyle, bent at posterior end; 2 parabasal bodies; in termite gut. M. decipiens G. In Kalotermes flavicollis. Genus Torquenympha Brown. Small; pyriform or top-form; axostyle; radially symmetrical; 8 radially arranged parabasal bodies; nucleus anterior; in termite gut. T. octoplus B. (Fig. 130, I). 15-26/x by 9-13^; in Reticulitermes hesperus. Family 3 Hoplonymphidae Light 2 flagellar tufts; each arises from a plate near anterior end of slender body which is protected by a highly developed pellicular armor. Genus Hoplonympha Light. Slender fusiform, covered with thick, rigid pelUcular armor; each tuft of flagella arises from a plate connected with blepharoplasts at anterior end; nucleus near anterior extremity, more or less triangular in form; in termite gut. H. natator L. (Fig. 131, a, 6). 60-1 20m by 5-12//; in Kalotermes sirnplicicornis. Genus Barbulanympha Cleveland. Acorn-shaped; small, nar- row, nuclear sleeve between centrioles; number of rows of flagella greater at base; large chromatin granules; numerous (80-350) parabasals; axostylar filaments 80 350; flagella 1500-13,000; different species show different number of chromosomes during mitosis; in gut of Cryptocercus punctulatus. Four species. B. ufalula C. (Figs. 57; 131, c). 250-340// by 175-275/x; 50 HYPERMASTIGINA 283 ^T>i ^;., > ''*M Fig. 131. a, b, Hoplonympha natator, X450 (Light); c, Barbula- iiympha ufalula, X210 (Cleveland et al.); d, Urinympha talea, X350 (Cleveland et al.); e, Staurojoenina assimilis, X200 (Kirby); f, Idio- nympha perissa, X250 (Cleveland et al.);g, Teratonympha viirabilis, X200 (Dogiel). chromosomes; flagellated area 36-4 l^u long; centriole 28-35/i long. B. laurabuda C. 180-240// by 135-170^; 40 chromosomes; flagellated area 29-33// long; 24-28/x long. Genus Rhynchonympha Cleveland. Elongate; number of flagel- lar rows same throughout; axial filaments somewhat larger and 284 PROTOZOOLOGY longer, about 30; 30 parabasals; 2400 flagella; in Cryptocercus punctulatus. R. tarda C. (Fig. 132,/). 130-215/x by 30 70m. Genus Urinympha Cleveland. Narrow, slender; flagellated area, smaller than that of the two genera mentioned above; flagella move as a unit; about 24 axial filaments; 24 parabasals; 600 flagella ; in gut of Cryptocercus punctulatus. U. talea C. (Fig. 131, d). 75-300m by 15-50^. Family 4 Staurojoeninidae Grassi 4 flagellar tufts arise from the anterior end. Genus Staurojoenina Grassi. Pyriform to cylindrical; anterior region conical; nucleus spherical, central; 4 flagellar tufts from anterior end; ingest wood fragments; in termite gut. S. assimilis Kirby (Fig. 131, e). 105-190^ long; in Kaloter^nes minor. Genus Idionympha Cleveland. Acorn-shaped; axostyles 8-18; fine parabasals grouped in 4 areas; pellicle non-striated; nucleus nearer anterior end than that of Staurojoenina; flagellated areas smaller; in gut of Cryptocercus punctulatus. I. perissa C. (Fig. 131,/). 160-275^ by 98-155m. Family 5 Kofoidiidae Light Flagellar tufts composed of 8-16 loriculae (permanently fused bundles of flagella); without either axostyle or parabasal body. Genus Kofoidia Light. Spherical; between oval nucleus and bases of flagellar tufts, there occurs a chromatin collar; wood fragments as food; in termite gut. K. loriculata L. (Fig. 132, a, h). 60-140^ in diameter; in Kalotermes simplicicornis. Family 6 Trichonymphidae Kent The body is divisible into three regions; rostellum with caps, flagellated region behind rostellum and non-flagellated area at posterior end; flagellated area 1/3-2/3 of body length; surface of anterior portion differentiated into 1-2 thick ectoplasmic layers, densely traversed by numerous flagella; an "axial core" or "head organ" at anterior tip; no cytostome; a single nucleus; flagella numerous and long, arranged in longitudinal rows ; multiplication by simple longitudinal fission; inhabitants of termites and wood- roach. HYPERMASTIGINA 285 Fig. 132. a, b, Kofoidia loriculata, Xl75, X300 (Light); c, Tricho- nympha campanula, Xl50 (Kofoid and Swezy); d, T. agilis, X410 (Kirby); e, Eiicomonymphaimla, X350 (Cleveland etal.); f, Rhyncho- nympha tarda, X350 (Cleveland et al.). Genus Trichonympha Leidy (Leidyonella Frenzel; Gymno- nympha Dobell; Leidyopsis Kofoid et Swezy). Anterior portion consists of nipple and bell, both of which are composed of 2 layers; a distinct axial core; nucleus central; flagella located in longi- 286 PROTOZOOLOGY tudinal rows on bell; in termite gut. Many species. Cleveland and his associates (1934) observed that encystment takes place in species inhabiting the wood-roach, Cryptocercus 'punctulatus and that it occurs only at the time of moulting of the host roach, namely once a year. T. campanula Kofoid et Swezy (Figs. 56; 132, c). 144-313^4 by 57-144;u; wood particles are taken in by posterior region by a method of Rumbler's "import" (Cleveland). In Zootcrmopsis angusticollis, Z. nevadensis and Z. laticeps. T. agilis Leidy (Fig. 132, d). 55-1 15m by 22-45yu; in Reticuli- termes flavipes, R. lucifugus, R. speratus, R. flaviceps, R. hesperus, R. tibialis. T. grandis Cleveland. 190-205iu by 79-88m; in Cryptocercus punctualatus. Genus Pseudotrichonympha Grassi. 2 parts in anterior end as in Trichonympha; head organ with a spherical body at its tip and surrounded by a single layer of ectoplasm; bell covered by 2 layers of ectoplasm; nucleus lies freely; body covered by slightly oblique rows of short fiagella; in termite gut. P. ^rassu Koidzumi. In Coptotermes formosanus; spindle-form; 200-300/x by 50-120m. Genus Deltotrichonympha Sutherland. Triangular; with a small dome-shaped "head"; composed of 2 layers; head and neck with long active fiagella; body fiagella short, arranged in 5 longitudinal rows; fiagella absent along posterior margin; nucleus large oval, located in anterior third; cytoplasm with wood chips; in termite gut. One species. D. operculata S. Up to 230^ long, 164/x wide, and about 50^ thick; in gut of Mastotermes darwiniensis; Australia. Family 7 Eucomonymphidae Cleveland All or most of body covered with fiagella that arise from basal granules arranged in nearly longitudinal rows; fiagella in 2 dif- ferent groups, and never in 3 groups as in Trichonymphidae; with- out peri-nuclear arrangement of parabasals. Genus Eucomonympha Cleveland. Body covered with fiagella arranged in 2 (longer rostral and shorter post-rostral) zones; rostral tube very broad, filled with hyaline material; nucleus at base of rostrum; in gut of Cryptocercus punctulatus. E. imla C. (Fig. 132, e). 100-165^ by 48-160^; attached forms more elongate than free individuals. HYPERMASTIGINA 287 Family 8 Teratonymphidae Koidzumi Genus Teratonympha Koidzumi {Cyclonympha Dogiel). Large and elongate; transversely ridged, and presents a metameric ap- pearance; each ridge with a single row of flagella; no cytostome; anterior end complex, containing a nucleus; reproduction by longitudinal fission; in termite gut. T. mirahilis K. (Fig. 131, g). 200-300^ or longer by 40-50ai; in Reticulitermes speratus of Japan. References Cleveland, L. R. 1925 The effects of oxygenation and starva- tion on the symbiosis between the termite, Termopsis, and its intestinal flagellates. Biol. Bull., Vol. 48. and others 1934 The wood-feeding roach, Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts and Sci., Vol. 17. Dogiel, V. 1922 Untersuchungen an parasitischen Protozoen aus dem Darmkanal der Termiten. III. Trichonymphidae. Arch. soc. Russe Protist., Vol. 1. Janicki, D. v. 1910, 1915 Untersuchungen an parasitischen Flagellaten. I, II. Zeitschr. wiss. Zool., Vols. 95, 112. KiRBY, H. 1926 On Staurojoenina assimilis sp. nov., an intestinal flagellate from the termite, Kalotermes minor Hagen. Uni. Calif. Publ. Zool., Vol. 29. 1932 flagellates of the genus Trichonympha in termites. Ibid., Vol. 37. KoFoiD, C. A. and Olive Sw^ezy 1919, 1926 Studies on the parasites of termites. Ibid. Vols. 20, 28. Koidzumi, M. 1921 Studies on the intestinal Protozoa found in the termites of Japan. Parasit., Vol. 13. Kudo, R. 1926 Observations on Lophomonas hlattarum, a flagel- late inhabiting the colon of the cockroach, Blatta orienialis. Arch. f. Protistenk., Vol. 53. Sutherland, J. L. 1933 Protozoa from Australian termites Quart. Jour. Micr. Sci., Vol. 76. Chapter 16 Class 2 Sarcodina Butschli THE members of this class do not possess any definite pellicle and, therefore, are capable of forming pseudopodia (p. 40). The term 'amoeboid' is often used to describe their appearance. The pseudopodia serve for both locomotion and food-capturing. The peripheral portion of the body shows no structural dif- ferentiation in Amoebina, Proteomyxa, and Mycetozoa. Internal and external skeletal structures are variously developed in other orders. Thus, in Testacea and Foraminifera, there is a well- developed test or shell that usually has an aperature, through which the pseudopodia are extruded; in Heliozoa and Radiolaria, skeletons of various forms and materials are developed. The cytoplasm is, as a rule, differentiated into the ectoplasm and the endoplasm, but this differentiation is not constant. In Radiolaria, there is a perforated membranous 'central capsule' which marks the border line between the two cytoplasmic layers. The endoplasm contains the nuclei, food vacuoles, various granules, and contractile vacuoles. The majority of Sarcodina are uninucleate, but numerous species of Foraminifera and Mycetozoa are multinucleate. In the family Paramoebidae, there occurs a peculiar 'secondary nucleus.' The Sarcodina are typically holozic, but in a few cases holo- phytic. Their food organisms are Protozoa, small Metazoa and Protophyta, which present themselves conspicuously in the cytoplasm. One or more contractile vacuoles are invariably pres- ent in forms inhabiting the fresh water, but absent in parasitic forms or in those which live in the salt water. Asexual reproduction is usually by binary (or rarely multiple) fission, budding, or plasmotomy. Definite proof of sexual re- production has been given in a comparatively small number of species. Encystment is common in the majority of Sarcodina, but is unknown in a few species. The life-cycle has been worked out in some forms and seems to vary among different groups. The young stages are either amoeboid or flagellate, and on this account, it is sometimes very difficult to distinguish the Sarcodina and the Mastigophora. In some forms the mature trophic stage 288 SARCODINA, PROTEOMYXA 289 may show an amoeboid or flagellate phase owing to differences in environmental conditions. The Sarcodina are divided into two subclasses as follows: With lobopodia, rhizopodia, or filopodia Subclass 1 Rhizopoda With axopodia Subclass 2 Actinopoda (p. 356) Subclass 1 Rhizopoda Siebold The name Rhizopoda has often been used to designate the entire class, but it is used here for one of the subclasses, which is further subdivided into five orders, as follows : Without test or shell With radiating pseudopodia Order 1 Proteomyxa With rhizopodia; forming Plasmodium Order 2 Mycetozoa (p. 296) With lobopodia Order 3 Amoebina (p. 304) With test or shell Test single-chambered; chitinous Order 4 Testacea (p. 323) Test 1- to many -chambered; calcareous Order 5 Foraminifera (p. 344) Order 1 Proteomyxa Lankester A number of incompletely known Rhizopods are placed in this group. The pseudopodia are filopodia which often branch or anastomose with one another. In this respect the Proteomyxa show affinity to the Mycetozoa. Flagellate swarmers and encyst- ment occur commonly. The majority of Proteomyxa lead para- sitic life in algae or higher plants in fresh or salt water. Pseudoplasmodium-formation Family 1 Labyrinthulidae Solitary and Heliozoa-like With flagellate swarmers . . . Family 2 Pseudosporidae (p. 290) Without flagellate swarmers . Family 3 Vampyrelhdae (p. 290) Family 1 Labyrinthulidae Haeckel Small fusiform protoplasmic masses are grouped in network of sparingly branched and anastomosing filopodia; individuals encyst independently; with or without flagellate stages. Genus Labyrinthula Cienkowski. Minute forms feeding on various species of algae in fresh or salt water; often brightly colored due to the chlorophyll bodies taken in as food. 290 PROTOZOOLOGY L. cienkowshii Zopf (Fig. 133, a). Attacks Vaucheria in frosli water. L. sp. Renn. Renn (1934, 1936) found in the diseased leaf tissue of the eel-grass, Zostera marina, whose leaves showed 'spotting and darkening,' a species of Labyrinthula; fusiform with termi- nal, often branching, filopods; frequently in network by associa- tion of many individuals; infected host cell is completely destroyed; Atlantic coast. Genus Labyrinthomyxa Duboscq. Body fusiform; amoeboid and flagellate phases, variable in size; flagellate stage penetrates the host cell membrane; in plants. L. sauvageaui D. (Fig. 133, b-e). Fusiform body 7-1 l/z long; pseudoplasmodium-formation; amoeboid stage 2.5-14/i long; flagellate stage 7-18^ long; parasitic in Laminaria lejolisii at Roscoff, France. Family 2 Pseudosporidae Berlese Genus Pseudospora Cienkowski. Body minute; parasitic in algae and Mastigophora (including Volvocidae); organism nourishes itself on host protoplasm, grows and multiplies into a number of smaller individuals, by repeated division; the latter bifiagellate, seek a new host, and transform themselves into amoeboid stage; encystment common. P. volvocis C. (Fig. 133, /, g). Heliozoan form about 12-30^ in diameter; pseudopodia radiating; cysts about 25/x in diameter; in species of Volvox. P. parasitica C. Attacks Spirogyra and allied algae. P. eudorini Roskin. Heliozoan forms 10-12^ in diameter; radiating pseudopodia 2-3 times longer; amoeboid within host colony; cysts 15/x in diameter; in Eudorina elegans. Genus Protomonas Cienkowski. Body irregularly rounded with radiating filopodia; food consists of starch grains; division into bifiagellate swarmers which become amoeboid and unite to form pseudoplasmodium; fresh or salt water. P. aniyli C. (Fig. 133, h-j). In fresh water. Family 3 Vampyrellidae Doflein Filopodia radiate from all sides or formed from a limited area; flagellate swarmers do not occur; the organism is able to bore through the cellulose membrane of various algae and feeds on SARCODINA, PROTEOMYXA 291 protoplasmic contents; body often reddish because of the forma- tion of carotin; multinucleate; multiplication in encysted stage into uni- or multi-nucleate bodies; cysts often also reddish. Genus Vampyrella Cienkowski. Heliozoa-like; endoplasm Fig. 133. a, Labyrinthula cienkoivskii, X200 (Doflein); b-e, Laby- rinthomyxa sauvageaui (b, c, flagellate forms, XlOO; d, e, amoeboid forms, X500) (Duboscq); f, g, Pseudospora volvocis, X670 (Robert- son); h-j, Protomonas amyli (Zopf); k, 1, Vampyrella lateritia, X530 (k (Leidy), 1 (Doflein)); m, n, Nuclearia delicatula, X300 (Cash). vacuolated or granulated, with carotin granules; numerous vesic- ular nuclei and contractile vacuoles; midtinucleate cysts, some- times with stalk; 50-700/1 in diameter. Several species. V. lateritia (Fresenius) (Fig. 133, k, I). Spherical; orange-red 292 PROTOZOOLOGY except hyaline ectoplasm; feeds on Spirogyra and other algae in fresh water. On coming in contact with an alga, it often travels along it and sometimes breaks it at joints, or pierces individual cell and extracts chlorophyll bodies by means of pseudopodia; multiplication in encysted condition; 30-40^ in diameter. Genus Nuclearia Cienkowski. Subspherical, with sharply pointed fine radiating pseudopodia; actively moving forms vary in shape; with or without a mucous envelope; with one or many nuclei; fresh water. A^. delicatula C. (Fig. 133, m, n). Multinucleate; bacteria often adhering to gelatinous envelope ; up to 60ju in diameter. N . simplex C. Uninucleate; 30;u in diameter. Genus Arachnula Cienkowski. Body irregularly chain-form with filopodia extending from ends of branches; numerous nuclei and contractile vacuoles; feeds on diatoms and other micro- organisms. A. impatiens C. (Fig. 134, a). 40-350/i in diameter. Genus Chlamydomyxa Archer. Body spheroidal ; ectoplasm and endoplasm well differentiated ; endoplasm often green-colored due to the presence of green spherules; numerous vesicular nuclei; 1-2 contractile vacuoles; secretion of an envelope around the body is followed by multiplication into numerous secondary cysts; cyst wall cellulose; in sphagnum swamp. C. montana Lankester (Fig. 134, 6, c). Rounded or ovoid; cyto- plasm colored; about 50/x in diameter; when moving, elongate with extremely fine pseudopodia which are straight or slightly curved and which are capable of movement from side to side; non-contractile vacuoles at bases of grouped pseudopods; in active individual there is a constant movement of minute fusi- form bodies (function?); when extended 100-150/x long; total length 300)U or more; fresh water among vegetation. Genus Rhizoplasma Verworn. Spherical or sausage-shaped; with anastomosing filopodia; orange-red; with a few nuclei. R. haiseri V. (Fig. 134, d). Contracted form 0.5-1 mm. in diameter; with 1-3 nuclei; pseudopodia up to 3 cm. long; ex- tended body up to 10 mm. long; originally described from Red Sea. Genus Chondropus Greeff. Spherical to oval; peripheral portion transparent but often yellowish; endoplasm filled with green, yellow, brown bodies; neither nucleus nor contractile vacuoles SARCODINA, PROTEOMYXA 293 Fig 134. a, Arachnula impatiens, X670 (Dobell); b, c, Chlamy- domyxa moniana: b, X270 (Cash); c, X530 (Penard); d, Rhizoplasrna kaiseri, X30? (Verworn); e, Bionnjxa vagans, X200 (Cash); f, Penardia mutabilis, X200 (Cash); g, Hyalodiscus rubicundus, X370 (Penard). 294 PROTOZOOLOGY observed; pseudopods straight, fine, often branched; small pearl- like bodies on body surface and pseudopodia. C. viridis G. Average diameter 35-45ju; fresh water among algae. Genus Biomyxa Leidy {Gymno'phrys Cienkowski). Body form inconstant; initial form spherical; cytoplasm colorless, finely granulated, capable of expanding and extending in any direction, and of projecting filopodia which freely branch and anastomose; cytoplasmic movement active throughout; numerous small con- tractile vacuoles in body and pseudopodia; with one or more nuclei. B. vagans L. (Fig. 134, e). Main part, of various forms; size varies greatly; in sphagnous swamps, bog-water, etc. B. cometa (C.). Subspherical or irregularly elHpsoidal; pseudo- podia small in number, formed from 2 or more points; body 35- 40m, or up to 80m or more; pseudopodia 400m long or longer. Cienkowski maintained that this was a 'moneran.' Genus Penardia Cash. When inactive rounded or ovoid; at other times expanded; exceedingly mobile during progression; endoplasm chlorophyll-green with a pale marginal zone; filopodia, branching and anastomosing, colorless; nucleus inconspicuous; one or more contractile vacuoles, small; fresh water. P. mutabilis C. (Fig. 134,/). Resting form 90-100m in diameter; extended forms (including Pseudopodia) 300-400m long. Genus Hyalodiscus Hertwig et Lesser. Discoid, though outhne varies; endoplasm reddish, often vacuolated and sometimes shows filamentous projections reaching body surface; a single nucleus; ectoplasmic band of varying width surrounds the body com- pletely; closely allied to Vampyrella; fresh w^ater. H. ruhicundus H. et L. (Fig. 134, g). 50-80m by about 30m; polymorphic; when its progress during movement is interrupted by an object, the body doubles back upon itself, and will move on in some other direction; freshwater ponds among surface vegetation. References Calkins, G. N. 1926; 1933 The biologij of the Protozoa. Phila- delphia. DoFLEiN, F. and E. Reichenow 1929 Lehrhuch der Protozoen- kunde. Jena. KtJHN, A. 1926 Morphologie der Tiere in Bildern. H. 2; T. 2. Rhizopoden. SARCODINA 295 Cash, J. 1905 The British freshwater Rhizopoda and Heliozoa, Vol. 1. London. DoBELL, C. 1913 Observations on the life-history of Cienkow- ski's Arachnula. Arch. f. Protistenk., Vol. 31. DuBOSCQ, 0. 1921 Labyrinthomyxa sauvageaui n. g., n. sp., pro- teomyxee parasite de Laminaria lejolisii Sauvageau. C. r. soc. biol., Paris. Vol. 84. Leidy, J. 1879 Freshwater Rhiozpods of North America. Re- port U. S. Geol. Survey. Vol. 12. RosKiN, G. 1927 Zur Kenntnis der Gattung Pseudospora Cien- kowski. Arch. f. Protistenk., Vol. 59. ZoPF, W. 1887 Handhuch der Botanik (A. Schenk). Vol. 3. Chapter 17 Order 2 Mycetozoa de Bary THE Mycetozoa had been considered to be closely related to fungi, being known as Myxomycetes, or Myxogasteres, the 'slime molds.' Through extended studies of their development, de Bary showed that they are more closely related to the Protozoa than to the Protophyta, although they stand undoubtedly on the border-line between these two groups of microorganisms. The Mycetozoa occur on dead wood or decaying vegetable matter of various kinds. The most conspicuous part of a mycetozoan is its Plasmodium which is formed by fusion of several myxamoebae, thus producing a large multinucleate body (Fig. 135, a). The greater part of the cytoplasm is granulated, although there is a thin layer of hyaline and homogeneous cytoplasm surrounding the whole body. The numerous vesicular nuclei are distributed throughout the granu- lar cytoplasm. Many small contractile vacuoles are present in the peripheral portion of the Plasmodium. The nuclei increase in number by division as the body grows; the division seems to be amitotic during the growth period of the plasmodium, but is mitotic prior to the spore-formation. The granulation of the cytoplasm is due to the presence of enormous numbers of granules which in Calcarinea are made up of carbonate of lime. The Plas- modium is usually colorless, but sometimes yellow, green, or reddish, because of the numerous droplets of fluid pigment present in the cytoplasm. The food of Mycetozoa varies among different species. The great majority feed on decaying vegetable matter, but some, such as Badhamia, devour living fungi. Thus the Mycetozoa are holozoic or saprozoic in their mode of nutrition. Pepsin has been found in the plasmodium of Fuligo and is perhaps secreted into the food vacuoles, into which proteins are taken. The plasmodium of Badhamia is said to possess the power of cellulose digestion. When exposed to unfavorable conditions, such as desiccation, the protoplasmic movement ceases gradually, foreign bodies are extruded, and the whole plasmodium becomes divided into numerous sclerotia or cysts, each containing 10-20 nuclei and 296