AN INTRODUCTION TO THE STUDY OF THE PROTOZOA / ' , AN INTRODUCTION TO THE STUDY OF THE PROTOZOA WITH SPECIAL EEFERENCE TO THE PARASITIC FORMS BY E. A. MINCHIN, M.A., PH.D., F.R.S. PROFESSOR OF PBOTOZOOLOOY IN THE UNIVEBBITY OF LONDON a&Su, KOV (j.a6ovcn ILLUSTRATED SECOND IMPRESSION LONDON EDWARD ARNOLD 1922 [All rightt reterved} PREFACE THIS book, as its name implies, is intended to serve as an intro- duction to the subject with which it deals, and not in any way as a complete treatise upon it. The science of " protozoology," as it is now generally termed, covers a vast field, and deals with an immense series of organisms infinitely varied in form, structure, and modes of life. In recent years the recognition of the importance of the Protozoa to mankind in various ways, and especially from the medical point of view, has focussed attention upon them, and has brought about a great increase of our knowledge concerning these forms of life. To set forth adequately and in full detail all that is now known about the Protozoa would be a task that could not be attempted in a volume of this size, but would require a work many times larger. The aim of the present work is essentially didactic — that is to say, it is intended to furnish a guide to those who, having at least some general knowledge of biology, desire a closer acquaintance with the special problems presented by the Protozoa. First and foremost, it attempts to define the position of these organisms in Nature, and to determine, as far as possible, in this way exactly what should be included under the term " Protozoa," and what should be excluded from the group. Secondly, its function is to guide the student through the maze of technicalities necessarily surrounding the study of objects unfamiliar in daily life, and requiring, consequently, a vocabulary more extensive than that of common language ; and with this aim in view, care has been taken to define or explain fully all technical terms, since confusion of thought can be avoided only by a clear understanding of their exact significance and proper application. Thirdly, it aims at introducing the student to the vast series of forms comprised in the Protozoa and their systematic classification, based on their mutual affinities and inter-relationships, so far as these can be inferred from theif structural peculiarities and their life-histories. And, incidentally, attention has been drawn specially to those parts of the subject where the Protozoa throw vi PREFACE great light on some of the fundamental mysteries of living matter- as, for example, sex — and a special chapter dealing with the physiology of the Protozoa has been added. In so wide a field it is almost necessary to exercise some favour- itism in the choice of objects, and greater stress has been laid in this work upon the parasitic forms, both on account of the many interesting biological problems which they present, and also because they come into closer relationship with the practical needs of human life than the non-parasitic species. The author wishes, however, to point out clearly that he is not a medical man, but one who approaches the study of the parasitic Protozoa solely from the standpoint of a naturalist who is more concerned, so to speak, with the interests of the parasite than with those of the host. Conse- quently, purely medical problems — such as, for example, the symptoms and treatment of diseases caused by trypanosomes and other Protozoa — are not dealt with in this book, since the author deems it no part of his task to attempt to instruct medical men concerning matters with which they are better acquainted by their training and experience than himself. The needs of medical men have, however, been specially kept in view, and the author hopes that the book will succeed in supplying them with useful informa- tion, at least from a general zoological or biological standpoint. In a science, such as protozoology, which is growing actively and receiving continually new additions, and in which njost of the data are based upon an elaborate and delicate technique, there are necessarily many controversial matters to be dealt with. In such cases the points at issue have been reviewed critically, and the author has, wherever possible, attempted to give a lead by indicating more or less decisively what is, in his opinion, the most probable solution of the problem under discussion. Such judgments, how- ever, are not intended to be put forward in a dogmatic or polemical spirit, since the author recognizes fully that any conclusion now reached may be upset entirely by fresh evidence to the contrary. The vast literature of the Protozoa would, if cited in full, easily fill by itself a volume of the size of the present one. It has been necessary, therefore, to restrict the limits of the bibliography as much as possible, both by selecting carefully the memoirs to be cited and by abbreviating their titles. The works selected for reference comprise, first, comprehensive treatises which deal with the subject, or with some part of it in a general way, and in which full references to older works will be found ; secondly, classical memoirs on particular subjects, also containing, as a rule, full bibliographies ; and, thirdly, such memoirs of recent date as have PREFACE vii been deemed worthy of citation. In the many cases where the same authors have published several works on a given subject, only the last of them is cited — for example, the volume of researches published recently by Mathis and Leger (473) covers the ground of the earlier memoirs published by these authors, which are therefore not cited ; similarly, the memoir upon amoebae by Nagler (95) covers the earlier work of Hartmann and Nagler upon Amoeba diploidea. Since it was quite impossible to make the bibliography in any way exhaustive, the aim has been to make it, like the rest of the book, introductory to the subject. It is hoped that any reader who, desirous of pursuing further some special subject, consults the references cited will find in them and in the further works quoted in them the means of acquiring complete information with regard to modern knowledge concerning all the points in question. The following classes of memoirs are not cited, however, in the bibliography, unless there was some special reason for doing so : faunistic works, papers describing new species, and writings of a polemical character. New memoirs on Protozoa are being published continually, so rapidly, and in so many different periodicals (some of them very difficult to obtain), that the author fears he may himself have overlooked many such, especially of those publications which have appeared very recently, while the book was in course of preparation. For such omissions, some of which have already come under his notice, he can but apologize, and at the same time promise that they shall be rectified in future editions, if the patronage of those interested in the subject enables further editions of this book to be published. The present edition does not, however, profess to deal with works published later than 1911. In order to further the object of making this book a guide to the technicalities of the subject, the plan has been adopted of printing in heavier black type in the index the numbers of those pages on which the term cited is fulty explained, or, in the case of taxonomic names, is referred to its place in the systematic classification. In this way the index can be used as a glossary by anyone wishing to ascertain the significance of a technical term, or, though necessarily to a more limited extent, the systematic position of a genus, family, or order of the Protozoa. All that is necessary for this purpose is to look up the word in the index, and then to turn to the page or pages indicated by black type. The author has, in a few cases, modified the technical terminology in current use, or has made additions to it. The adjective in general use relating to chromatin is " chromatic," with its various deriva- viii PREFACE lives (" achromatic," etc.) ; since, however, these adjectives have a totally different meaning and use in optics, they have been altered to chromaiinic, etc., in so far as they relate to chromatin. New terms used in this book are chromidiosome (p. 65, footnote), endosome (p. 73), as an equivalent to the German Binnenkorper, and gregarinUla (p. 169). In conclusion, it is the author's pleasant duty to return thanks to those of his colleagues who have kindly rendered him assistance in his task. He is especially indebted for much help and many valuable suggestions and criticisms to Dr. H. M. Woodcock, whose unrivalled knowledge of recent bibliography has been throughout of the utmost assistance ; and to Dr. J. D. Thomson and Miss Muriel Robertson for many helpful discussions upon matters of fact or theory. Dr. A. G. Bagshawe, Professor J. B. Farmer, F.R.S., Mr. W. F. Lanchester, Dr. C. J. Martin, F.R.S., and Dr. P. Chalmers Mitchell, F.R.S., have kindly read through some of the chapters, and have given valuable advice and criticism. In justice to these gentle- men, however, it should be stated that they are in no way responsible for any of the theoretical opinions put forward by the author. The majority of the figures have been specially drawn, from the original sources, or from actual preparations by Mr. R. Brook-Greaves and Miss Mabel Rhodes, to both of whom, the author's best thanks are due. LISTER INSTITUTE OF PREVENTIVE MEDICINE, CHELSEA, S.W., July I, 1912. CONTENTS I. INTRODUCTORY — THE DISTINCTIVE CHARACTERS OF THE PROTOZOA AND OF THEIR PRINCIPAL SUBDIVISIONS . - 1 — 12 II. THE MODES OF LIFE OF THE PROTOZOA ... 13 — 28 The Four Types of Nutrition, 13-15 ; Problems of Parasitism 15-28. HI. THE ORGANIZATION OF THE PROTOZOA — EXTERNAL FORM AND SKELETAL STRUCTURES .... 29 — 39 IV. THE ORGANIZATION OF THE PROTOZOA (continued) — THE PROTO- PLASMIC BODY - ..... 40 — 44 V. THE ORGANIZATION OF THE PROTOZOA (continued) — DIFFERENTIATIONS or THE ECTOPLASM AND ENDOPLASM ... 45 — 64 A. Ectoplasmic Organs — (1) Protective, 45 ; (2) Kinetic and Locomotor, 46 ; (3) Excretory, 60 ; (4) Sensory, 61. B. Endoplasmic Organs, 62. VI. THE ORGANIZATION OF THE PROTOZOA (continued) — THE NUCLEAR APPARATUS — CHROMATIN, NUCLEUS, CHROMIDIA, CENTRO- SOMES, AND BLEPHAROPLASTS - - 65 — 99 VII. THE REPRODUCTION OF THE PROTOZOA- - . - 100 — 124 Types of Fission, 100 ; Division of the Nucleus, 101 ; Division of the Cell-Body, 122. VIII. SYNGAMY AND SEX IN THE PROTOZOA - - 125 — 161 Nature of the Sexual Process, 125 ; Occurrence of Sexual Phenomena and their Importance in the Life of the Organism, 128 ; Maturation and Reduction, 142 ; Examples of Syngamy and Reduction in Protozoa, 147 ; Theories of the Origin and Significance of the Syngamic Process, 154. / S i .S" i x CONTENTS CHAPTER PAGES IX. POLYMORPHISM AND LIFE-CYCLES OP THE PROTOZOA - 162 — 186 A. Polymorphism, 162-176 ; B. Life-Cycles. 177-185. X. THE GENERAL PHYSIOLOGY OF THE PROTOZOA 186 — 211 (1) Nutrition and Assimilation, 187; (2) Respiration, 195; (3) Excretion and Secretion, 197 ; (4) Transmutation of Energy, 199 ; (5) Reactions to Stimuli and Environments, 201 ; (6) Degeneration and Regeneration, 208. XI. SYSTEMATIC REVIEW OF THE PROTOZOA: THE SARCODINA 212 — 256 A. Rhizopoda — I. Amoebaaa, 218 ; II. Foraminifera, 231 ; III. Xenophyophora, 237 ; IV. Mycetozoa, 239. B. Aotinopoda— V. Heliozoa, 244 ; VI. Radiolaria, 249. XII. SYSTEMATIC REVIEW OF THE PROTOZOA : THE MASTIQOPHORA 257 — 279 I. Flagellate, 257; II. Dinoflagcllata sou Pcridinialos, 276; III. Cystoflagellata seu Rhynchoflagellata, 278. XIII. THE H.JBMOFLAGELLATES AND ALLIED FORMS 280 — 322 I. Trypanosoma, 283 ; II. Trypanoplasma, 309 ; III. Crithidia, 312 ; IV. Leptomonas, 313 ; V. Leishmania, 316 ; VI. Prowa- zekia, 319. XIV. THE SPOROZOA : I. THE GREOARINES AND COCCIDIA - - 323 — 355 I. Gregarinoidea, 326 ; II. Coccidia, 341. Comparison of the Life-Cycles of Gregarines and Coccidia, 354. XV. THE SPOROZOA: II. THE H.SMOSPORIDIA - - 356 — 397 (1) Haemamcebae, 357 ; (2) Halteridia, 365 ; (3) Leuoocytozoa, 369 ; (4) Hfflmogregarines, 371 ; (5) Piroplasms, 378 ; Affinities of the Hsemosporidia, 388 ; of the Telosporidia, 395. XVI. THE SPOROZOA: III. THE NEOSPORIDIA - 398 — 429 I. Myxosporidia, 399 ; II. Actinomyxidia, 409 ; III. Micro- sporidia, 411 ; IV. Sarcosporidia, 419 ; V. Haplosporidia, 423. Incertce Sedis, 425. XVII. The INFUSORIA - 430—461 I. Ciliata, 430; II. Acinetaria, 455. CONTENTS xi CHAPTER PAGES XVIII. AFFINITIES AND CLASSIFICATION OF THE MAIN SUBDIVISIONS — DOUBTFUL GROUPS ... 462 — 474 General Phylogeny of the Protozoa, 463. Spirochaetes, 466; Chlamydozoa, 470. BIBLIOGRAPHY - - 475 — 504 INDEX ... . . 505—517 AN INTRODUCTION TO THE STUDY OF THE PROTOZOA CHAPTER I INTRODUCTORY — THE DISTINCTIVE CHARACTERS OF THE PROTOZOA AND OF THEIR PRINCIPAL SUBDIVISIONS THE Protozoa are a very large and important group of organisms, for the most part of minute size, which exhibit a wide range of variation in structural and developmental characters, correlated with the utmost diversity in their modes of life. Nevertheless, however greatly adaptation to the conditions of life may have modified their form, structure, or physiological properties, a certain type of organization is common to all members of the group. The most salient feature of the Protozoa is their unicellular nature ; that is to say, the individual in this subdivision of living beings is an organism of primitive character, in which the whole body has the morphological value of a single " cell," a mass of protoplasm containing nuclear substance (chro matin) concentrated into one or more nuclei. However complex the structure and functions of the body, the organs that it possesses are parts of a cell (" organellae "), and are never made up of distinct cells ; and at least one nucleus is present, or only temporarily absent, as a constant integral part of the organism. The unicellular nature of the Protozoa, though a constant character, cannot, however, be used by itself to define the group, since it is also a peculiarity of many other distinct types of simple living things. As an assemblage of organisms of primitive nature from which, in all probability, the ordinary plants and animals have originated in the remote past by divergent processes of evolution, the Protozoa have always possessed very great interest from the purely scientific and philosophical point of view. Of recent years, however, they have also acquired great practical importance from the relations that have been discovered to exist between Protozoa of parasitic habit and many diseases of man and animals. Hence the study of the Protozoa has received an immense impetus, and has been 1 2 THE PROTOZOA cultivated zealously even by many who are not professed biologists, with the result that our knowledge of these organisms has made very great strides in the last two decades, and is advancing so rapidly that it becomes increasingly difficult for any single person to keep pace with the vast amount of new knowledge that is pub- lished almost daily at the present time. While the attention that is now focussed upon the Protozoa has led to a most gratifying increase of scientific and medical knowledge concerning particular forms, it tends frequently to a certain vague- ness in the notions held with regard to the nature and extent of the group as a whole. This is owing largely to the fact that many are now attracted to the study of the Protozoa whose aims are purely practical, and who investigate only a limited number of species in minute detail, without having an adequate foundation of general knowledge concerning other forms. Hence i«; is important to attempt to frame a general definition of the Protozoa, or at least to characterize these organisms in such a way as to enable a dis- tinction to be drawn between them and other primitive forms of life. This object may be attained logically in two ways — either by considering the distinctive characters of the group, or by enu- merating the types of organisms which constitute it ; in more technical phraseology, by determining either the connotation or the denotation of the term Protozoa. To attempt this task will be the object of the present chapter. The name Protozoa was first used in 1820* as an equivalent of the German word Urthiere, meaning animals of a primitive or archaic type. This fitting designation superseded rapidly the older term Infusoria (Infiisionsthierchen), used to denote the swarms of microscopic organisms which make their appearance in organic infusions exposed to the air. The word Infusoria is now em- ployed in a restricted sense, as the name of one of the principal subdivisions of the Protozoa (pp. 12 and 430). The first attempt at a scientific definition of the Protozoa was given by von Siebold, who defined them, from a strictly zoological standpoint, as unicellular animals. This definition, or a modifica- tion of it, is still the one given, as a rule, in zoological textbooks ; and from this time onwards the animal kingdom was subdivided universally into the Protozoa and the Metazoa. The Protozoa, as organisms in which the individual is a single cell, are regarded as those which come first (irpwros) in the ascending scale of animal life, or in the course of organic evolution ; the Metazoa, in which the individual is an organism composed of many cells, come after a) the simpler forms of life in rank and time. * For the detailed history of the growth of scientific knowledge of the Protozoa, see Biitschli (2), pp. i-iviii. DISTINCTIVE CHARACTERS OF THE PROTOZOA 3 Siebold's generalization was a great step in advance, introducing clear and orderly ideas into the place of the chaotic notions pre- viously held, and setting definite limits to the group Protozoa by excluding from it various types of organisms, such as Sponges, Rotifers, etc., which had hitherto been classed as Protozoa, but which were now referred definitely to the Metazoa. Nevertheless Siebold's definition presents many difficulties, especially when con- sidered from a wider standpoint than that of the zoologist. This will be apparent if the two words of the definition given above, " unicellular animals," be considered critically. 1. " Unicellular." — Accepting the standpoint of the cell -theory, it has already been noted that many other organisms besides Protozoa must be regarded as single cells. Moreover, it is found that many organisms which must be classed as Protozoa appear constantly in a multicellular condition ; such are the well-known genus Volvox and its allies, besides examples of other orders. In all cases of this kind, however, the constituent cells are morphologically equivalent, and are to be regarded as complete individuals more or less inde- pendent, showing as a rule no differentiation, or, if any, only into reproductive and vegetative individuals ; and the multicellular organism as a whole is to be regarded as a colony of unicellular individuals primitively similar but secondarily differentiated, it may be, in relation to special functions. Such multicellular Protozoa present, in fact, a perfect analogy with the colonial forms seen in many groups of animals higher in the scale, especially the Coelentera, where also the members of a colony, primitively equivalent and similar amongst themselves, may become differentiated secondarily for the performance of distinct functions by a process of division of labour among different individuals. It is not possible to con- found the multicellular Protozoa with the Metazoa, in which the organism is not only composed of many cells, but exhibits also cell-differentiation based on mutual physiological dependence of the cells on one another, leading to the formation of distinct tissues ; that is to say, aggregations or combinations of numerous cells, all specialized for the performance of a particular function, such as contraction, secretion, and so forth. The essential feature of the Protozoa, as contrasted with the higher animals or plants, is to be sought in the independence and physiological completeness of the cell-individual. The Metazoa are tissue-animals, in which the primitive individuality of the cell is subordinated to, or has a restraint imposed upon it by, tho corporate individuality of the cell -aggregate. In the Protozoa the cells are complete individuals, morphologically and physiologically of equal value. If, however, as few will doubt, the Motazoa have been evolved from sirnt>le unicellular ancestors, similar to the i THE PROTOZOA Protozoa, then there must have existed an unbroken series of transitions between these two types of living beings. Hence, as in all attempts to classify living beings, sharp verbal distinctions between Protozoa and Metazoa are rendered possible only through the extinction of intermediate forms, or by ignoring such forms if known to exist. It is expedient rather to recognize distinct types of organization characteristic of the Protozoa and the Metazoa respectively, and to compare and contrast them, than to attempt to limit these groups by precise definitions. 2-. " Animals." — This part of the definition raises more difficulties than their cellular nature. In the higher forms of life the distinc- tion between animals and plants is an obvious and natural one ; it is by no means so in the lower organisms. In the ranks of the simplest living creatures, those of animal nature are not marked off by any sharply defined structural or other features from those of vegetable nature, and cannot be separated from them in any scheme of classification which claims to be founded upon, or to express, the true natural affinities and relationships of the objects dealt with. As will be explained more fully in the next and subsequent chapters, the distinction between animal and vegetable is, at its first appeal - ance, nothing but a difference in the mode in which the organisms obtain their living. Forms that are obviously closely allied in all their characters may differ in this respect, and in some cases even one and the same species may nourish itself at one time as a plant, at another as an animal, according to circumstances. In short, the difference between plant and animal is primarily a distinction based upon habits and modes of life, and, like all such distinctions, does not furnish characters that can be utilized for systematic classifica- tion until the mode of life has continued so long, and the habit has become so engrained, as to leave an impress upon the entire structural characteristics of the organism. The Protozoa cannot therefore be defined strictly and con- sistently as organisms of animal nature, for, though the vast majority of them certainly exhibit animal characteristics, it is impossible to exclude from the group many which live temporarily or permanently after the manner distinctive of the vegetable kingdom, and which are plants, to all intents and purposes, leading on in an unbroken series to the simplest algae. For this reason it has been proposed to unite all the simplest and most primitive forms of life in one " kingdom " under the title Protista (Protistenreich, Haeckel), irrespective of their habit of life and metabolism, whether animal or vegetable. The kingdom Protista is then to be considered as equivalent in systematic value to the animal and vegetable kingdoms, which in their turn are restricted in their application to true animals and plants as ordinarily DISTINCTIVE CHARACTERS OF THE PROTOZOA understood. The term Protista thus unites under a single systematic category the vast assemblage of simple and primitive living beings from which the animal and vegetable kingdoms have taken origin, and have developed, by a continuous process of natural evolution, in different directions in adaptation to divergent modes of life. The conception of a Protistan kingdom separate from the animal and vegetable kingdoms is open to the objection that it contains organisms which are indubitably of animal or vegetable nature respectively. The relations of the Protista to other living things may be repre- sented graphically by the accompanying dia- gram (Fig. 1), where the circle represents the Protista, the two triangles the annual and vegetable kingdoms respectively. It is seen that the separation of the Protista as a systematic unity cuts across the ascending series of evolution ; to express it figuratively, it is a transverse cleavage of the phylogenetic "tree." A truly natural classification of living things, however, is one which expresses their genetic affinities and follows their pedigrees and lines of descent ; it should represent a vertical cleavage of the ancestral tree. Judged by this standard, the kingdom of the Protista can only be regarded as a convenient makeshift or compromise, rather than as a solution of a difficult problem — that, namely, of giving a natural classification of the most primitive forms of life. FIG. 1. — Graphic representa- tion of the relation of the animal and vegetable king- doms to the kingdom of the Protista (Protiaienreich). The Protozoa are represented by the portion of the triangle representing the animal kingdom which lies within the circle representing the Protista. Whether the kingdom Protista be accepted or not as a natural and valid division of living beings, it is imperative to subdivide it further, not only on account of its vast extent and unwieldy size, but also because it comprises organisms very diverse in nature, requiring for their study the application of methods of technique and investigation often entirely different in kind. Hence in actual practice the Protista are partitioned among at least three different classes of scientific workers — zoologists, botanists, and bacteri- ologists— each studying them by special methods and to some extent from different points of view. It is necessary, therefore, to consider from a general standpoint the principal types of organization comprised in the kingdom Protista, and we can recognize at the outset two chief grades of structure, bearing in mind always that transitional forms between them must exist, or at least must have existed. In the first grade, which is represented by the Bacteria and allied groups of organisms, a type of organization is found which is probably the more primitive, though by many regarded as the 6 THE PROTOZOA result of degeneration and specialization. These organisms do not conform to the type of structure of the cell, as this word is usually understood, since they do not exhibit, speaking generally, a division of the living body substance into a nucleus distinct from the cytoplasm ;* but the chromatin is distributed through the proto- plasmic body in the condition of scattered lumps or granules (" chromidia "), and in many cases it constitutes, apparently, the whole or a very large proportion of the substance of the body. Fro. 2. — Amoeba proteus. A, An individual in active . movement ; the arrows indicate the direction of the currents in the protoplasm ; at r is seen a pseudo- podium which is nearly completely retracted and has assumed a mulberry- like appearance ; c.v., contractile vacuole ; /., fsecal matter extruded at the end of the body posterior in movement ; the nucleus is obscured by the opacity of the protoplasm. B, An individual in the act of capturing ita prey (P1), an Infusorian (Urocentrum) ; two pscudopodia have flowed round it, as shown by the arrows, and met at the point c, enclosing the prey ; another Infusorian (P2) is seen in a food vacuole in the body ; N., nucleus ; other letters as in A. After Leidy (226), magnified 200 diameters. Further, the body in organisms of the bacterial type is of definite form, limited in many cases by a rigid envelope or cuticle, and special organs of locomotion are either absent or present in the form of so-called " flagella," structures perhaps different in nature from the flagella of truly cellular organisms. But the most remark- able and significant feature of organisms of the bacterial type is seen in the many different modes of metabolism and assimilation * The significance of the terms " nucleus," " cytoplasm," " chromidia," etc., w ill be explained more fully in subsequent chapters. DISTINCTIVE CHARACTERS OF THE PROTOZOA 7 seen to occur amongst them. Although their metabolism is in general distinctly of a vegetative or saprophytic type, it often exhibits peculiarities not found in any true plant.* In the second grade of the Protista, the organism possesses the characteristics of a true cell, in that the body shows a differentia- tion of the living substance into two quite distinct parts — the cytoplasm, or general body-protoplasm, in which is lodged at least one nucleus, a body representing a concentration and organiza- tion of the chromatin-substance. In some cases the nuclear sub- stance or chromatin may be in the scattered, chromidial condition /////// I \\Vi\\ W % Y#/wMl\\vv x v\i* / /// / ' • j i \\ ^fv. FIG. 3. — Actinosphcerium eichhornii. ect., Ectoplasm ; end., endoplasm ; c.w.1, a contractile vacuole at its full size ; c.v.2, a contractile vacuole which has just burst ;/£«., f.v., food vacuoles ; D., a large diatom engulfed in the protoplasm ; pg., pseupopodia ; N., one of the numerous nuclei. After Leidy (226), magni- fied 250 diameters. during certain phases of the life-history, but such a condition is comparatively rare and probably always temporary. The body- protoplasm may be limited by a fircm envelope, or may, on the other hand, be naked, in which case the body-form may be quite in- definite. Organs of locomotion, when present, are of various kinds ; and these organs may serve also for the capture and ingestion of food. And, finally, the metabolism is always one of the four types * For a summary and review of different modes of metabolism among bacterial organisms, see article " F«rmentationr " in Thorpe's " Dictionary of Applied Chemistry " (Longmans). 8 THE PROTOZOA described in more detail in the next chapter — namely, animal-like (holozoic), plant-like (holophytic), fungus-like (saprophytic), or at the expense of some other living organism (parasitic). The cellular organisms that constitute the second or higher grade of the Protista are commonly partitioned between botanists and zoologists as Protophyta (unicellular algae and fungi) and Protozoa respectively. It has been «-0 a t-« pointed out already, however, that this J/— ar- -P -N FIG. 4. — Euglena spi- rogyra. ces.. (Esopha- gus ; st., stigma ; c.r., reservoir of the con- tractile vacuole; P,P, paramylum - bodies ; N., nucleus. After Stein. FIG. 5. — Trichomonaa eberthi, from the intestine of the common fowl, ftt., Anterior flagella, three in number; p.fl., posterior flagellum, forming the edge of the undulating membrane ; chr.L, " chromatinic line," forming the base of the undulating membrane; chr.b., " chromatinic blocks " ; bl., blepharoplast f rom which all four flagella arise ; m., mouth-opening ; AT., nucleus ax., axostyle. After Martin and Robertson. DISTINCTIVE CHARACTERS OF THE PROTOZOA method of subdividing them is purely arbitrary and artificial ; it leads to the result that many forms are claimed by both sides, and are always to be found described in both botanical and zoological treatises. It is nevertheless convenient for many reasons to retain the group Protozoa, even though we are obliged to include in it some forms which are plants in every sense of the word. The systematist who desires to give a rigidly logical definition of the Protozoa is, then, confronted with a dilemma : either to exclude from it forms with plant-like metab- olism which naturally belong to it, or, by admitting such forms, to impair the universal applicability of the definition given. Such difficulties arise in every attempt to apply I/^^BH^H'' rigid verbal definitions to natural groups of living things ; they are the direct outcome of the fact that all organisms have undergone and are undergoing a process of evolution, whereby they adapt themselves to new conditions of life and acquire new characters, as a result of which any two forms now distinct are or have been, connected by intermediate forms. -N FIG. 6. — Trypanosoma rcmaki of the pike. A, Slender form (" van parva "). B, Stout form (" var. magna "). After Mirichin, x 2,000. FIG. 7. — Qregarina polymorpha, parasite of the digestive tract of the mealworm ; " syzygy " of two individuals attached to one another. In each individual, N., nucleus; pr., proto- merite, or anterior segment of the body; d., deutomerite, or posterior segment. After Schneider. 10 THE PROTOZOA The attempt, therefore, made in the following paragraph to give a diagnosis of the Protozoa must not be regarded as a definition of the group in the rigidly logical sense, but merely as the construc- tion of a general type, the characters of which are liable to a certain amount of variation in special cases — a compromise between the claims of logic and the versatility of Nature. The Protozoa, then, are Protista in which the organization is of the cellular type, with nucleus distinct from the cytoplasm. They are uni- cellular, in the sense that the cell constitutes an entire individual, 0«y~~^ia$Mff which may exist singly and in- dependently or in the form of cell- colonies ; but in the latter case the cells are not subordinated to the individuality of the entire cell- -f.e aggregate by the formation of /-' FIG. 8. — Slentor roeselii, fully expanded. one o{ tions. In some cases, however, certain organs can be asserted to have a sensory function, exhibited in sensitiveness either to impressions of touch or light. Thus, in many Flagellates the flagella appear to be tactile as well as locomotor in function, and in Ciliata tactile cilia occur, especially in the creeping hypotrichous forms. Sensitiveness to light is a marked feature of many Protozoa, even .of quite undifferentiated forms, such as amoebae. Rhumbler (34) has shown that many amoebae cease feeding in a strong light, and even disgorge food that they have taken in when suddenly subjected to the intense illumination necessary for microscopic study. This characteristic is, however, most marked in the holo- phytic species, to which light is a necessity for their plant-like metabolism. In the holophytic Flagellates a red pigment-spot, or stigma, is found constantly, situated close to the anterior end of the the Dinoflagellata, to show the large stigma (at.), in front of which is a lens (1.). After Schiitt (386). 62 THE PROTOZOA body (Fig. 4, st.). The belief that the stigma is the seat of light- perception receives support from the fact that in some cases it is found associated with lens-like structures, which evidently serve to concentrate light upon it and act as dioptric elements, as in Pouchetia (Fig. 31). B. Endoplasmic Organs. The bulk of the endoplasm in proportion to that of the whole body varies greatly in different Protozoa. In Flagellata, for example, the protoplasmic body must be considered as consisting almost entirely of endoplasm, the ectoplasm furnishing only the delicate periplast and myonemes. Similarly, in motionless para- sitic forms, such as the Coccidia or the " coelomic " Gregarines (p. 326, infra), the body within the cuticle is entirely endoplasm. On the other hand, in Ciliata, in which the ectoplasm may give rise to a number of different structures, the endoplasm is often a rela- tively restricted region of the body. In these examples that have been cited, the ectoplasm and endoplasm are probably stable layers, and their relative proportions are consequently more or less constant for a given phase of the life-history ; but in amoeboid forms, as already pointed out, ectoplasm and endoplasm are interchange- able, and the amount of each layer present in an organism varies with the extent of its body -surface ; that is to say, the proportion of ectoplasm to endoplasm is greatest when the amoeba is moving actively and throwing out many pseudopodia, and least when it is in a resting condition and has assumed the spherical form. As stated above, the endoplasm is a fluid, granular substance, which contains various enclosures connected with the nutritive function, and also the nucleus or nuclei. Hence it may be re- garded as the seat of trophic and reproductive functions. The nuclear apparatus will be dealt with in a separate chapter, since it belongs, strictly speaking, neither to the ectoplasm nor the endo- plasm, though commonly lodged in the latter. In this chapter only the structural elements connected with the function of food ingestion and assimilation will be described. The contents ef the endoplasm vary greatly, according to the mode of life of the organism. In saprophytic and most parasitic forms no special organs are found in connection with the nutritive function, the food being simply absorbed in a soluble condition at the surface of the body, probably by the aid of enzymes secreted by the organism, but not by any recognizable organs. In holozoic and holophytic forms, however, special organs, differing widely in each case, are present for the assimilation or elaboration of food. 1. In holozoic Protozoa the organs of assimilation take the form oifood-vacv0les, minute droplets of fluid in which the solid particles 63 ingested as food are suspended and gradually digested. In some cases, however, and especially when the prey is relatively large, no distinct fluid vacuole can be made out surrounding it, but the food appears to be simply lodged in the endoplasm itself ; the vacuole is " virtual." When the digestion is completed, the in- soluble faecal residues are cast out of the body. In Protozoa in which the body consists of naked, non-corticate protoplasm, the food is ingested, and the faecal remains are expelled, at any point on the surface of the body. In corticate Protozoa, on the other hand, in which the body is limited by a resistant envelope or cuticle of a certain strength and thickness, food can- not be ingested at any point, but is taken in through a special aperture, a cell-mouth or cytostome. In such cases the organs of food-capture are either flagella or cilia, and by their action the food is wafted into the mouth. Primitively the mouth is a superficial aperture in the cuticle, opening into the endoplasm by means of a longer or shorter tube, the oesophagus or cytopharynx. In the Peritricha (p. 433), however, the mouth and oesophagus are, as it were, carried into the body at the end of an in-sinking of the ecto- plasm, which forms a long tube or vestibule, comparable in its mode of formation to the stomodaeum of the Metazoa. In any case the food-vacuoles are formed at the bottom of the oesophagus, in the endoplasm. The mode in which the vacuoles arise, and the processes of digestion and defsecation, are discussed in a subsequent chapter (p. 189, infra). 2. In holophytic forms assimilation is carried on by cell-organs of the same nature as those found in the green cells of ordinary plants. Of primary importance are the chromatophores, or chromo- plasts, bodies containing chlorophyll or allied pigments by means of which the organism is enabled to decompose carbon dioxide in the sunlight, setting free the oxygen and utilizing the carbon for build- ing up the living substance. The chromatophores vary greatly as regards size, form, and number present in the cell-body. Other bodies of constant occurrence are pyrenoids, small glistening cor- puscles which appear to serve as centres for the formation or storage of starch or similar substances of amyloid nature produced in the process of anabolism (see infra, p. 188). In any Protozoa, whatever their mode of nutrition, the endo- plasm contains usually various enclosures, which can be classed generally as metaplastic — that is to say, as products of the upward (anabolic) or downward (catabolic) metabolism of the living sub- stance. Instances of anabolic products are the grains of starch or of the allied substance, paramylum, found in the holophytic forms, and the reserve food-materials — fat, " paraglycogen," and other substances — often stored up in considerable quantity in prepara- 64 THE PROTOZOA tion for developmental changes, especially in the female gamete, in a manner analogous to yolk-grains in an ovum. Instances of bodies resulting from catabolic activity are waste-products of various kinds in the form of granules, crystals, pigment-grains, etc., often present in great numbers, and giving the endoplasm an opaque and coarsely-granular appearance. A familiar instance of such waste- products is seen in the grains of melanin-pigment formed in the bodies of the malarial parasites (Fig. 156) as a result of the absorp- tion and decomposition of the haemoglobin of the red blood-cor- puscle. Many bodies present in the protoplasm of Protozoa may be con- sidered as originally of metaplastic nature and origin, but as utilized secondarily for various functions. Such are the oil-drops in the intracapsular protoplasm of Radiolaria (p. 251), which appear to have a hydrostatic function, and also to serve as reserve food- material in the development. It is also highly probable that both internal and external skeletons originated simply as excretions in the first instance — that is to say, as waste - products of the metabolism which have been utilized for the function of support, and subsequently adapted and modified in accordance with the special requirements of the organism. Finally, as bodies of hydrostatic function, though not to be included necessarily under metaplastic products, are the peculiar gas-vacuoles of Arcella, bubbles of gas which can be secreted, absorbed, and formed again, as circumstances may require, in and by the living protoplasm. Bibliography. — For references see p. 477. CHAPTER VI THE ORGANIZATION OP THE PROTOZOA (Continued)— THE NUCLEAR APPARATUS— CHRO MATIN, NUCLEUS, CHROMIDIA, CENTROSOMES, AND BLEPHAROPLASTS OF all the parts or organs of the cell-body, there is none of greater importance for the life and activities of the organism than the so-called nucleus, a term which, understood literally, means simply a kernel or central portion of the body, and conveys no idea of the true nature of the structure in question or of its significance for the life of the organism. The cell-nucleus, in all its various modifications, of form and structure, is essentially and primarily a collection of grains and particles of a peculiar substance which has received the name chromatin, on account of its characteristic tendency to combine with certain colouring matters and dyes. A nucleus may consist, perhaps, in some cases of little more than a single mass of chromatin, or of several such masses clumped together. In most cases, how- ever, the chromatin is combined with other substances which may be termed comprehensively achromatin, and which are built up with the chromatin in such a way- as to produce a complicated nuclear structure, as will be described in detail presently. The chromatin-substance is not necessarily, however, concen- trated entirely in the nucleus in all cases. In many Protozoa, especially amongst the Sarcodina, as, for example, Arcella (Fig. 32), Difflugia, and many other genera, the cell-body contains, in addi- tion to one or more nuclei, extranuclear granules of chromatin, termed chromi'dia,* which may be scattered in the cytoplasm * The term " chromidia," in the German form " Chromidien," was coined by Hertwig (66) to denote the extranuclear grains of chromatin, and the whole mass of them in the cell-body was spoken of as a " Chromidialnetz." Subsequent authors, however, have used the word in its singular form, " chromidium," in a collective sense, to denote the entire mass of chrcmidia present in a cellular organ- ism, and not, as might have been expected, to mean the individual grains or particles of chromatin which constitute the chromidial mass. In order to avoid confusion, it is proposed in this work to use the term chromidiosome to denote the smallest chromatin-particles oi which the chromidial mass is made up, and which grow and multiply by division like other elementary living bodies. It is clear, however, that the chromidiosomes of which the chromidial mass scattered in the cytoplasm is built up are in no way different in kind from the minutest granules of chromatin contained in the nucleus. The term "chromidiosome" must there- fore be applied to the ultimate, individual grain or particle of chromatin, alike whether it be lodged inside or outside a nucleus. 65 f> 66 THE PROTOZOA throughout the cell, or may be aggregated in certain regions of the body to form " chromidial masses " or " chromidial nets." It is even found that in some species a true nucleus may be absent temporarily during some phases of the life-cycle, all the chromatin being then in the form of chromidia, from which nuclei arise by a process of condensation and organization of the chromatin in com- bination with achromatinic elements. Such a condition may be regarded as a temporary reversion to a more archaic and ancestral condition, since, as has been pointed out already (Chapter I.), the Protista of the lower or bacterial grade of organization do not possess, speaking generally, a true nucleus, but only scattered grains of chromatin. Hence the chromidial condition of the chromatin may be ranked as an earlier and more primitive state, from which the strictly cellular grade of organization has been evolved by concentration of some or all of the chromatin to form a nucleus. In the tissue-cells of Metazoa, as a general rule, and in many Protozoa, the chromatin is concentrated entirely in the nucleus or nuclei, and chromidia do not occur. Whatever view be taken as to the primitive or secondary nature of the chromidial condition (a question upon which individual opinions may differ considerably), the following facts can be stated definitely with regard to the chromidia. In some cases the chromidia can be observed to arise as extrusions of chromatin from the nucleus, which either casts off a certain amount of chromatin into the cyto- plasm, while preserving its individuality, or may undergo complete fragmentation, becoming resolved entirely into chromidia, and ceasing to exist as a definite nucleus. In other cases, chromidia arise from pre-existing chromidia, by growth and multiplication of the chromidiosomes, thus keeping up a chromidial mass or stock which is propagated from cell to cell through many generations, independently of the nuclei present in addition to them in the cell. The chromidial mass itself may -vary considerably in structure in different cases or at different seasons ; the chromidiosomes may be arranged in clumps, strands, or trabeculae, on a protoplasmic framework, and the mass is often vacuolated and contains substances other than chromatin. In Difflugia, Zuelzer (85) has shown that in the autumn the chromidial mass assumes a vacuolated or alveolar structure, and in each alveolus grains are formed of a carbohydrate substance allied to glycogen, which functions as reserve food- material for the organism during the reproductive processes initiated at that season. On the other hand, as chromidia arise from nuclei, so nuclei may arise from chromidia. In many Protozoa, as, for example, A rcella (Fig. 32), the formation of so-called " secondary " nuclei (which, however, do not differ from other nuclei except in their mode of THE NUCLEUS 67 origin), by concentration of ehromidia into a clump or mass which acquires gradually the structure and organization of a true nucleus, is a frequent and normal occurrence in the life-cycle, as will be seen in subsequent chapters. Those who regard the chromidial condition as the more primitive will see in the formation of secondary nuclei from chromidia the ontogenetic recapitulation of the phylo- genetic origin of the nucleus as a structural element of the cell-body. From the foregoing it is seen that nuclei, in the Protozoa, do not necessarily arise from pre-existing nuclei ; the generalization " Omnis nucleus e nucleo," though it probably holds good universally for the cells of. Metazoa, cannot be maintained for Protozoa if the term " nucleus " be taken in its strict sense. On the other hand, there B FIG. 32. — Arcella wlgaris, to show formation of secondary nuclei from the chro- midia. A, Ordinary type of individual, with two nuclei and a ring of chromidia ; B, example in which secondary nuclei are being formed in the chromidial ring. Nl, Primary nucleus ; N'2, secondary nucleus in process of formation ; chr., chromidial ring ; o. aperture of the shell. After R. Hertwig (65). is no evidence that chromatin, within or without the nucleus, can ever arise de novo or in any way except from pre-existing chromatin, the particles of which grow and multiply as the result of processes of assimilation such as constitute the most essential characteristic of the living substance generally. There is no doubt, however, that chromatin may itself give rise to other substances of achromatinic nature, and probably of simpler constitution, by a process of breaking down of its complex sub- stance ; and also that there may be present in the cell various substances very similar to chromatin in their properties and charac- teristics, representing stages in the building-up of the complex material of the chromatin-substance. In one or the other of these two ways it is possible to account for bodies in the cell known by various names, such as " metachromatinic grains," " chromatoid 68 THE PROTOZOA grains," and so forth — bodies which are often mistaken for true cliromatin, but which must be carefully distinguished from it, just as metaplastic bodies are to be distinguished from protoplasm. Among such bodies must be mentioned more especially the so-called " volutin-grains,"* which have attracted much attention of recent years, and which occur in various bacterial or unicellular organisms. The volutin-grains resemble chromatin in showing affinities for so-called " nuclear stains," which they hold more firmly than the chromatin itself, when treated with reagents that extract the stain. According to Reichenow (78), volutin is a nucleic acid combination which is to be regarded as a special reserve-material for the forma- tion of the nucleo-proteins of the chromatin-substance ; during phases of the life-cycle in which the chromatin in the nucleus increases in quantity, the volutin in the cytoplasm diminishes, and, conversely, when the quantity of chromatin is stationary, the volutin-grains increase in number. Volutin-grains are thus seen to be bodies of totally different nature from chromidia, with which they are often confused on account of their similar appearance and staining reactions ; chromidia are formed, typically, as extrusions from the nucleus into the cytoplasm ; volutin-grains, on the other hand, are formed in the cytoplasm, and represent, as it were, a food-substance which is absorbed by the nucleus in the growth and formation of the chromatin. In some cases, however, the meta- chromatinic grains may represent chromidial extrusions from the nucleus which are breaking down or being modified into other substances ; compare, for example, the extrusion of vegetative chromidia, which degenerate into pigment, from the nucleus of Actinosphcerium during a depression-period (p. 209). The occurrence in the cell-body of volutin and other substances which resemble chromatin very closely may often render extremely difficult the task of identifying and distinguishing the true chro- matin, especially when it is not concentrated into a definite nucleus, but is scattered in the cytoplasm in the form of chromidial grains. The test upon which reliance is most usually placed for the identi- fication of chromatin is its staining properties, and especially its readiness for combining with basic aniline dyes and certain other colouring matters. But this test is extremely inadequate and un- reliable ; on the one hand, as has been stated above, there are substances, such as volutin, which are coloured by " nuclear " stains more intensely than the true chromatin itself ; on the other hand, in cellular organisms which possess true nuclei containing undoubted chromatin, the staining reactions of the nuclei may be strikingly different in different cases. A good example of each of * The name " volutiu " was coined by A. Meyer in 1904, and is derived irom the fact that the substance was first studied by him in Spirillum volutans. THE NUCLEUS 69 these statements is furnished by the trypanosomes parasitic in vertebrate blood : on the one hand, these parasites often contain in their cytoplasm so-called " chromatoid grains," probably of the nature of volutin (Swellengrebel , 514), which stain in a similar manner to the nucleus ; on the other hand, the nuclei of the parasites react to stains in a manner very different from the nuclei of the blood-cells amongst which they live. In short, it is not possible to name any stain or class of stains which can be relied upon either to combine with chromatin alone, or to stain chromatin in the same manner and to the same degree, at all times and in all cases* (compare Fig. 33). When, therefore, the adjectives " chromatinic " and , x " ~ ^ NV " achromatinic " are used in the course of this work, it must be clearly understood that these terms signify that the bodies or substances to which they are applied con- sist or do not consist, as the case may be, of chromatin, and not that they stain or do not stain with certain dyes. As regards the chemical nature of chro- matin, it is characterized by containing protein-substances more complex in com- FIG. 33. — Diagram to reprc- position than any other part of the cell ; it «nt in a graphic manner .. , / . f _ ., . . .the action of colouring is not possible to say definitely, however, matters that stain chro- whether it is to be regarded as a single chemical substance or as a combination or mixture of several. Its most salient feature is its variability ; judged by microchemical tests, no two samples of cKromatin can be considered identical in composition, whether from different cells or even from the same cell at different times. Certain substances, especially phosphorus-compounds, are espe- cially characteristic of nucleo-proteins, but it is not possible at the present time to define or identify chromatin by its chemical properties or composition. All experience at the present time tends to show that the final test for the identification of chromatin in the cell' is its relation to the vital activities and life-history of the organism. The term "chromatin" is thus to be regarded as denoting a biological or physiological, but not a chemico-physical, unity. A given body * Methyl-green, acidulated with acetic acid, has sometimes been indicated as a most distinctively nuclear stain ; but Hertwig (64) has shown that in the nuclei of ActinospJicerium this stain colours the plastin-framework, and not the chro- matin, and this author casts doubt on the alleged value of this stain as a reagent for demonstrating chromatin in the nucleus. matin. The circle drawn with an uninterrupted line is supposed to represent a theoretically perfect chromatin - stain, which would stain chromatin always, and nothing else but chromatin ; the circles drawn with interrupted lines represent the action of chromatin stains actu- ally ; they will stain chro- matin as a general rule, thojigh not in variably, but they will also stain other things which are not chro- matin. 70 THE PROTOZOA or grain in the cell cannot be definitely identified as chromatin, in all cases, by any chemical or physical test, but only by its relation to the life and development of the organism as a whole, and more especially to the function of reproduction and the phenomena of sex, as will be shown more fully by means of concrete instances in subsequent chapters. The sum of modern knowledge with regard to the vital activities of living bodies and the life-histories of organisms, whether plants or animals, Protozoa or Metazoa, indicates that the chromatin exercises a regulative and determina- tive influence over the functions and properties of the cell-body. Direct experimental proof of the all -importance of the nucleus for the life of the cell is obtained by cutting Protozoa into pieces, some containing portions of the nucleus, others consisting of cytoplasm alone (p. 210, infra). Those pieces that contain nuclear substance are able to regenerate the lost parts of the body and to perform all the functions of life, and in particular those of assimilation, growth, and reproduction ; those, on the contrary, that contain no portion of the nucleus rapidly lose the power of assimilation, and are unable to regenerate the body, to grow or to reproduce; and though they remain for a time irritable and capable of movement, they soon lose these properties. There are a number of facts which indicate that in the physiological activities of the cell the chief function of the nucleus is the formation of ferments ; it is therefore all-important in regulating the assimilative processes of the living substance (p. 194). The conception of chromatin as the directive and regulative centre of the cell-body renders intelligible a number of phenomena con- nected with it, such as the elaborate mechanisms which, as will be described in the next chapter, are gradually evolved and perfected for the exact partition of the chromatin in the reproduction of the cell by division, and the relation of chromatin to the Sexual process. Further, the extremely variable nature of the chromatin-substance becomes at once intelligible on this view of its relation to the specific characters and properties of the organism ; for since every species of living being — perhaps, even, every in- dividual of the same species — differs to a greater or less extent from every other : then, if such differences are determined by the chromatin, it follows that the chromatin must also differ to a corresponding degree in each case, and that consequently uni- formity of character in different samples of chromatin cannot be expected to occur. Hertwig (67, 92) considers that a certain quantitative relation of nucleus and cytoplasm is necessary in any cell for the normal continuance of the vital functions. This nucleo-cytoplasmic ratio (" Kernplasma-Relation ") is subject to variations at different THE NUCLEUS 71 periods of life-history, but is the -same, normally, for corresponding phases of the life of the cell ; it can be influenced by external con- ditions, such as food and temperature, and also by internal factors, undergoing changes in a regular manner, in harmony with changing functional conditions of the cell. In cultures of a given species at a lower temperature, multiplication is slower and the organisms grow larger and possess larger nuclei ; with increase of temperature the reverse takes place (compare p. 206, infra). It has also been observed that, in long-continued cultures of Protozoa, periods of active assimilation and multiplication arc followed by periods of depression, during which assimilation and reproduction are at a standstill, even in the midst of abundant nutriment (see especially Calkins, 5). The depression-periods are characterized by an in- crease of the nuclear substance relatively to the cytoplasm, a " hyperchromasy " of the cell, which may lead to the death of the individual unless compensated by the elimination and absorption of part of the nuclear substance (p. 209, infra) ; when the balance has been thus restored, the organism becomes normal and feeds and multiplies again. From this conception of a definite relation between the mass of the nucleus, or rather of the chromatin, and that of the cytoplasm, Hertwig has deduced a number of important consequences to which reference will be made in subsequent chapters. The influence exerted by the chromatin upon the life of the organism may be manifested in two ways, which may be termed, for convenience, actual and prospective, respectively. In the first case it regulates the metabolism and functions, both trophic and kinetic, of the cell in which it is contained, and is then commonly termed, vegetative chromatin, or trophochromqtin. In the second case it may be dormant and inactive in the cell that contains it, remaining latent, as it were, until carried on to future generations in. the course of cell-reproduction ; at a later period the whole or a part of this latent chromatin may become active, determining the nature and properties of the offspring, and thus serving as the vehicle for hereditary transmission of the characters of antecedent generations. Such temporarily dormant chromatin is commonly termed genera- tive chromatin, or idiochromatin. It is probable that in all Protozoa the cell-body contains chromatin both in the active and inactive state, the one regulating the vital functions of the living body, the other remaining dormant, in reserve for future generations. The validity of this conception, according to which the chromatin present in an organism is regarded as being either vegetative or generative in function, must be tested by 'its capacity to account for the facts of the development and life-cycle which will be con- sidered more fully in subsequent chapters. There are no means of recognizing and distinguishing vegetative and generative chro- 72 THE PROTOZOA matin except by their respective relations to the life-cycle, at certain periods of which, as will be seen, the nuclear apparatus is entirely reconstituted, effete vegetative chromatin being eliminated from the organism, either cast out or absorbed, and its place taken by reserve generative chromatin. It is only necessary to remark that some authorities speak of vegetative and generative chromatin as if they were two distinct kinds of substance, whereas they are probably to be considered rather as two phases or states of one and the same chromatin. Vegetative chromatin is that which is in a state of functional activity, and which thereby tends to become exhausted and effete in its vital powers, exhibiting in consequence the phenomena of " senility." Generative chromatin, on the con- trary, by remaining inactive, conserves its " youth " unimpaired, and constitutes a reserve from which the worn-out vegetative chromatin can be replaced. Generative chromatin of one genera- tion may become vegetative chromatin in the next. As regards their distribution in the cell-body, in some cases vegetative and generative chromatin cannot be distinguished by the observer as separate structural elements, but are mixed up together in the same nucleus ; in other cases, however, they occupy distinct situations in the body. Thus, in Sarcodina it is common for the vegetative chromatin to be lodged in the principal nucleus or nuclei, while the generative chromatin occurs in the form of chromidia, as in Arcdla (Fig. 32), or vice versa. In the Infusoria there are two kinds of nuclei, which are shown by their behaviour to consist, the one of vegetative, the other of generative chromatin. Chromidia, when present in the cell, may also differ in kind, being in some cases extrusions from the nucleus of purely vegetative chromatin, in process of elimination, while in other cases, as already mentioned, the chromidia, or a part of them, represent the generative chromatin (see p. 150, infra). The nuclei of Protozoa exhibit great variety of structure and form as compared with the relatively uniform structure of the nuclei of Metazoa. As stated already, the constituent substances or structural elements in any nucleus may be distinguished broadly as chromatinic and achromatinic : the former consisting of the chromatin, the primary and essential element never absent in any nucleus ; the latter comprising ^various accessory structures, an- cillary to the chromatin, and not all of them invariably present in any given nucleus. Amongst the principal achromatinic con- stituents of nuclei in general must be mentioned the following : (1) linin, occurring in the form of a framework, which stains feebly or not at all by chromatin-stains, and which presents the appear- ance of a delicate reticulum or network, the optical expression of an alveolar structure ; (2) a fluid enchykma or nuclear sap, filling 73 the interstices of the linin-frame\vork ; (3) plastin, a substance which has staining reactions different to those of chromatin, and which occurs in lumps or masses forming the ground-substance of the nucleoli or karyosomes presently to be described. The whole nucleus is commonly enclosed in a membrane, but this structure is probably formed in different ways in different cases, and may be absent. In addition to these various constituents, there are commonly present also in con- nection with nuclei bodies of kinetic nature! Such are the centrosomes or centrioles, which appear to control, or at least to act as centres for, the move- ments which the various parts of the nucleus perform during the process of reproduction by division. The structure and appear- ance of nuclei depend chiefly on the manner in which the chromatin is distributed. Two principal types of structure may be distinguished : in the first the chromatin is concentrated into a single mass or grain, or, if other grains are present in the nucleus, they are smaller and relatively insignificant in size ; in the second a number of grains are present which are m'ore or less equal in size. In the condition with a single, or one greatly preponderating, mass of chromatin, the nuclear space is not as a rule filled by it, but presents the appear- ance of a vesicle containing the chromatin-mass at or near its centre ; consequently such nuclei are commonly termed " vesicular " in type, and the chromatinic mass may be termed generally, and without further determination of its precise nature, an endosome ('* Binnenkorper "). When, on the other hand, the chromatin is in the form of numerous grains, they are generally distributed more or less evenly throughout the nuclear cavity ; such nuclei are termed "granular." FIG. 34. — Cyclical vegetative changes in the resting nuclei of TricJwsphccrium sieboldi. A, Stage with finely-meshed chromatic network and large karyosome (see p. 76) ; B, the meshes of the network widening, the karyosome budding off blocks of chromatin into it ; C, the same prduess carried farther ; D, coarse network con- taining scanty chromatin at the nodes, karyosome wanting ; E to G, the chro- matin increases greatly in quantity, covering the linin-framework — in G the meshes of the network are becoming finer ; //, the network has become fino- meshed again ; 7, a karyosome is being formed by condensation of the chro- matin at certain points, leading to the condition of A again. After Sclmudinn, X 2,250. 74 THE PROTOZOA Every transition from the^one type of structure to the other may be found in the nuclei of Protozoa ; in a vesicular nucleus the prin- cipal mass of chromatin may break up into smaller grains which become distributed throughout the nuclear cavity ; in a granular nucleus some or all of the grains of chromatin may be clumped together, and become fused to form a principal or single mass of chromatin. Such changes may take place during successive periods of activity of one and the same nucleus (Fig. 34). It is usual to speak of the condition of the nucleus as " resting " when it is hot actually undergoing the process of reproduction by division ; but it must be borne in mind that, so long as the cell is in a state of physiological activity of any kind, the nucleus also shares in this activity, and, strictly speaking, cannot be said to be resting. The activity of the nucleus is expressed in continual changes in its structure and rearrangements of its chromatin-substance and other constituents. In the gregarine Porospora gigantea, L6g«r and Duboscq (72) have observed changes taking place rhythmically in o o m A • FIG. 35. — Successive stages of the karyosomo (sec p. 76) of Porospora gigantea, showing the transformation of a hollow into a homogeneous karyosome by expulsion of a vacuole of clear viscous fluid into the nuclear cavity, where it forms a little mass of chromatin in front of the micropyle. .After Ldger and Duboscq (72). the living condition (Fig. 35) ; compare also Chagas (48'5). Hert- wig (64) has shown that the structure of the nucleus of Actino- spheerium can be correlated with the functional activities of the cell. Thus a condition with the chromatin all concentrated to form a central endosome is found prior to division of the nucleus, and is also found when the animal is being starved ; on the other hand, when it is supplied with abundant nutriment and is feeding actively, the chromatm-grains spread over the whole nuclear space. Since, however, abundant food also leads to frequent nuclear division, the condition with the chromatin concentrated at the centre also occurs during active cell-metabolism, as well as during hunger-periods. In the simplest condition of the nucleus the grain or grains of chromatin are lodged in a space or vacuole, containing a clear fluid or nuclear sap, but not enclosed by a definite membrane. Nuclei of this simple type of structure are seen in some of the primitive forms, such as the small amoebae of the Umax-type, in which the THE NUCLEUS 75 nucleus consists of a large mass of chromatin suspended in the nuclear sap. In some cases no other structural elements can be made out ; in others the nuclear sap contains granules of peripheral chromatin varying in size from the most minute and scarcely visible particles to distinct grains. For a simple nucleus of this type the term " protokaryon " has been proposed ; it is just such a nucleus as may be imagined to have arisen by a concentration of chromidiosomes at one spot in the cell-body, and in many cases such nuclei can be seen to be formed actually in this manner. The kinetonucleus of trypanosomes may be considered as a nucleus of this type in which the single mass of dense chromatin fills almost or quite completely the space in which it lies. In other cases there may be a clump of chromatin-grains more or less equal in size, filling the nuclear cavity, as in the nucleus of haemogregarines. When there are numerous grains of chromatin, those placed super- ficially may be united to form a limiting layer which may be termed a "false" or " chromatinic " membrane, in distinction to a true nuclear membrane, which is an achromatinic structure. Even in nuclei of the most simple type, however, substances or structures accessory to the chromatin are probably always present. In the first place, it is very probable that the grain or grains of chromatin do not lie loosely and freely in the nuclear vacuole, but are suspended in it, in all cases, by a delicate achromatinic frame- work, presenting the appearance of a fine network or reticulum, at the nodes of which the chromatin-grains are lodged. It is true that in many of the minute and primitive forms no such framework has been made out, and is believed by many observers to be absent ; but on that view it is difficult to account for the definite position of the chromatin, its changes of position during division, and the frequent appearance, during this process of an achromatinic spindle, phenomena that may be noted even in the simplest cases. The achromatinic framework is often very fine and delicate, and its substance stains feebly or not at all with the colouring matters commonly employed in microscopical technique ; hence it is very probable that it has often been overlooked in cases where it is really present. When there is but a single mass of chromatin, or one grain very much larger than all the others, the achromatinic reticulum presents the appearance of very delicate threads of linin radiating from the principal mass of chromatin to the periphery. When, on the other hand, there are numerous grains more or less equal in size, the retioulum is seen as fine lines passing from each grain of chromatin to each of the grains adjacent? to it. In all probability the apparent " threads " of the reticulum are but the optical expression of the walls or partitions separating alveoli, and there is no reason for considering the achromatinic reticulum or 76 THE PROTOZOA limn framework as different in any essential point from the alveolar framework of the general protoplasm, with which, in nuclei that lack a true membrane, it is perfectly continuous. Hertwig (66) regards the oytoplasmic framework as achromatinic substance in intimate combination with chromatin ; the nuclear framework, on the other hand, as pure achromatinic substance (linin) from which the chromatin has become separated out and organized into special structures' independent of the framework in which they are lodged. Similarly, the nuclear sap filling the nuclear space and the inter- stices of the reticulum must be identified with the enchylema of the body-protoplasm. As compared with the alveolar structure of the general protoplasm, that of the achromatinic nuclear frame- work is characterized chiefly by the larger size of the alveoli, and, consequently, the greater distinctness of the apparent reticular structure. A true nuclear membrane, when present, is probably formed in all cases from the achromatinic framework. In the nuclei of Actino- spficerium, according to Hertwig (64), the membrane is a super- ficial condensation of the achromatinic reticulum. The membrane may attain to a considerable thickness and appear doubly-con- toured in optical section, separating the nuclear framework com- pletely from the extranuclear protoplasm ; but it is always a structure very readily absorbed and re-formed, and it appears to present no obstacle to the passage of substance from the nucleus into the cytoplasm, or vice versa. Awerinzew (47), on the other hand, regards the nuclear membrane as a product of the cytoplasm. In addition to the achromatinic framework, plastin is commonly, if not invariably, present in the form of masses or bodies which receive different names, according as they consist of pure plastin or of plastin impregnated to a greater or less extent with chromatin. In the vesicular type of nucleus, the endosome may perhaps consist, in some caseSj of pure chromatin, but in most cases, if not always, it is composed of a matrix or ground-substance of plastin in which the chromatin is lodged. An endosome of this kind is termed a karyosome, or chromatin-nucleolus ; as a rule it has the form of a rounded mass, occupying the centre of the nucleus, sometimes of more than one such mass, but in a few cases it may have the form of a crescent or cap (" calotte ") closely applied to the nuclear mem- brane. In the granular type of nucleus, on the other hand, there may be one or more masses of pure plastin containing no chromatin ; such a body is termed a nucleolus simply, or a " plastin-nucleolus." In the nuclei of the tissue-cells of Metazoa, true nucleoli occur almost invariably ; in the nuclei of Protozoa, however, pure plastin- nucleoli are not of common occurrence, but have been described in a few instances — for example, in the hsemogregarine-nucleus THE NUCLEUS 77 (Reichenow, 78). As a general rule in the Protozoa, the plastin-sub- stance is found as the matrix of karyosomes, but also as that of other masses of chromatin, such as the chromosomes of the dividing nucleus (see next chapter). Goldschmidt (41) observed that the formation of generative chromidia in Mastigella (p. 265) was pre- ceded by the extrusion of plastin from the nucleus into the cyto- plasm, to serve as a matrix for the chromatin which passed out from the nucleus subsequently. In Actinosphcerium, Hertwig has shown that a karyosome or chromatin-nucleohis, present during certain states or phases of nuclear activity, may give off its chro • matin-substance into the nuclear framework (re ticulum), leaving the plastin-matrix as a body which is then seen to consist of a reticular framework similar in structure to the achromatinic reticulum of the nuclear framework, but distinguished from it by smaller meshes (alveoli) enclosed by thicker walls, as well as by its different staining properties. Certain phases of the development of Actinosphcerium are further characterized by the formation in the nucleus of numerous small plastin-nucleoli, each consisting of a single vesicle (alveolus) of plastin containing nuclear sap. Thus, a nucleus in its full complication of structure, and apart from the centrosomic elements, to be discussed presently, consists of the following parts : (1) An achromatinic framework or nuclear reticulum ; (2) a true membrane, formed from the achromatinic framework, and separating the nuclear contents from the surround- ing cytoplasm ; (3) nuclear sap, pervading the entire nuclear cavity ; (4) plastin, in the form of one or more bodies or masses which may consist either of pure plastirt (nucleolij or of plastin impregnated with chromatin (karyosomes) ; and (5) the chromatin, which may be present either in the form of granules lodged at the nodal points of the reticulum, and scattered evenly or unevenly throughout the nuclear framework, or may be concentrated in a karyosome, or may combine both these two modes of distribution in various ways. Achromatinic framework and nuclear sap may be considered as a part of the general body-protoplasm, enclosed within the nuclear space, and set apart from the cytoplasm as a special nucleoplasm ; plastin, on the other hand, is probably to be regarded as a product derived from the chromatin itself, either as a secretion or as a modification of its substance, to form a cement-like material or matrix in which true chromatin is carried. The two primary con- stituents of a nucleus are chromatin and protoplasmic framework. Nuclei, whatever their structure, are, as a general rule, bodies of spherical or ovoid form ; but in some cases, especially amongst Infusoria, the nuclei exhibit very varied forms in different species. The nucleus may then be sausage-shaped, or in the form of a horse- shoe, or resemble a string of beads ('' moniliform "), or be branched 78 THE PROTOZOA in a complicated manner. In the remarkable Acinetan Dendrosoma radians a colony is formed by budding, which resembles super- ficially a hydroid colony, each hydranth being represented by the head of an Acinetan individual with suctorial tentacles ; the branched nucleus is continuous throughout the whole colony, pass- ing uninterruptedly from one individual to another. Typically the cell-body contains a single nucleus, but in many Protozoa two or more nuclei occur constantly. When there are more nuclei than one, they may be all alike and quite undifferentiated, or they may show differences in size, structure, and function. In many Sarcodina multiple nuclei without differentiation are found to occur constantly in certain species ; for instance, two in Amoeba binudeata and Arcdla ; several, perhaps a dozen or so, in Difflugia (Fig 16) ; from twenty to forty up to some five hundred in Actinosphcerium (Fig 3) ; so also in Pdomyxa ; and in the large plasmodia of Mycetozoa many thousands of nuclei are found. Differentiation of nuclei, when it occurs, may be related to various causes. In trypano- somes and allied forms two nuclei occur con- stanfcly— a principal nucleus, or tropkonudeus, (Esox lucius). so called because it appears tjo regulate the a.fl., Anterior flagel- general metabolism and trophic activities of lum; n, kinetonu- 7, ., , , ., , . , . , . . cleus ; N, trophonu- the cell- body ; and a kinetonudqus, which is in cleus;p./Z., posterior special relation to the organs of movement, I^t*2££ fla8ella> and undulating membrane. As a rule ting membrane, and the kinetonucleus is smaller, in some cases very continued beyond it minute an(i nas a dense compact structure, as a very short free ' flagellum posteriorly, while the trophonucleus has a vesicular struc- ture ' kut m otiier cases (TjryPan°Plasma) the kinetonucleus is the larger of the two (Fig. 36). A nuclear differentiation of totally opposite character is seen in the Infusoria, where two nuclei of different sizes, hence termed " macronucleus " and " micronucleus," are constantly present ; the behaviour of these two nuclei in relation to sexual phenomena and reproduction (vide p. 153, infra) shows that the macronucleus is composed of vegetative chromatin, while the micronucleus contains the reserve generative chr.omatin. In some cases — for example, in Myxosporidia (p. 403) — nuclei of different sizes occur in relation to sexual differences. In some Protozoa — the so-called " Monera " of Haeckel — the nucleus has been stated to be wanting entirely ; but this statement THE NUCLEUS 79 is probably based on incomplete or erroneous observation, or on defective technique. In all Protozoa that have been examined in recent times, at least one nucleus has been found to occur without exception, though in some phases of the development the nucleus may temporarily disappear and resolve itself into chromidia. There now remains for consideration the question of the centro- some, the centre of the kinetic activity of the nucleus. Of all the questions connected with the nuclear apparatus, those relating to the centrosome are the most difficult to handle in a general manner, largely on account of the minuteness of the bodies dealt with, and the consequent difficulty of ascertaining their structure and com- position, even their presence, in many cases. Hence, in the litera- ture of the centrosome, there is found considerable confusion in the terminology, different authors disagreeing entirely as to the precise structures to which the name centrosome should be applied, and opposed theories, which cannot be discussed adequately in a short space, have been put forward as to the nature and origin of the centrosome. As the focus of the kinetic activities of the nucleus, the centro- some is most apparent and recognizable when the nucleus is in process of reproduction by division, And much less so when the nucleus is in the so-called " resting state." Hence the study of the nucleus during the process of division is alone decisive as to the presence of a centrosome in any given case ; and since in many cases nuclear division appears to go on without centrosomes being present, it nlay be taken as equally probable that, in all such cases at least, no centrosome is present in the resting state of the nucleus. In many cases, however, the presence of a centrosome in, or in connection with, the resting nucleus can be ascertained clearly ; it may then lie either outside or inside the nucleus. When the centrosome lies outside the nucleus, as it usually does in the cells of Metazoa, it is found typically as a minute grain or pan: of grains (" diplosome ") close beside the nuclear membrane. Its presence may be indicated by the radiate structure of the surrounding protoplasm, giving the appearance of a system of rays centred on the centrosome ; but such radiations are absent as a rule during the resting state of the nucleus, and the appearance of rays is often the first sign of impending activity and division of the nucleus. In many cases the centrosome is found lying in a mass of clear protoplasm termed archoplasm, a substance which differs, apparently, from the rest of the cytoplasm only in being free from granulations of all kinds. Archoplasm may, in short, be regarded simply as pure cytoplasm, and it appears either perfectly homogeneous, or traversed by striations which radiate from the centrosome, through the archoplasm, and even beyond its limits; 80 THE PROTOZOA the ttriations themselves being the optical expression of a radiate arrangement of the protoplasmic alveoli (meshes of the " retic- ulum "), indicating lines of force or tension centred in the centro- Bome. in some cases it is probable that archoplasm showing radiate striations may be present without any centrosome. In Actinosphcerium Hertwig showed that rays were formed in the archoplasm before a centrosome had been formed, and heralded its appearance. When the centrosome lies within the nucleus, it is found most frequently, in Protozoa, within a plastin-body or karyosome, a position which it may retain permanently during botl}. the resting and dividing conditions of the nucleus. The simple nuclei of the protokaryon-type probably contain in most cases a centro- somic gram lodged in the karyosome. In a few cases, however, an intranuclear centrosome occurs without a karyosome, or outside the karyosome if one is present. On the other hand, there are many examples of the occurrence of extranuclear centrosomes in Protozoa ; but these are for the most part cases in wliich the centro- some is in relation, not only to the kinetic functions of the nucleus, but also to those of other cell-organs, as will be described presently. Nuclei containing centrosomes have been termed '' centronuclei " by Boveri. The centrosome is seen, as a general rule, under the form of a minute gram, or centriole. This is the form in which it occurs invariably when it has an intranuclear position, lodged within the karyosome. But when it occurs outside the nucleus, it exhibits structural peculiarities which may vary at different periods, and it often presents cyclical changes corresponding to different phases of the activity of the nucleus. Thus, in Aclinosphccrium, Hertwig (64) describes the centrosome at its first appearance as a relatively large body of spongy structure, formed at one pole of the nucleus from extruded portions of the achromatinic reticulum (Fig. 37, A — E). At this stage, in which the centrosome is termed a centrosphere, it lies in a patch of archoplasm, and is the centre of a well-marked system of radiations. The centrosphere then gives rise, by con- densation of its substance, to two centrioles, or to one which divides, and at the same time the archoplasmic radiations become fainter and disappear (Fig. 37, F, G). The centrioles then take part in the division of the nucleus, and when this process is complete they again become spongy centrospheres, which go through the same series of successive changes that have already been described. Ana- logous cyclical changes of the centrosome have also been described in other cases, and have led to a conflict of opinion as to whether the term " centrosome " should be applied to the whole centrosomic complex, as it may be termed, or to the centrioles, of which many THE NUCLEUS 81 may be present. It is simplest in theory, and probably correct in fact, to regard the centriole as the primary, in many cases the sole, constituent of ths centrosome — an element which may be capable, to a greater or less extent, of changes in size and structure, and which multiplies by division. To the primary centrosome or FIG. 37. — Actinosphcerium eichhorni: formation of the centrosome. ^.Concentra- tion of the nuclear reticulum towards one pole of the nucleus, near which the cytoplasm appears free from granulations, forming the archoplasm ; B, G, D, passage of a portion of the nuclear reticulum to the exterior to form the " spongy centrosome " lying in the archoplasm ; E, spongy centrosome with striations passing from it through the archoplasm to the nucleus ; F, G, tho centrosome passes back again to the vicinity of the nucleus and undergoes a reduction of substance — the archoplasm also diminishes tem- porarily in quantity ; H, division of the centrosome. After Hertwig (64). centriole there may be added adventitious elements of protoplasmic or nuclear origin, thus forming a centrosomic complex which may attain a size relatively considerable in some cases. So far the centrosome has been discussed only in its relation to 6 82 THE PROTOZOA the kinetic activities of the nucleus, a function which may be re- garded as its primary and most characteristic role. It may act also, however, as the centre of other kinetic functions of the cell- body, especially in relation to motile organs such as flagella ; it then appears as the so-called " basal granule," from which the flagella take origin. The basal granule appears as a thickening at the base of the flagellum. It rnay be continued farther into the cytoplasm, or connected with the nucleus, by means of one or more root-like processes known as the rhizoplast. A centrosome which is in relation to a motor cell-organ is termed generally a blepharoplast. The rhizoplast may .-~"f' have various origins ; in some cases it represents the centrodesmose (p. 103) which connects the bleph- aroplast with the nuclear centro- some, or the remains of such a connection ; in other cases it repre- sents the remains of the nuclear spindle of the previous nuclear division, as in the swarm-spores of Stemonitis flaccida (Jahn, 69) and Fio. 38. — Mastigina setosa, after Gold- schmidt (41). ra., Nucleus from which the long flagellum arises ; the body is full of diatoms and other food- bodies. The surface of the body has a covering of short bristle-like processes. FIQ. 39. — Connection of the flagellum and the nucleus in Mastigina setosa. A and B, As seen in the living state ; C, after fixation and staining. After Goldachmidt (41). the collar-cells of Heterocosla (Robertson, 79) ; while in some instances it may be formed by outgrowth of root-like processes, of no special cytological significance, from1»he blepharoplast. The relation of the nuclear to the kinetic apparatus is best studied in the Flagellata, where three principal conditions may be distinguished as follows : 1. The cell-body contains but a single centrosome, which functions also as a blepharoplast ; these two names, then, denote two different phases of activity of one and the same body, whieh is a centro- THE NUCLEUS 83 5-S n. some when it is active in relation to the division of the nucleus, and a blepharoplast when it is in connection with flagella or other motile organs during the resting state of the nucleus. In this, probably the most primitive state of things, there are, further, two different structural conditions found to occur in different cases. First, the centrosome - blepharoplast may be within, or closely attached to, the nucleus ; secondly, it may be quite independent of the nucleus, and some distance from it in the cell- body, during the resting state of the nucleus. In the first case — of which an example is seen in Mastigina (Figs. 38, 39), paralleled by collar - cells in the Leuco- soleniid type of calcareous sponges — the flagellum ap- pears to arise directly from the nucleus; in the second case, exemplified by Mas- tigdla (Fig. 40), and by collar-cells of the Clathrinid type, the flagellum takes origin quite independently of the resting nucleus. In both cases alike, the flagel- lum generally disappears v FlQ w. — Masiigdla vitrea, after before division ef the nucleus \ Goldschmidt (41). n, Nucleus, begins; the blepharoplast \ almost obscured by the mass of , ' \ food-bodies of various kinds in becomes the centrosome, I the cytoplasm. divides, and initiates the division of the nucleus ; the new flagella of the daughter- cells grow out from the two daughter - centrosomes dur- ing or after division of the nucleus, and in either case, when the two daughter-cells are completely formed, their centro- somes, as blepharoplasts, remain as the basal' granules from which the flagella arise. 2. The cell-body contains more than one body of centrosomic nature — namely, a definitive centrosome, in relation to the single nucleus, and, in addition to this, one or more blepharoplasts in relation to motile organs. Then, when division of the cell takes place, one of two things may happen. 84 THE PROTOZOA In the first place, the flagellum or flagella may disappear, together with their blepharoplasts ; the nuclear centrosome divides into two, which control the division of the nucleus in the usual way, and the centrosome of each daughter-nucleus divides again into two, one of which is the definitive centrosome, the other the blepharo- plast, of the daughter-cell. The new flagella may either grow out from the daughter-centrosomes before they divide, and be carried off, as it were, by the product of division which becomes the FIG. 41. — Stages in the division of Spongomonas aplendida, to show different wa3rs in which the daughter-flagolla arise. Compare the stages of S. uvella (Fig. 42). A, Resting condition ofc- plasts. C, Daughter-flagolla arising at the poles of the nuclear spindle ; the flagella of the parent have disappeared. D, Nucleus completely divided ; one pair of daughter-flagella are seen arising from the karyosome of a daughter- nucleus, in which the blopharoplasts are still enclosed ; in the other daughter- nucleus the blepharoplasts have become distinct and the fiagolla are given off from them. E, Similar stage ; the two pairs of blepharoplasts, from which the flagella arise, are quite independent of the two daughter-nuclei. After Hartmann and Chagas (62), magnification about 2,400 diameters. blepharoplast (Fig. 41, C, D, E ; Fig. 42, C), or they may not arise from the blepharoplasts until a later period, after they have separated off from the definitive centrosomes (Fig. 42, D, E, F). The examples figured show that these differences in the origin of the flagella may occur as developmental variations in one and the same species. In the second place, the blepharoplasts and flagella may persist throughout the division of the cell ; then either the old flagellum and blepharoplast are retained by one daughter-cell, while the other THE NUCLEUS 85 forms a new blepharoplast from its ccntrosome, and subsequently a new flagcllum ; or the blepharoplast of the parent cell divides independently to form the blepharoplasts of the daughter-cells (Fig. 43). In this last type, the blepharoplast, though obviously a body of centrosomic nature, acquires a more or less complete independence of the definitive centrosome, and becomes a distinct cell-organ, permanent for at least a certain number of cell-genera- tions ; it may multiply and undergo various structural complica- tions, to be described presently. FIG. 42. — Stages in the division of Spongomonas uvella. A, Resting condition of the cell ; two flagella arise, each from one of a pair of blepharoplasts (diplo- some) ; the nucleus contains a large karyosomo, in which the centriole is lodged, and a few irregular grains of peripheral chromatin in the nuclear cavity. Ji, Early stage of mitosis ; an achromatinic spindle is formed with the centrioles at the poles, ono contriole (on the right) having already divided into two ; the chromatin, both peripheral and central, has united to form a dense equatorial plate in which separate chromosomes cannot be discerned ; the Hagella have disappeared, together with their blepharoplasts. C, Similar stage in which the daughter-flagella are growing out precociously from the centrioles, ono on the left, two on the right. D, Later stage in which the equatorial plato has split into two daughter-plates, but no flagella have as yet grown out from the centrioles, of which there are two at each pole. E, I >i\ isnon of the nucleus nearly complete ; no flagella. F, Nucleus completely divided, daughter-nuclei in process of reconstruction ; from each a pair of blepharoplagte lias been budded off, still connected by a centrodesmose with tho contriole contained in the karyosome ; a pair of daughter-flagella has ai-irtcn from OHO pair of blepharoplasls, but not as yet from the other. After Havt.iuanii and Chagus (02), magnification about 2,400. 3. In certain flagellates — for example, trypano-somes and allied forms ('' Binucleata ") — the cell-body contains two nuclei, as already noted : a trophonucleas and a kiiu-tonucleus. To what extent these nuclei are provided with centrosomes is at present a little doubtful ; probably thi.s point is one which varies in different cases (compare Wenyon, 84). There are, however; three chief possi- bilities : (a) There may be but a single centrosome, that of the kinetonucleus, which acts both as blepharoplast and division-centre FIG. 43. — Stages in the division of Pclytomdla agilis. A, Resting condition of the cell ; the four flagella arise from four blopharoplasts which are connected by a rhizoplast with the nucleus ; in the nucleus is seen a large karyosome, which contains the centriole and is surrounded by a peripheral zone of chromatin-grains in a nuclear reticulum. B, Early stage of mitosis ; the karyo- some is dividing to form a bar of chromatin occupying the axis of the achro- matinic spindle, at the equator of which a plate of chromosomes is formed by the peripheral chromatin of the last stage. C, Later stage ; the karyosome has divided completely, forming two masses at the poles of the spindle con- nected by a centrodesmoso. D, The spindle has become elongated, and the equatorial plate has split ; the centrioles are seen connected by the centro- desmose. E, Division advancing ; the polar masses have become cap-shaped, and the daughter-plates of chromosom%s have fused into conical masses ; centrioles and centrodesmoso still visible. F, Division of body beginning. O, Centrodesmose broken through, the two daughter-nuclei separate. //, /, J, Division of cell complete, one daughter-cell only represented, to show the reconstitution of the daughter-nucleus ; the polar cap becomes the karyosome, enclosing the centriole ; the conical mass formed in Stage E by fusion of the chromosomes in the daughter-plates becomes resolved gradually into chromatin-grains again, and so forms the peripheral zone of the daughter- nucleus ; each daughter-cell has two of the four blepharoplasts and flagella of the parent, and the number is doubtless made up to four again by division after the daughter-cells are set free. After Aragao (45). THE NUCLEUS 87 for the cell ; then, when cell-division takes place, the kinetonucleus first divides, and the two products of its division place themselves on each side of the trophonucleus and act as its centrosomes, as described by Franca and Athias (56)* ; (b) the trophonucleus may have a centrosome of its own, lodged in the karyosome, in addition to the centrosome-blepharoplast in connection with the kineto- nucleus ; this is probably the most usual condition with two sub- ordinate variations, according as the centrosome-blepharoplast is lodged within the kinetonucleus, as in Leishmania tropica (Wenyon, 84), or is situated close beside it, as in most trypanosomes • in either case the kinetonucleus .and trophonucleus divide quite independently of one another, as commonly seen ; (c) it is possible, but perhaps not very probable, that in some cases there may be a blepharoplast for the flagellum distinct from the centrosomes of the two nuclei ; such a condition, perhaps, occurs in Trypanoplasma. In all cases alike, division is initiated by the centrosome from which the flagellum arises; next the kinetonucleus, and lastly the tropho- nucleus, divide. The various forms of flagollar insertion described in the foregoing para- graphs admit of a simple and uniform phylogenetic explanation. Starting with a non-flagellated organism in which a simple protokaryon contains a single centriole (Fig. 44, Oa), we may suppose the flagellum at its first origin to grow out from the centriole in the nucleus (0b). No such condition is actually known amongst flagellates, though it may be compared to the origin of the axo podia from a central grain in an Actinophrys-type of Heliozoon (see below) ; in the flagellates the centrosome-blepharoplast always, ap- parently, moves out of the nucleus, either remaining in close proximity to it (la) or becoming quite independent of it (lh), the two variations of the first type. The secondctype may be derived by division of the centrosome-blepharo- plast to form the definitive centriole and the blepharoplast ; the latter may also remain in close proximity to the nucleus (2a) or become quite independent of it (2b). The third type may be supposed to arise from the hypothetical primitive condition (#") by supposing that, not the blepharoplast-centrosome alone, but the whole nucleus, divides to form two nuclei of unequal size and distinct function, the trophonucleus and kinetonucleus, each with its own centriole (3a, 3b). The centriolo of the kinetonucleus, which is at the same time the blepharoplast, may either remain within the kinetonuclous (3b) or come out of it (3C) ; its relations to the kinetonucleus are parallel to those of the centro- some-blepharoplast to the nucleus in types la and lh. Or, on the other hand, the centrosome-blepharoplast may divide into a definitive kinetonuclear centrosome and a true blepharoplast (3"). The condition with only a single centriole for both the nuclei may, if it exists, be derived from 3a or 3b by supposing that the trophonuclear centrosome becomes atrophied. When a blepharoplast exists independently of the nuclear apparatus, it may retain the form of a single grain or basal granule of the flagellum, when this organ is single, or it may multiply to * The statements of Franca and Athias are not, however, confirmed by Lebedeff (468), and it may be doubted whether any species of trypanosomc or other " binu- cleate " exists which has but a single division-centre in the cell. form two or more grains when there are numerous flagella. Thus, in £op&omONa9, which shows the extreme of complication, there are numerous basal granules corresponding to the tuft of flagella (Fig. 45). Each basal granule in this case is divided into a proximal Fio. 44. — Diagrammatic representation of the possible phylogenetic origin of the different types of flagellar attachment in Hagcllates. For the saUe of sim- plicity it is supposed that the animal lias but a single llagollum. O1, Non- tlagellatcd cell with a centriole in the nucleus ; O1', in a cell like the last a flagellum arises from the centriolo ; 1;1, condition with a fiagcllum arising close beside the nucleus ; I1', condition with the blepharoplast quite .separate from the nucleus ; 2a, division of the single centriole into a definitive centro- some and a blcpharoplast, which becomes quite independent (21') of the nucleus ; 3*, division of both nucleus and centriole to form distinct kinetic and trophic nuclei, each with its own centriole ; 3b, the kinetonuclcar centriole remains within the nucleus ; 3C, the kinetonuclear centriole becomes distinct from the nucleus ; 3d, condition with a single centriole in the cell ; 3C, condition with a blepharoplast distinct from the eentrioles of the two nuclei. and a distal granule, and the pairs of granules are arranged in a ring, interrupted at one point ; the tuft of flagclla takes origin from the distal granules of the ring. When the nucleus divides, the daughter-centrosomes give rise to new rings of blepharoplasts, THE NUCLEUS 89 from which daughter-tufts of flagella grow out ; the old tufts, with their rings of blepharoplasts, persist for some time after the new ones have been formed (Fig. 45, C), but ultimately they degenerate and disappear. The ring of blepharoplasts in Lophomonas is supported on the edge of a membranous structure, or " calyx," which in its turn is surrounded by a peculiar striated body, the " collar " of Grassi, or " parabasal apparatus " of Janicki (Fig. 45, d.). Janicki (71) has found a corresponding parabasal apparatus in other flagellates, especially in Tricfionymphidce ; the significance 90 THE PROTOZOA of this peculiar structure remains for the present problematical. In the spores of Derbesia, Davis (" Annals of Botany," xxii., pp. 1-20, plates i. and ii.) has described a condition very similar to that of Lophomonas — namely, a double ring of blepharoplasts, which, however, fuse together to form a ring of homogeneous appearance. The blepharoplast-graina are given off from the nucleus. Centrosomic bodies may be related, not only to flagella, but also to pseudopodia, especially in those cases in which the pseudopodia FIG. 46. — Adinophrys sol, showing the axial filaments of the pseudopodia centred on the nucleus. N, Nucleus ; ps., pseudopodia ; ax., axial filament ; c.v., contractile vacuole ; f.v., food-vacuole. After Grenacher. have become specialized in form and movement, as in the Heliozoa. In this group the relationship of the nuclear apparatus to the pseudopodia exhibits two types of arrangement, which are analo- gous to the two arrangements described above in Mastigina and Mastigetta respectively, and which may be explained by supposing that in the one case the kinetic centre lies within, in the other case without, the nucleus itself. Thus, in Actinophrys (Fig. 46) the numerous pseudopodia are all centred on the single nucleus, in which the centrosome is contained. A variation of this type is described by Schaudinn (43), in the peculiar multinucleate form THE NUCLEUS 91 Camptonema nutans, in which a pseud opodium arises directly from each nucleus (Fig. 47).* In Acanthocystis (Fig. 18) an example is seen of the second type, the evolution of which can be traced in the actual development ; in the buds of Acanthocystis a centriole is contained in the karyosome of the nucleus, but during the growth of the bud into the adult condition the centriole passes out of the nucleus, and becomes the so-called "central grain" of the adult, a corpuscle which occupies the centre of the body, and upon which * In Aclinosphcerium, however, there is no relationship between the pseud opodia and the nuclei. From the researches of Hertwig (64), it is evident that in this form the centrosomes are lost altogether during the vegetative life, and are formed only in certain phases of the development (p. 115). 92 THE PROTOZOA the axial filaments of the pseudopodia are centred, while the nucleus is displaced to one side and becomes excentric in position ; when the cell enters upon division, the central grain becomes the centro- some(Fig. 64). From the condition seen in Acanthocystis, it is not difficult to explain tho state of things which has been described by Zuelzer (86) in the remarkable form Wagner ella (p. 246). Here also the buds formed possess each a single nucleus containing a centriole ; in this condition they may multiply by fission with mitosis, in which the centriole functions as a centrosome. When the buds develop into the adult form, a centriole is extruded from the nucleus to form the central grain. The organism attaches itself, and the body becomes divided into three regions — head, stalk, and basal plate (Fig. 48). The nucleus travels down into the basal plate, while tho central grain remains in the head and functions as the kinetic centre of tho pseudopodia, becoming very complicated in structure. It consists of a controsome surrounded by a sphere, which is perhaps of the. nature of archoplasm, but is stated to be rich in plastin ; when the pseudopodia are extended tho sphere shows well- marked radial striations. From the centrosome minute granules are budded off. which pass along the atriat ions of the sphere to its surface, and from these granules arise the delicate axial filament* of the pseudopodia ; the basal granules are therefore comparable to the ring of blepharoplasts in Lophomonas. When the pseudopodia are retracted, the basal granules lie within the sphere, immediately surrounding the centrosome, and the radial striations of the sphere vanish. The coatrosome itself varies in structure at different times, going through cyclical changes, but usually shows a distinct central granule or centriole. When WagnereUa divides by fission, the central grain and the nucleus divider independently, and the central grain does not act as a centrosome for the dividing nucleus, which contains its own centriole. In this form, therefore, the central grain, though centrosomic in origin and nature, loses its primitive relation to the division of the nucleus, amd becomes specialized exclusively as a kinetic centre for the organs of locomotion, a course of evolu- tion perfectly parallel to that which has been traced above for the blepharo- plasta in their relation to flagolla. While there can be but little doubt as to the centrosomic nature of the blepharoplasts or basal granules of the flagella, and of the central grains on which the pseudopodia of the Heliozoa are centred, the true nature of the basal grains of cilia, on the other hand, is less certain. The majority of those who have studied them in Ciliata are of opinion that they have nothing to do with centro- somes (compare Maier, 73, and Schuberg, 44, and see p. 443, infra) ; but there are certain observations which indicate that the basal granules of the cilia have a connection with (Collin, 50), or an origin from (Entz, 53), the nuclear apparatus, in which case they may be of the same nature as the multiple blepharoplasts of such a form as Lophomonas. Hertwig (66) considers that the basal grains of the cilia may be of centrosomic nature, and that, if they have no connection with the nucleus, they afford support for the view that centrosomes can arise from the cytoplasm as well as from the nuclear framework. In view of the great structural similarity between cilia and flagella in other respects, it seems THE NUCLEUS 93 hardly likely that the basal granules would be of a different nature in the two cases. The whole question of the nature of the basal granules has been discussed in a recent memoir by Erhard (54). FIG. 48. — Wagnerdla borealis, Mercschk. A, Whole specimen seen under a low magnification: //., head containing the central grain ; P, stalk ; N., nucleus contained in the basal plate of attachment. B, Enlarged view of the head, after fixation and staining with iron-haema- • toxylin : c., cuticle of the stalk ; p*., pseudopodia ; ax., axial filaments of the pseudopodia, each arising from a ba^al granule ; c.y., central grain. After Zuelzer (86). Few problems in cytology have been more discussed than the question of the nature and origin of the centrosome, and three opposed views have been put forward which may be termed, re- spectively, the achromatinic theory, the nucleolo - centrosomic theory, and the nuclear theory. 94 THE PROTOZOA According to the achromatinic theory, the centrosome is " an individualized portion of the achromatinic nuclear substance " (Hertwig, 66), a kinetic centre on which the movements of the framework are focussed. The essential and primary constituent of the centrosome is the centriole, and so long as the centrosome remains intranuclear, as in perhaps the majority of Protozoa, it consists of the centriole alone. When, however, the centrosome becomes extranuclear, as in many Protozoa and almost universally H Flo. 49. — Pa.ram.teba eUhardi : stages of the life-cycle. A, Amoeba in the vegetative stage: N., nucleus; n.k., " Nebenkern " ; d., ingested diatom. B, C, D, Stages in the multiplication of the encysted amoeba ; in B the Nebenkern has divided up, the nucleus is still undivided ; in C the nucleus has divided up into a number of daughter-nuclei, each of which has paired with a daughter- Nebenkern ; in D the body has divided into a number of daughter-cells, each containing a nucleus and a Nebenkern. E, A free-swimming flagollula, derived from one of the daughter-cells in D, and containing a nucleus and a Nebenkern. F , 0, H, /.Four stages of the division of a nagellula ; in F the Nebenkern is dividing ; in O the two halves of the Nebenkern have placed themselves on each side of the nucleus, which is preparing for division ; //, stage of the nuclear spindle with the two halves of the Nebenkern at each pole ; in / the nuclear division is nearly complete, and the body is beginning to divide. After Schaudinn (81), all figures magnified about 500 diameters. in the cells of the higher animals and plants, accessory cytoplasmic elements may be added to the centriole to form a centrosomic complex. A point still undecided, on the theory that centrosomes are of achromatinic origin, is whether or no these bodies can be formed, in some cases, in the cytoplasm also, as maintained by some authorities. On Hertwig's view, mentioned above, that the achromatinic substance of the nucleus is identical in nature with the ground -substance of the general protoplasm, it follows that material for the formation of the centrosome must be present in the cyto- THE NUCLEUS 95 plasm no less than in the nucleus. Biitschli (3) considers it possible that the centrosome might have been originally a cytoplasmic structure, which had nothing to do with the nucleus, but became included in it when a nuclear membrane was formed. Attention must be drawn here to the remarkable genus Paramaeba (Fig. 49) founded by Schaudinn for the species P. eilhardi (see p. 228). In this form there is present beside the nucleus a body which was termed the " Neben- kern," consisting of a darkly-staining middle piece, at each end of which is a cap of clear substance. The Nebenkern has generally been considered to represent a centrosome, and Chatton (49) has put forward the suggestion that it may correspond to a karyosome or a portion of a karyoaame that has passed out of the nucleus with the centrosome. Recently, however, Janicki (71*5) has described two new species of Paramoeba, and puts quite a different interpretation upon the Nebenkern. He regards the middle piece as chro- matin, the clear caps as archoplasmic masses, each of which contains a centrosome ; and he considers the entire structure " as a second nucleus, as it were, fixed in division, in which the state of division has become the permanent form." He proposes to replace Schaudinn's term " Nebenkern " by the term " nucleus secundus," and considers it especially comparable to the " sphere " of Noctiluca (Fig. 65). Division of the nucleus and Nebenkern takes place quite independently of one anothe'r. On the nucleolo-centrosomic theory, the whole karyosome with the contained centriole, as found in many Protozoa, is compared with the complex extranuclear centrosome of the higher organisms. It is clear, however, that the karyosome consists chiefly of plastin which is impregnated to a greater or less extent with chromatin, and in which the centriole is imbedded. As Chatton (49) has pointed out, the three elements which compose the karyosome are independent of each other. When the centriole and chromatin have left the karyosome, the plastin-mass remaining behind is homologous in every way with the nucleolus of the metazoan cell, and the only element common to both the karyosome of Protozoa and the centrosome of Metazoa is the centriole. The nuclear theory of the centrosome is associated especially with the names of Schaudinn and, in more recent times, of Hart- mann and Prowazek (63). According to this view, the centrosome represents a second cell-nucleus, and every cell is to be regarded as primarily binucleate. The starting-point of the evolutionary series would be such a form as Amceba binudeaia, which possesses two similar and equivalent nuclei. In the next stage of evolution one of the two nuclei became specialized more for kinetic, the other for trophic, functions ; examples of this stage would be furnished by Paramceba (Fig. 49), with its nucleus and " Nebenkern," and by a trypanosome, with its trophonucleus and kinetonucleus, the Nebenkern of the first and the kinetonucleus of the second repre- senting the kinetic nucleus. The central grain of the Heliozoa or the extranuclear centrosome of the Metazoa would represent the final stage of evolution, namely, a kinetic nucleus deprived of all 9<> THE PROTOZOA chromatin -elements ; while the cell-nucleus proper would represent the trophic nucleus deprived of all kinetic elements. On the other hand, the condition in amoebae and similar or- ganisms, where the cell appears to contain but a single nucleus which includes the kinetic centres, is explained by supposing that here the kinetic nucleus is encapsuled in the trophic nucleus, and is represented by the karyosome with its centriole ; hence the supporters of this theory term the type of nucleus characterized by a large karyosome an " amphinucleus " or " amphikaryon," and, in their descriptions of such nuclei, they speak of the outer nucleus (peripheral zone of chromatin) and the inner nucleus (karyosome). The reasons against homologizing the karyosome and the extra- nuclear centrosome have been stated already. Against the theory of binuclearity it may be urged further — First, that to regard the protokaryon-type of nucleus seen in the most primitive forms of. Sarcodina and Flagellata as a secondary condition is a complete inversion of what is, to all appearance, the natural series of evolu- tion of the nuclear apparatus ; secondly, that the binucleate con- dition of trypanosornes and allied forms is clearly, by comparison with other Flagellates, a specialized condition ; the trophonucleus of trypanosomes also contains a karyosome and centriole, and would therefore be an " amphikaryon," on this theory ; thirdly, that the binuclear theory still leaves the centriole as a kinetic centre of achromatinic origin, which is present in both trophonucleus and kinetonucleus of trypanosomes, in both nucleus and central grain of Heliozoa (Wagnerdla), etc. All that the binuclear theory is capable of explaining is the secondary elements of the extra- nuclear centrosomic complex. That the centriole is a body of intranuclear origin and formation is shown clearly by its presence in nuclei of the primitive karyosomatic type which arise, not by division of pre-existing nuclei, but by aggregation and organization of clumps of chromidia. It should be added that, in its most recent exposition by Hartmann (61), the theory of binuclearity has undergone considerable modification and restriction. Having considered now the structure and composition of the nucleus in its principal types and morphological variations, it remains to attempt to establish a more precise conception as to what exactly is meant by a nucleus. It is evident, in the first place, that the essential component of a nucleus, never absent, is chromatin ; but it is equally clear, in the second place, that a simple mass, or several such masses, of chromatin, do not by themselves constitute a nucleus in the true sense of the word. The word " chro- matin" connotes an essentially physiological and biological con- ception, as it were, of a substance, far from uniform in its chemical THE NUCLEUS 97 nature, which has certain definite relations to the life-history and vital activities of the cell. The word " nucleus," on the other hand, as many authorities and more recently Dobell (52) have pointed out, is essentially a morphological conception, as of a body, con- tained in the cell, which exhibits a structure and organization of a certain complexity, and in which the essential constituents, the chromatin-particles, are distributed, lodged, and maintained, in the midst of achromatinic elements which exhibit an organized arrangement, variable- in different species, but more or less constant in the corresponding phases of the same species. If this standpoint be accepted, and the nucleus be regarded as an essentially morpho- logical conception, it seems to me remarkable that Dobell, in his valuable memoir on the cytology of the bacteria, should apply the term " nucleus " to a single grain of chromatin, or to a collection of such grains, and should speak of a nucleus "in the form of ehromidia scattered through the cell," or " in the form of a discrete system of granules (ehromidia)," phrases which are self-contra- dictory on the principles that he himself has laid down. We are confronted, nevertheless, with a considerable difficulty when we attempt to state exactly what amount of organization and structural complexity is -essential to the morphological concep- tion of a nucleus. If, as is probable in phylogeny, and certainly occurs frequently in ontogeny (compare Fig. 32), the nucleus arises from a primitive chromidial condition of scattered, unorganized chromatin, at what point does the mass cease to be a chromidium and become a nucleus ? This is a question very difficult to answer at present, a verbal and logical difficulty such as occurs in all cases where a distinction has to be drawn between two things which shade off, the one into the other, by infinite gradations, but which does not, nevertheless, render such distinctions invalid, any more than the gradual transition from spring to summer does away with the distinction between the seasons. Hartmann and his school consider the possession of a centriole as the criterion of a nucleus (see Nagler, 76) ; but it cannot be considered as established, in the present state of knowledge, that all nuclei have centrioles or centrosomes. All that can be said is that, as soon as a mass or a number of particles of chromatin begin to concentrate and separate themselves from the surrounding protoplasm, with formation of distinct nuclear sap and appearance of achromatinic supporting elements, we have the beginning at least of that definite organiza- tion and structural complexity which is the criterion of a nucleus as distinguished from a chromidial mass. In the first chapter of this book a distinction was drawn between organisms of the " cellular " grade, with distinct nucleus and cytoplasm, and those of the " bacterial " grade, in which the 7 98 THE PROTOZOA cliromatin does not form a distinct nucleus. In all Protozoa there is a true nucleus in at least the principal stages of the life-history, and it is obvious that the recognition of a cellular grade, charac- terized by the possession of a true nucleus, postulates that the first origin and evolution of the nucleus must be sought amongst those organisms which have been classed, speaking broadly, as the bacterial grade. We may expect, therefore, to find in organisms which stand on the plane of morphological differentiation which characterizes the bacteria the early stages of the evolution of the nucleus from the primitive chromidial condition, and even cases in which the condition of a true nucleus has been reached. The matter cannot be discussed further here, where it must suffice to establish the existence of true nuclei in Protozoa ; but Dobell (52) has described an interesting series of conditions which may be regarded as stages in the evolution of nuclei amongst bacterial organisms. Since the possession of a true nucleus has been regarded here as the criterion of the cellular grade of organization, it is necessary to discuss briefly the meaning and application of the term " cell." By many, perhaps most modern writers, the cell has been regarded as the elementary vital unit, than which there exists nothing simpler amongst living beings. In this sense the word " cell " becomes synonymous with the term " micro-organism," "protist," or any other word used to denote living beings of the most primitive type : " tout ce, qui vit n'est que cellules " (Delage and Heiouard, 6). The word " cell'" was, however, applied originally to the elements that built up the tissues of animals and plants. At first, as the word cell implies, it was used to denote only the enclosing membrane or framework ; but when it became apparent that the membrane was of secondary importance, it was transferred to the contained stuff, and so came to signify a structural element in which the living substance, protoplasm, is differentiated into two distinct parts — nucleus and cytoplasm. If the term " cell " is not to become so vague and indefinite in its significance as to be absolutely meaning- less, it is best to restrict its application to living organisms which have reached this degree of differentiation. Dobell considers that all Protista are nucleated organisms ; in the preceding paragraphs reasons have been advanced against accepting this proposition as a statement of fact, and from the point of view of phylogenetic speculation, I, at least, find it difficult to believe that the earliest form of life could have been an organism in which the living sub- stance was differentiated ab initio into distinct nucleus and cyto- plasm. In my opinion the cell, as defined above — that is to say, an organism in which the living substance, protoplasm, has become THE NUCLEUS 99 differentiated into two parts, a nucleus, in the morphological sense, distinct from the cytoplasm — does not represent the primary and universal form of the living organism or unit, but is to be con- sidered as a stage in the evolution of living beings, a stage which many living beings have not reached. Thus a bacterial type of organism, in which the chromatin is scattered through the proto- plasmic body in the form of chromidial granules, and which there- fore does not possess a true nucleus, is not to be regarded as a cell, but as representing a condition antecedent to the evolution of the true cellular type of structure. In all Protozoa, on the other hand, the entire plan of the organization is founded on the type of the cell, which is to be regarded as the starting-point in the evolu- tion of the entire animal and vegetable kingdoms (compare Min- chin, 75). This point will be discussed further in a. subsequent chapter (p. 464). Bibliography. — For references see p. 477. CHAPTER VII THE REPRODUCTION OF THE PROTOZOA THE methods by which reproduction is effected amongst the Protozoa vary greatly in matters of detail, as will be seen ; but the obvious diversity in method throws into greater relief the under- lying unity in principle. In Protozoa, as in Protista generally, reproduction takes place always by means of some form of fission — that is to say, division or cleavage of the body into two or more parts, which are set free as the daughter -individuals. An essential part of the process is the partition amongst the daughter-individuals of at least some part of the chromatin-substance possessed by the parent. Hence fission of the cell-body as a whole is always pre- ceded by division of the nucleus ; and if chromidia are present, they also are divided amongst the products of the fission of the body. On the other hand, division of the nucleus is not necessarily followed at once by division of the body. Considering the methods by which fission is effected from a general standpoint, we may distinguish three chief typss of repro- duction, each of which may show subordinate variations : 1. Division of the nucleus, or, if there are two differentiated nuclei, division -of each of them, is followed by division of the body ; this is the commonest and most typical mode of reproduction, inown as simple or binary fission. 2. Division of the nucleus or of each of two differentiated nuclei is not followed immediately by corresponding divisions of the body, but may be repeated several times, and so give rise to a multi- nucleate condition of the body, which may be — (a) Temporary, and soon followed by cleavage of the body into as many daughter-individuals as there are nuclei or pairs of dif- ferentiated nuclei ; this method is known as multiple fission (Fig. 127) ; or it may be — (b) Permanent, giving rise to a multinucleate body which is termed a pfcuMMNfMMh Then division of the body may take place at any time by cleavage of the body into two or more multinucleate parts ; this process is known as plasmot&my. Ultimately, however, in all cases a plasmodium breaks up by multiple fission into uni- 100 THE REPRODUCTION OF THE PROTOZOA 101 nucleate individuals at the end of a longer or shorter vegetative existence during which it may have multiplied frequently by plasmotomy. The process of fission must now be considered in more detail, beginning with — 1. Division of the Nucleus. — As in the case of the cell-body as a whole, the division of the nucleus is effected in various ways. Probably the most primitive type is that in which the nucleus becomes resolved into chromidia, from which, again, secondary daughter-nuclei are reconstituted. This type of division may be termed "cmomidial fragmentation." It is of comparatively rare occurrence, but examples of it are found among Sarcodina and Sporozoa. In Echinopyxis two daughter-nuclei are formed in this way (Hertwig, 66, p. 8). In other cases numerous daughter-nuclei may arise, as in the formation of the nuclei of the microgametes in Coccidium (Fig. 50), where the parent nucleus gives off into the cytoplasm a fine dust of chromidial particles which travel to the surface of the cell and become concentrated at a number of spots to form the daughter-nuclei. True nuclear division, in which the parent and daughter-nuclei retain throughout the process their individuality and distinctness from the cytoplasm, must be distinguished clearly from the above- mentioned process of chromidial fragmentation. Fn the vast majority of cases the nucleus divides into two halves by simple or binary fission, which, as already stated, may be repeated several times before cell-division takes place ; but in a few cases the j tunleus divides simultaneously into a number of portions by m Uiple fission. In the cells of Metazoa true nuclear division alone occurs, and may follow one or the other of two sharply-marked types, termed comprehensively direct and indirect. In direct division the nucleus is constricted simply into two parts, without circumstance or ceremony. In indirect division, on the other hand, the nucleus goes through a complicated series of changes, following each other in a definite order and sequence, the whole process being known as karyokinesis or mitosis. In spite, however, of the intricate nature of karyokinetic division, and the variations in matters of detail that it exhibits in different cases, the whole process is perfectly uniform in its general plan, and admits of being described without difficulty in generalized terms. Such a description is found in every textbook of biology at the present time, and need not be repeated here ; it will be sufficient to analyze briefly the more important events that take place. In the process of karyokinesis, the achromatinic elements of the nucleus furnish the active mechanisms, while the chromatin-sub- 102 THE PROTOZOA stance appears to be the passive subject of the changes that are effected. With the achromatinic nuclear elements, extranuclear cytoplasmic substances, such as archoplasm, may collaborate. After Fro. 60. — Formation of microgametes in Coccidium schubergi. A, Full-grown microgametocyte, with finely-granular cytoplasm and large nucleus con- taining a conspicuous karyosome ; freed from the host-cell. B, The nuclear membrane has disappeared, and the chromatin, in the form of minute chro- midial granules, is passing out into the cell. C, The chroniidia have collected at the periphery of the body ; the karyosome is left at the centre, and has become pale through loss of chromatin-substance. D, The chromidia, seen on the surface of the body, are collecting together into irregular streaks and clumps. E, The chromatin -streaks of the preceding stage are collecting together into patches. F, The patches of chromatin of the preceding stage have become dense and closely pacfced. 0, H, The patches of chromatin take on a definite form as the future nuclei of the miccogametes. /, Two flagella grow out from close to each microgamete-nucleus, and by their activity the microgametes, consisting almost entirely of chromatin, break loose from the body of the gametocyte and swim away. J, Three micro- gametes, more highly magnified ; in each, two flagella arise from the thicker find ; one of the flagella (the shorter) becomes free at once, the other (the longer) runs along the body and becomes free at the hinder end. n., Nucleus, k, karyosome, of the microgametocyte ; n', n', nuclei of the microgametes. After Schaudinn (99); A—E magnified 1,000, F—I magnified 1,500, J magnified 2,250. disappearance, as a rule, of the nuclear membrane the achromatinic substance, or the combination of achromatinic and archoplasmic THE REPRODUCTION OF THE PROTOZOA 103 elements, assumes a characteristic bipolar form, like a spindle. At each pole of the spindle a centrosome or centriole is to be found, as a general rule. The two centrosomes have arisen by division of the originally single centrosome, and may remain for some time connected by a fibril or by a system of fibrils, forming what is often termed a " central spindle," but is better named a centrodesmose. The axis of the achromatinic spindle is formed by the centrodesmose, if it persists, and the remainder of the spindle is constituted by the so- called ' ' mantle-fibres ' ' running from pole to pole. The mantle-fibres are derived from the achromatinic reticulum of the nucleus and the archoplasm ; they are probably hi most cases the optical expression of an arrangement of the protoplasmic alveoli in longitudinal rows, under the influence of tensions or forces centred at the poles of the spindle. Such an arrangement of the alveoli produces the optical appearance of fibrils connected by cross-junctions, the apparent fibril being formed by thickened walls of alveoli hi line with one another, while the cross-junctions are the trsasverse walls between consecutive alveoli. On this view the apparent fibrils of the achro- matinic spindle are in reality merely the indication of lines of force in the protoplasmic framework ; but some authorities consider that in certain cases at least true fibrils are formed, which may be isolated from each other and without cross-connections (Hertwig, 64). The spindle-fibres, whether real or apparent, are centred at the poles of the spindle on the centrosomes, from which other striations may radiate out in all directions through the archo- plasmic masses (" attraction-spheres "), and extend into the sur- rounding cytoplasm. While the achromatinic spindle-figure is in process of formation, the chromatin of the nucleus has gone through a series of changes which may differ in different cases, but which result in the forma- tion of a number of masses of chromatin termed chromosomes. The number, size, and shape, of the chromosomes vary greatly in dif- ferent species, but in Metazoa these characters are generally con- stant for the corresponding phases of the same species. Each chromosome, when formed, consists of a great number of minute grains of chromatin, chromidiosomes, cemented together in a matrix or ground-substance of plastin. The chromosomes arrange them- selves at the equator of the achromatinic spindle in the form of a plate, hence termed the equatorial plate. The nucleolus disappears, being absorbed or cast out, and does not contribute to the karyo- kinetic figure, but a part at least of its substance probably furnishes the plastin ground-substance of the chromosomes. At this phase, when the achromatinic spindle is fully formed, with the plate of chromosomes at its equator, the actual partition of the chromatin between the two future daughter-nuclei usually begins, 104 THE PROTOZOA though in some cases it is accomplished at an earlier stage ; it takes place in one of two ways, known respectively as equating and re- ducing division. In equating division each chromosome divides into two daughter-chromosomes, a process which, in the finished and perfect karyokinesis of the higher organisms, is effected by a longi- tudinal splitting of the chromosome, and which may be interpreted as a simple division into two of each of the component chromidio- somes (compare Fig. 60). In reducing division, on the other hand, the individual chromosomes do not divide, but are sorted out, half of them going to one pole of the spindle, and eventually to one daughter-nucleus, the other half to the other ; with the result, finally, that each daughter-nucleus has half the number of chromo- somes possessed originally by the parent nucleus. Equating division is the usual type of karyokinesis seen in ordinary cell- multiplication ; reducing division, on the other hand, is seen only in certain phases of the maturation of the germ-cells, as explained in the next chapter. In either type of division, whether equating or reducing, the equatorial plate of chromosomes as a whole divides into two daughter-plates, which separate from one another and travel towards the poles of the achromatinic spindle. As the daughter-plates move away from each other, an achromatinic framework appears between them, in which a longitudinal striation or fibrillation is seen in line with, and continuing that of, the achromatinic spindle. Hence the achromatinic spindle as a whole consists now of the older terminal portions passing from the poles to the daughter-plates, and a new median portion passing between the two daughter-plates ; the two terminal portions constitute together what may be termed conveni- ently the " attraction-spindle," the median portion the "separation- spindle." As the daughter-plates travel further apart, the separa- tion-spindle elongates more and more ; the attraction-spindle, on the other hand, becomes shorter, usually to such a degree that the daughter-plates are brought close up to the poles of the attraction- spindle, which consequently is obliterated and disappears. When full separation of the daughter-plates is attained, the separation- spindle breaks down and disappears gradually, the middle part alone persisting in some cases ; the chromatin of the daughter-plates becomes rearranged to form the daughter-nuclei, going through a scries of changes similar to those by which the chromosomes arose from the parent-nucleus, but in inverse order. A nuclear mem- brane is formed round each daughter-nucleus, and the process is complete. In the Metazoa, direct and karyokinetic division stand out as the sole types of nuclear division, in sharp contrast and without inter- mediate or transitional forms of the process. In Protozoa, on the THE REPRODUCTION OF THE PROTOZOA 105 contrary, every possible form of nuclear division is found, from the most simple and direct to karyokinesis as perfect as that seen in the Metazoa. The nuclear division-processes of Protozoa are there- fore exceedingly interesting as furnishing object-lessons in the gradual evolution of the mechanism of nuclear division ; but the extreme diversity in these processes makes it very difficult to deal with them in the Protozoa in a general and comprehensive manner in a short space and without excessive detail. Speaking generally, the indirect nuclear division seen in Protozoa differs from that of the higher organisms in a number of points which indicate that it stands on a lower grade of evolution. As regards the achromatinic elements, the nuclear membrane is usually persistent throughout the process of division, a circumstance which enables a sharp dis- tinction to be drawn between the portions of the division-mechanism derived from the nuclear framework and the cytoplasm respectively. In many cases it is then seen that the cytoplasm does not take any share in the process at all, but that the nucleus divides in a per- fectly autonomous manner, spindle and centrioles remaining intra- nuclear throughout the whole process. As regards the chromatin, the chromosomes when formed are often irregular in form, size, and number ; they often appear imperfectly separated from one another ; they are not always arranged in a definite equatorial plate, but may be scattered irregularly along the spindle ; and they do not always split in the exact manner characteristic of the nuclear divisions of the higher organisms, but divide irregularly and often transversely. The principal types of nuclear division in Protozoa will now be described with the aid of a few selected examples. We may begin with those in which the division of the nucleus is autonomous, without co-operation of cytoplasmic elements. Division has often been asserted to be direct in cases in which subsequent research has revealed a more elaborate type; never- theless, many typical cases of amitosis occur among Protozoa. In some nuclei of the vesicular type, the chromatin appears to be concentrated entirely in the karyosome, which may contain a centriole also, and when the nucleus divides the karyosome becomes dumb-bell-shaped, and is finally constricted into two halves, the entire nucleus following suit ; as an example of this, almost the simplest conceivable type of nuclear division, may be cited the nuclei of the Microsporidia and allied organisms (Fig. 173, p. 416). A type similar in the main to that just described, but slightly more advanced in structural complication, is exemplified by the division of the nucleus in the schizogony of Coccidium (Fig. 51 , F — M ) ; here there is a peripheral zone of chromatin and a more distinct nuclear membrane. After division of the karyosome, the peripheral 106 THE PROTOZOA chromatin is halved irregularly ; no definite chromosomes are formed, but the grains of peripheral chromatin form clumps and masses of various shapes and sizes. A definite achromatinic spindle FIG. 51. — Formation of the karyosome and division of the nucleus in the schizont of Coccidium schubergi. A, Nucleus of the sporozoite, with scattered grains of chromatin but no karyosome. B, C, D, Nuclei of young schizonts in which larger grains of chromatin collect together at the centre to form the karyo- some. E, Nucleus of older schizont with complete karyooome. F, Nucleus of full-grown schizont. 0 — M , Division of the nucleus of the schizont ; the chromatin of the nucleus becomes aggregated into larger clumps and the karyosome becomes dumb- bell-shaped, •» with masses of chromatin at each pole (G and H) ; the two daughter-karyosomes, at first connected by a fila- ment ox centrodesmose, travel apart, taking the polar clumps of chromatin with them (/) ; the centrodesmose breaks through and disappears, and the two daughter-nuclei travel apart, with formation of an intermediate body on the filament between them (J — L) ; finally the connecting filament breaks down and the daughter-nuclei separate (M). k1, Karyosome ; Jfc2, P, daughter- karyosomes ; t'., intermediate body. After Schaudinn (99), magnified 2,250. FIG. 52. — Direct division of the nuclei in the oocyst of Coccidium schubergi. A, The resting nucleus ; B, C, D, clumping together of the chromatin-granules preparatory to division ; E, F-, 0, the nucleus elongates and becomes dumb- bell-fahaped ; H, the nucleus has just divided into two halves. After Schaudinn (99), magnified 2,250. also does not become differentiated. As the daughter-karyosomes, connected by a centrodesmose, travel apart, half the peripheral chromatin follows one karyosome, half the other. This method of THE REPRODUCTION OF THE PROTOZOA 107 division is a very common one in the nuclei of Protozoa, and may show a further advance towards a true mitosis in that the peri- pheral chromatin may shape itself into more or less definite chromosomes, as in Euglena. Examples of granular nuclei which divide in the direct method are seen in the division of the nucleus of the oocyst of Coccidium (Fig. 52) to form the nuclei of the sporoblasts (see p. 349, infra) and in the corresponding divisions of the nuclei of hsemogregarines (Fig. 53). In these two cases the presence of a centriole in the nucleus is douotful, but is affirmed by Hartmann and Chagas (89) for haemo- gregarines ; a true nuclear membrane, FIG. 53. — Direct division of the nucleus in the zygote of Hcemogregarina stepanowi. $ , Degenerating male elements attached to the zygote ; N., divid- ing nucleus of the zygote, two successive stages (A and B). After Eeichenow (78). however, appears to be absent, and this form of division is not much advanced beyond the condition of chromidial frag- mentation. In the macronucleus of Infusoria (Fig. 54), in which a distinct membrane is present, the division is also direct, and centrioles are stated to be absent as a general rule ; in some cases, however, true centrioles appear to be wiakoff, in Leuckart and present (Nagler). When centrioles are Nitsche's Zoologische Wand- absent, the achromatinic framework of the nucleus appears to be principally active in the division. In some cases the division of the macronucleus of Infusoria is not into two equal halves, but may take the form of budding off a smaller daughter-nucleus from the main mass. Remark- able instances <>i nuclear budding of this kind are seen in the Acinetaria, where it is related to the formation of buds by the parent individual. In some cases (Fig. 55), the nucleus may form a con- macronucleus (N) dividing without mitosis, the micro- nucleus (n) dividing mito- tically. c.v.1 Old, and c.t>.2, new, contractile vacuoles. After Biitschli and Sche- 108 THE PROTOZOA siderable number of buds simultaneously, each of which becomes the nucleus of a daughter-individual budded off from the parent. The simplest types of mitosis show but little advance on the processes of direct division that have just been described. Taking first the vesicular type of nucleus with a large karyosome (" proto- karyon "), the first stage in the process is the division of the karyo- some, as in Coccidium ; its centriole divides first, then the karyo- some becomes constricted and divides, the two halves often plainly connected by the centrodesmose formed by the division of the cen- trioles. Next an achromatinic spindle is formed between the two daughter-karyosomes, and chromosomes make their appearance, derived partly (perhaps in some cases entirely) from the peripheral zone of chromatin, partly from the chromatm contained in the karyosome. A good example of this mode of division has been described by Aragao (87) in an amosba named by him A. diplomitotica from the fact that two types of mitosis occur in this species. In the first type (Fig. 56, A—G), the little rod -like chromo- somes are not arranged FIG. 55. — Budding in Podophrya gemmipara. The • j /* -. ,n4.n,,^^ macnmucleus of the parent has sent off a number m a definite equatorial of outgrowths, which extend into the buds and plate, but are scattered give rise to the nuclei of the daughter-individuals irregularlv along the about to be budded off. N1, Parent-nucleus ; . ?« N2, nuclei of buds. After Hertwig. spindle ; some travel to- wards one pole, some towards the other, and, after separation into two groups in this manner, the chromosomes of each group fuse together to form an apparently solid mass of chromatin, representing the daughter- plates ; these masses of chromatin follow each their respective karyosomes as they travel apart, and when the nucleus is finally constricted into two daughter-nuclei, the chromatin-masses break up again into their constituent chromosomes, which become dis- tributed in the peripheral zone and karyosome of the daughter- nuclei, where they can be distinguished plainly even during the resting state (Fig. 56, A). In the second type of mitosis seen in A. diplomitotica (Fig. 56, H — K), the chromosomes arrange themselves in a definite equatorial THE REPRODUCTION OF THE PROTOZOA 109 plate, which divides into two equally definite daughter-plates com- posed of distinct chromosomes ; whether this division is brought about by splitting of the individual chromosomes is not clear. When the nucleus is finally constricted into the two daughter- nuclei, the chromosomes are at first aggregated close beside their respective karyosomes, but soon distribute themselves in the manner already described. The simple types of mitosis described in the two foregoing para- graphs are examples of the so-called " promitosis " (Nagler, 05) Fia. 5(5. — The two raethuds of nuclear division in Amoeba diplomitotica. A, Resting nucleus ; R — 0, first method ; // — K, second method. In F and G only one of the two halves of the nuclear figure is drawn. After Aragao (87). seen commonly in nuclei of the protokaryon-type. The nuclear membrane in this type is a negligible quantity ; it may be scarcely or not at all developed in the resting nucleus, and when a distinct membrane is present it may vanish entirely during the mitosis, as in the form just described. In any case, however, the entire mitosis goes on within the nuclear space. The chromosomes may .show every possible condition in different cases, from complete invgu- larity in form, number, arrangement, and mode of division, to the 110 THE PROTOZOA formation of a definite equatorial plate which splits into two daughter-plates. The most striking and salient feature of this type of mitosis is furnished by the relatively huge " polar masses," con- sisting of the daughter-karyosomes with their contained centrioles. In the division of the nucleus of Arcella (Fig. 57), however, the karyosome first breaks up into fine grains of chromatin, from which the polar masses and the equatorial plate are formed. The karyo- some, as has been pointed out in the previous chapter, consists of three distinct elemente — namely, plastin, chromatin, and centriole Fio. 57. — Nuclear division in Arcella vulgaris: karyo kinesis of one' of the two principal nuclei. A, Spireme-stage, resulting from disruption of, the karyosome ; B — D, formation of an equatorial plate of minute chromosomes (?) which split ; E, antiphase ; F, the two daughter-nuclei shortly after division. After Swarczewsky (101), magnified 2,250. — each independent of, and separable from, the others. In proportion as the karyosome loses its plastin and chromatin elements, and becomes reduced to the centriole alone, so the primitive promitosis will approach more and more to the type of an ordinary mitosis. Such a reduction of the karyo- some could take place during the mitosis if, as happens frequently, the whole of the chromatin p contained in the karyosome passed out to join the peripheral chromatin in forming the chromosomes, the plastin-substance at the same time furnishing the required ground-substance of the chromosomes (Fig. 58). On the other hand, the karyosome may disappear from the- resting nucleus also ; Chatton (49) has brought together a number of instances of nuclei showing a gradual reduction of the karyosome in different species, and the evolution of a granular type of nucleus in which the chromatin is scattered through the achromatinic framework, leaving the centriole free or but slightly encumbered by other elements in the nuclear cavity. When a nucleus of this type divides by mitosis, a most typical and perfect karyokinetic THE REPRODUCTION OF THE PROTOZOA 111 figure may be produced, as ill Euglypha (Figs. 59, 60), only differing from that of Metazoa in that the whole mitosis takes place within the nuclear membrane, and consequently without any co-operation of cytoplasmic elements. Chatton proposes for a mitosis of this type^the term " mesomitosis," as distinguished from the more ad- vanced type, or " metamitosis," in which a collaboration of cyto- plasmic and nuclear elements is effected, and the entire karyokinetic Fio. 58. — Division of H cematococcus pluvialis. A, Resting condition, the nucleus with a conspicuous karyosome and fine grains of chromatin in an achromatize reti- culum ; B, C, preparations for nuclear division, the chromatin passing from the karyosome into the nuclear reticulum ; D, further stage, the karyosome in disruption and chromosomes beginning to bo formed ; E, nuclear spindle ; F, division of the nucleus complete, the karyosomes reconstituted in the daughter-nuclei, the cell-body beginning to divide ; 0, division of the cell, the daughter-nuclei of the normal resting type. After Reichenow (97-5). figure lies free in the cytoplasm after disappearance of the nuclear membrane. Before passing on, however, to this more advanced type, account must be taken of the more simple types of mitosis seen in granular nuclei. Instructive examples of the division of nuclei, in which the chromatin is not concentrated into a karyosome, but distributed evenly throughout the achromatinic framework, are seen in the nuclei 112 THE PROTOZOA •i'.v FIG. 59. — Division of Euglypha alveolate, as seen in the living animal. A, Condition of the animal when about to divide. The protoplasmic body shows three zones : (1) At the fundus of the shell is clear proto- plasm containing the nucleus (N. ) and the reserve shell- plates (s.p.) ; (2) the middle region is occupied by granular protoplasm containing ingested food-materials {/.)and the contractile vacuole (c.v.) ; (3) near the mouth of the shell is a zone of hyaline protoplasm from which the pseudopodia (ps.) arc given off. B, Early stage of division, about twenty minutes later than A. The proto- plasm is streaming out of the shell-mouth to form the body of a daughter- individual, into which the reserve shell-plates are passing and arranging them- selves at its surface to form a daughter-shell. In the nucleus chromosomes are beginning to be formed. C, About twenty-five minutes later than B. The body of the daughter and its shell are further advanced in formation ; in the nucleus of the parent the equatorial plate is forming, and the two centrosomes are becoming visible on the two flattened sides of the nucleus (the centrosomes are probably derived from the division of the karyosome, no longer visible in the nucleus at this stage, or from a centriole contained in the karyosome). [Continued at foot of p. 113.J THE REPRODUCTION OF THE PROTOZOA 113 Fio. 60. — Details of the structural changes of the nucleus of Eurjlypha alveolata during karyokinesis, showing the formation of the chromosomes. A, Coarsely - meshed condition of the nucleus ; the chromatin-granules aggregated at the nodes of the reticulum. B, Later stage ; the nucleus beginning to show a fibrous structure as a result of the irregular clumps of chromatin-granules of the previous stage becoming arranged in linear series. B2, Some of the fibrils of this stage more highly magnified. C, Later stage ; the fibrils have become smoother and more parallel in arrangement. C2, Fibrils more highly magnified ; they consist, as in the last stage of darker and lighter parts (the former chro matin, the latter probably plastin) ; between the individual fibrils are cross-connections, more regular in this stage than in the last (remains of the nuclear reticulum). D, The fibrils have become shorter and thicker, and are bending up to form the U-shaped chromosomes. After Schewiakoff (100) ; magnification of A, B, C, and D, about 1,200 diameters. of ciliate Infusoria, such as Paramecium. The macronucleus divides without mitosis, as stated already, but the micronucleus exhibits a primitive type of mitosis (Fig. 61). When division begins, the Fia. 59 — continued: D, About fifteen minutes later than 0. The daughter-shell is now com- pletely formed, and the middle granular zone of the parent is passing over into it ; the nucleus of the parent has assumed its definitive orientation, with the centrosomes at the poles of an axis coincident with the longitudinal axis of the animal, and the equatorial plate is definitely formed. E, About thirty minutes later than D. The whole of the middle zone of the parent has passed over into the daughter-shell ; the flattened form of the nucleus is changing into an elongated spindle-form, and the equatorial plate is splitting to form the two daughter-plates. F, About five minutes later than E. The daughter-plates have travelled apart, and the division of the nucleus is beginning. 0, About five minutes later than F. The iii vision of the nucleus is com- plete, and one daughter-nucleus has passed ovr ••* into the body of the daughter- Euglypha. H, About twenty-five minutes later than G (about 125 minutes from the beginning). Some of the protoplasm of the middle zone flows back into the parent-shell, and each individual has its own contractile vacuole ; the two daughter-nuclei are reconstituted, and the karyosome has reappeared in each ; pseudopoclia are being protruded from the mouths of the shells ; the division is complete, and the animals are beginning to separate. After Schowiakoff (100) ; magnification about 470 diameters. 8 114 THE PROTOZOA amount of chromatin increases, and the nucleus becomes oval in form. The chromatin forms a number of chromosomes shaped like elongated rods or short threads, which arrange themselves at the equator. At the same time the achromatinic framework shows a longitudinal fibrillation or striation, the apparent fibrillae being centred in thickenings of the achromatinic framework which appear at the two poles of the nucleus within the persistent nuclear mem- brane, hence termed the " polar plates." Centrosomic grains are stated to be entirely absent, and their functions are performed by the polar plates. The nucleus continues to elongate, and the chromosomes divide transversely to their long axis to form the daughter-plates, which travel apart ; as they do so the fibrillated B G FIG. 61. — Stages in the division of the micronucleus of Para- mecium. A, B, Early stages ; O, spindle-stage with equa- torial plate of chromosomes ; D, spindle with the two daughter-plates; E — H, growth of the separation-spindle and separation of the two daughter-plates ; /, rcconstitu- tion of the daughter-nuclei, whioh are widely separated, but still connected by the greatiy elongated separation- ^ spindle, the central part of which shows a dilatation prior to its final absorption. After Hertwig. Figs A — E are drawn on a larger scale than the other figures. / separation-spindle appears between them. The nucleus as a whole now becomes dumb-bell-shaped ; the daughter-plates are lodged in the terminal swellings, while the rapidly-growing separation-spindle occupies the handle of the dumb-bell. The daughter-plates now break up and reconstitute the daughter-nuclei, but the connecting portion continues to elongate and to push the daughter-nuclei apart. It is clear that the separation is effected by intrinsic growth of the achromatinic framework constituting the separation-spindle, which is often curved up into a horseshoe-figure, and shows bending or twisting of its fibrils, as the result of the inert resistance of the sur- rounding cytoplasm. Finally, however, a limit of growth is attained ; the daughter-nuclei become constricted off completely from the connecting bond, which is absorbed and disappears. The nuclear membrane persists throughout the division. In all the forms of nuclear division dealt with so far, nuclear elements alone have been active in the process. A most instructive series, showing how extranuclear elements come to collaborate in THE REPRODUCTION OF THE PROTOZOA 115 the mechanism of division, is furnished by some examples of the Heliozoa, and especially by the nuclear divisions of Actinosphcerium, which have been the subject of extraordinarily thorough investiga- tion by Hertwig (64). In this form there are three different modes of karyokinesis, which, however, for present purposes may be classified under two heads : karyokinesis without and with centre - somes. In the ordinary nuclear division during the vegetative life of the organism, and also in the divisions by which the primary FIG. 62. — Actinosphcerium eichhorni : stages of the ordinary, vegetative nuclear division, without centrosomes, of free>living individuals (not encysted). A, B, Formation of the chromosomes within the nucleus, and of the proto- plasmic polar cones outside the nucleus ; G, spindle-stage with polar cones \p.c.), polar plates (p.p.), and equatorial plate of chromosomes (c.r.); D, stage with daughter - plates of chromosomes which have travelled towards the polar plates ; E — 0, division of the nucleus, reconstitution of the daughter-auclei, and disappearance of the polar cones. After Hertwig (64). cysts divide into the secondary cysts (p. 138), centrosomes are absent, but they are present in the two divisions which produce the two reduction - nuclei thrown off from each secondary cyst. In the ordinary karyokinesis of Actinosphverium (Fig. 62) an equatorial plate is formed composed of a large number of small, rod-like chromosomes, imperfectly separated from one another, which divide transversely. The spindle arises from the achromatinic framework of the nucleus, and terminates in two conspicuous polar »<#* •-^ ? ; i-.-.» •' ^ ;L*,<«M •«•ole of the nuclr'us ; B, two centrosomes and arclioplasmic cones, taking up positions on opposite sides of the nucleus, in which chromosomes are beginning to appear ; G. I), formation of the nuclear spindle and equatorial plate- of chromosomes ; E, division of the equatorial plate ; F, division of the nucleus beginning ; G, H, division of the nucleus and reconstitution of the danghter-nuclni ; one daughter-nucleus will degenerate and be rejected as a reduction-nucleus ; the neginniiiH of this is seen in //, where the upper darker daughter nucleus is the one which degenerates. After Hcrtwig (64). THE REPRODUCTION OF THE PROTOZOA 117 plates lying within the persistent membrane. External to the membrane are two large conical masses of archoplasm, termed the " polar cones." As in the micronucleus of Paramecium, the polar plates represent functionally the centrosomes, towards which the daughter- plates travel, and division of the nucleus is effected by growth of the separation-spindle. The archoplasmic polar cones appear to take little or no part in the mechanics of the division, since their apices maintain their distance from, one another, and the growth of the separation-spindle pushes the daughter-nuclei into their substance. The reduction-karyokinesis is heralded by the formation of a centrosome from the nucleus (Fig. 37 ; see p. 80, supra). The centrosomes are at first close to the nucleus, external to its mem- brane, but when the karyokinetic spindle is formed the centro- somes travel to the apices of the cones. From the centrosomes radiations extend through the polar cones, continuing the direction of the longitudinal striations of the intranuclear spindle, though separated from them by the intervening nuclear membrane. During the division the apices of the cones move apart to a slight extent, but the separation of the daughter-nuclei is still mainly the work of the separation-spindle, which pushes them into the polar cones and brings them close to the two centrosomes again ; hence the activity of the polar archeplasm can be but slight. The chromo- somes in the reduction -divisions are more distinctly separated from each other as the result, apparently, of a reduction in the amount of the plastin forming the ground-substance. The nuclear membrane persists throughout the whole process. In Actinophrys the karyokinesis appears to be of a type similar to that of Actinosphcerium, with persistent membrane, but with more activity in the extranuclear archoplasmic elements. In Acanthocystis (Fig. 64), however, the nuclear membrane disappears completely from the karyokinetic figure, and it is no longer possible, in consequence, to distinguish the parts of the achromatinic spindle which are of intranuclear and extranuclear origin respectively. Nuclear and cytoplasmic elements are in complete co-operation, a condition of things which has apparently been brought about and rendered possible by the extrusion of the centrosome from the nucleus in the first instance. From the foregoing examples, it is seen that amongst the Protozoa the material is to be found for illustrating the gradual evolution of the mechanism of karyokinetic division, from the starting-point of simple and direct division up to the most advanced type in which a perfect karyokinetic figure is formed by co-operation of nuclear and cytoplasmic substance. It is not necessary to suppose, how- ever, that the course of evolution has always been in the direction 118 THE PROTOZOA of that type of mitosis found in the cells of Metazoa ; it would be more reasonable to expect that in some cases at least other distinct types 'of division-mechanisms would have been evolved — side- \ s **£#- i3 W£»- _ , . .. . *'(>.? 7-* 5\* ?!&••:.*.'. •••••••:-. C-fp^ ^W&?°£ »>»?.vw ••;•' ^^~S-*^— • r> ^iSi? u*.JJV_iy^ ~ ^~\7f^ „• . v^ ^? ,"u^\ TS-.& vp Fio. 64. — Division of Acanthocystis aculeate. A, Resting state of the animal. N., Nucleus ; c., central grain ; a./., axial filaments of the pseudopodia, pa. ; ap., spicules. B, Pseudopodia withdrawn ; nucleus in the spircme-stage ; central grain dividing. C, Division of the central grain further advanced ; nucleus showing distinct chromosomes. D, Central grain completely divided into centrosomes, between which the nucleus is placed ; in the nucleus the membrane is becoming dissolved, the rcticulum is becoming modified in arrangement to form the achromatuvc spindle (or a part of it), and the chromo- somes are taking up their position in the equatorial plate. E, Complete nuclear spindle, with centrosomes, achromatinic spindle, and equatorial plate. F, Later stage with daughter-plates and division of the cell-body beginning. O. Division of the nucleus and of the cell-body nearly complete. After Schaudinn (82). THE REPRODUCTION OF THE PROTOZOA 119 branches, as it were, of the stem which culminates in the Metazoan type. Aa example of this is seen in the peculiar karyokinesis of Noctiluca (Fig. 65), in which the division is directed by a large " sphere," consisting of a mass of archoplasm containing the cen- trioles. The sphere divides and forms the axis of the karyokinetic figure, of whidh the nuclear portion is placed asymmetrically to one side. In considering this remarkable process of evolution, consisting in the gradual elaboration of a highly complicated mechanism for division of the nucleus, the question naturally arises, What is the object of a process so elaborate ? Or, if this method of posing the problem offends as being too teleological, we may alter the phrase- ology, and inquire, What is the result of the process ? The answer is perfectly obvious. The result effected by equating karyokinesis Fio. 65. — Stages in the nuclear division of Noctiluca miliaris. A, Early stage, the "sphere" (sph.) beginning to divide, the nucleus wrapping round it; 13, later stage, the sphere nearly divided, ths two poles of the nuclear- spindle in section attached to the two daughter-spheres ; C, section across B; the sphere contains a centriole (c. ), to which the chromosomes (chr.) are attached by achromatic fibrils. After Calkins (48). in its most perfected forms is an exact halving, both quantitative and qualitative, of the chromatin-substance of the nucleus — quanti- tative, by division of each chromatin-granule or chromidiosome, and the partition of the division-products equally between the two daughter-cells ; qualitative also, if we suppose that different chro- midiosomes may have different properties, and exert their own peculiar influence on the life and activities of the cell ; then, since each daughter-cell contains finally the sister-chromidiosomes of those contained in the sister-cell, the qualities of its chromatin are the exact counterpart of those of the sister-cell and also of the original parent-cell. Hence karyokinesis may be regarded as insuring the transmission to the daughter-cells of the distinctive properties of the parent-cell, unimpaired and unaltered. The whole process indi- cates clearly the immense importance of the chromatin-substance 120 THE PROTOZOA in the life of the cell. It is probable, also, that the elaboration of the process of karyokinetic division in Protista was an indispensable antecedent to the evolution of multicellular organisms, since for the formation of a tissue it is necessary that all the cells which build it up should be perfectly similar in their constitution and properties, and this condition could only be brought about, prob- ably, by karyokinetic division of the nuclei in the process of cell- multiplication. In the foregoing paragraphs we have dealt only with simple (binary) nuclear division, but, as already stated, in some cases the nucleus divides by multiple fission into a number of daughter- nuclei simultaneously. A simple instance of direct multiple division of a nucleus, in which, apparently, no centrioles are present, has been described by Lebedew (93) in the nuclei of Trachdocerca (Fig. 66 ; see also p. 448). In this form partitions are formed within the nucleus between the grains and masses of chromatin, and finally the nucleus becomes segmented into a mulberry -like mass of daughter - nuclei, which separate from one another. In most cases, prob- ably, of multiple fission the nucleus contains a centriole, FIG. on. — Four stages of direct multiple fission in j , , •,,. •, * • the nuclei of Trachdocerca pfcwwcojfan** After and the multiple fission Lebedew (93). is brought about in a manner analogous to the formation of a plasmodium by multiplication of the nucleus in a cell which remains undivided — that is to say, the centriole multiplies by fission a number of times without the nucleus as a whole becoming divided. Thus, in a nucleus of the simple protokaryon type, containing at first a single karyosome and cen- triole, division of these structures may take place within the mem- brane without the nucleus as a whole dividing, so that the nucleus contains finally two or more karyosomes, each containing a cen- triole. The karyosomes are ultimately set free from the nucleus, either by being budded off singly from it, or by the nucleus as a wholo breaking up ; then each karyosome becomes the foundation of a new nucleus. Division of this type, which may be termed a multiple promitosis, has been described by Zuelzer (86) in Wag- nerella. In cases where the division of the nucleus is of the karyo- kinetic type, repeated divisions of the centriole result in the forma- tion of a complicated multipolar mitotic figure, leading to a multiple division of the nucleus, as seen in the divisions of the nuclei in the male sporont of Aggregate, (Fig. 67), as described by Moroff (94). THE REPRODUCTION OF THE PROTOZOA 121 The presence of more than one centriole in a nucleus has led Hartmann (60) to formulate the theory that such nuclei are to be regarded as " polyenergid " nuclei.* Hartmann proposes to dis- tinguish a nucleus with a single centriole as a " monokaryon " from a polyenergid nucleus or " polykaryon " containing many cen- trioles ; he interprets many cases, in which a nucleus appears to become resolved into chromidia from which secondary nuclei are formed, as being really a setting free of monokarya from a complex polykaryon — an interpretation which certainly gets over the diffi- culty of the formation of centrioles in second- ary nuclei (see further, p. 255s infra). In conclusion, men- tion must be made of the theory of cell-divi- sion and of the causes which bring it about, put f orward by Hertwig (91, 92). This theory is based on the sup- position, of which men- tion was made in the previous chapter (p. 70, supra) — that for the normal performance of Fid. 67. — Multiple nuclear division in the male vital functions a cer- sporont of Aggregata jacquemeti. The nucleus, of which the outline has become irregular but is still tain quantitative re- visible, is surrounded by eight centrioles, from lation must be main- each of which striations pass towards and into the nucleus. After Moroff (94), magnified 750 tamed between the linear. nuclear substance and the cytoplasm. As a standard for the proportion of nuclear mass and cytoplasm (" Kernplasma-Norm "), the individual im- mediately after fission may be taken. Exact measurements made on Infusoria show that, while the body grows continuously in size from one division to the next, the nucleus at first diminishes slightly * The conception of " energids " is due to Sachs, who coined the term to denote " a single cell-nucleus with the protoplasm governed by it, so that a nucleus and the protoplasm surrounding it are to be conceived of as a whole, and this whole is an organic unity, both in the morphological and the physiological sense." Heitwig (GG) has criticized this conception, and 1ms shown its untonability in the case of Protozoa, which behave as single individuals whether they possess one nucleus or many. Hartmann, considering the centriole as the criterion of in- dividuality rather than the nucleus, has revived the energid theory in the manner described above. It leads him to regard an ordinary Metazoan karyokinesis as the division of a polykaryon, in which each separate chromosome represents a distinct nuclear element or monokaryou — a conclusion which appears to lead rather to a reductio ad absurdum of the theory. 122 THE PROTOZOA in size, and then grows slowly until the next division-period is reached. As a result of the slow " functional growth " of the nucleus, a disproportion between the mass of the nuclear substance and that of the cytoplasm is brought about, producing a condition of tension between the nucleus and the cytoplasm (" Kernplasma- Spannung "). When the tension reaches a maximum, the nucleus acquires the power of growing rapidly at the expense of the cyto- plasm, and this " division-growth " leads to the fission of the cell, restoring the standard balance of nucleus and cytoplasm. Relative increase of the nuclear substance retards the cell-division, and brings about increase in the size of the cell ; relative decrease of the nuclear mass has the opposite effect. 2. Division of the Cell. — A distinction has been drawn above between binary fission, or division of the body into two, and mul- tiple fission into many parts simultaneously. The daughter-indi- viduals produced u\ either case may be similar to the parent-indi- vidual in all respects except size, or may differ from it in lacking more or fewer of its characteristic parts and organs, which are then formed after the daughter-individuals are set free. In extreme cases one or more of the daughter-individuals may possess, when first liberated, no structure more elaborated than the essential parts of a cell, cytoplasm and nucleus or chromidia ; in such cases the daughter is termed a " bud," and the process of fission by which it arises is termed " budding " or gemination, distinguished further as " simple gemmation " when only one bud is formed at a time, and " multiple gemmation " when many arise simultaneously. In many cases of multiple gemmation the parent-organism does not survive the process, but breaks up almost completely into buds, leaving only a greater or less amount of residual protoplasm, which degene- ates and dies off ; budding of this kind is termed spondation. In binary fission, when the organism is of simple structure, as in the case of amoebae, the division is equally simple. After division of the nucleus, the two daughter-nuclei travel apart, and the body follows suit, by flowing, as it were, in two opposite directions, forming two smaller individuals each with a nucleus, and con- nected at first by a protoplasmic bridge, which soon snaps and is drawn in. The contractile vacuole, if present, is taken over by one of the two daughter-individuals, while the other forms a new vacuole ; in many cases the normal number of contractile vacuoles is doubled before division begins. In forms of more complicated structure, the division also becomes a more complex process. Where the body-form is definite, the plane of cleavage bears usually a constant relation to it. Thus, in Ciliata the division of the body takes place typically transversely to its longitudinal axis, except in the order Peritricha. In Flagel- THE REPRODUCTION OF THE PROTOZOA 123 >; .^OTP^m f vv' -S4 ii^tr^ c ^flfife ^ 6:^ ^ ^ ^3&£ »>^V-: v^J^V ^%;^^ "^"i-'^'-fe.^'^ • ^-^^ .^ .rit-f?:-- "-.•.rvi'^'PSv*: M >'* ?*$%&&'&£:, ^ ^ w •*») ? - V te^ ^•'•'"V'^ HI » i FKJ. 68. — Budding of Acanthocystia aculeate (compare Fig. 64, A). A, B, Division of the nucleus, in which the central grain takes no part ; C, extrusion of a bud ; D, three buds in process of extrusion, the nucleus of the parent dividing again ; E, free bud ; F, flagellula, and O, amoebula, produced from buds ; // and I, two stages in the extrusion of a centriole from the nucleus of a bud to form the central grain of the adult form. After Schaudinn (82). 124 THE PROTOZOA lata, on the other hand, the division of the body is usually longi- tudinal. In any case, the two products of fission may be equal or subequal in size, without perceptible difference of parent and young ; or they may be markedly unequal, in which case parent and offspring can be distinguished clearly. The various organs of the body may be doubled before division : either by splitting or new growth of one set ; or, if there are many organs of a particular kind present, such as the cilia and tricho- cysts of Ciliata, they may be simply shared between the two daughter-organisms ; or, finally, any given organ present in the animal before division may be retained by one of the two daughter- individuals, while the other forms the organ in qtfestion anew after division. Thus, in Ciliata one daughter-individual retains the old peristome ; the other forms a new one for itself. The greater the number of organs formed afresh in the daughter-individual, the more advanced is the transition from ordinary fission towards budding. In typical gemmation small portions of the parent-organism grow out, into which pass either nuclei, the products of the division of the parent-nucleus (Fig. 68), or of budding from the nucleus of the parent (Fig. 55), or chromidia, alone or together with a nucleus. Such buds may arise on the surface of the parent-body, or they may be cut off in the interior of the cytoplasm of the parent, and may remain for some time within its body. Endogenous budding of this kind is seen in the Neosporidia (p. 325), in the Acinetaria, where it is combined with nuclear budding, and in Arcella (Fig. 80) and some amoebae, where it is combined with formation of secondary nuclei from chromidia. Bibliography. — For references see p. 479. CHAPTER VIII SYNGAMY AND SEX IN THE PROTOZOA Hvirpi papaa. KI/-JT/H vffjieffffard, Kvvpt Qvarolaiv cnrex^?!*- IT is a matter of common knowledge that amongst all the higher animals and plants the phenomena of sexual generation and sexual differentiation are of universal occurrence. Reduced to its simplest terms, and stripped of all secondary complications, the sexual process in an ordinary animal or plant consists essentially of the following series of events : In the multicellular body certain cells are produced which may be termed comprehensively and universally the gametes. In the two sexes the gametes exhibit characteristic differences ; those of the male sex, the spermatozoa, are typically minute, active, and produced in large numbers ; those of the female sex, the ova, are, on the contrary, relatively bulky, inert, and produced in far fewer numbers. The gametes are set free from the body, or, at least, from the organs in which they arise, and each male gamete, if it finds a partner and if circumstances permit, unites with a female gamete ; their bodies fuse completely, cell with cell and nucleus with nucleus, and the product is a " fertilized ovum," or zygote, a single cell which proceeds to multiply actively by cell-division, the final result being a new multicellular individual. In the Protista belonging to what has been termed in the first chapter of this book the " cellular grade" — that is to say, in the Protozoa and the unicellular plants sexual phenomena are also of widespread, probably of universal, occurrence, and the process of sexual union differs only in unessential points from that seen in higher organisms. In the first place, since the individual in Protozoa is a single cell, the gametes themselves are also complete individuals, modifica- tions merely of the ordinary individuals of the species produced at certain periods or phases of the life-cycle. Secondly, the differentiation of male and female gametes rarely attains to the high degree seen in the Metazoa, and may be nil ; the two gametes may be perfectly similar in all perceptible features of structure or constitution, as, for example, Copromonas (Fig. 111). 125 126 THE PROTOZOA Sexual union of similar gametes is termed isoqamy ; of dissimilar, anisogamy. When the gametes are differentiated, then one gamete is generally smaller, more active, often with highly developed motor mechanisms, and without reserve food-material in the cytoplasm ; this is the microgamete, regarded as male. The other gamete, on the contrary, exhibits a tendency, more or less pro- nounced, to be large, inert, without motor mechanisms of any kind, and to store up reserve food-material in the cytoplasm — the macrogamete, regarded as female. The differentiation of the gametes is seen to be a specialization of two kinds of cell-individuals, the one rich in motile or kinetic protoplasm but poor in trophic substapce, the other rich in trophic protoplasm but poor in kinetic substance. In some cases the sexual differentiation may affect also the mother-cells of the gametes, the gametocytes, or may be thrown back still farther in the series of generations preceding the gametes ; in such cases a number of successive generations of gamonts exhibiting sexual differentiation terminate in a gameto- cyte generation from which the actual gametes arise. Thirdly, in the process of sexual union, or syngamy, as it may be termed comprehensively, the bodies of the two gametes do not always fuse completely ; in some cases the two gametes come together and merely interchange portions of their nuclear apparatus, remaining separate and retaining their distinct individuality. The nucleus which remains stationary in the one gamete then fuses .with the migratory nucleus derived from the other gamete. Examples of this type of syngamy are seen in the Infusoria (Fig. 77). The type of syngamy in which the two gametes fuse completely is sometimes termed copulation (or total karyogamy) ; that in which they remain separate and exchange nuclear material, is known as conjugation (or partial karyogamy), and the two sexual individuals themselves as conjugants (they should not, perhaps, be termed " gametes," strictly speaking, for reasons explained below) ; but the term " conjugation " is often used quite loosely for either type and lacks precision. These differences in the sexual process between Protozoa and the higher organisms enable us to give a wider significance, and at the same time a more precise definition, to the word " syngamy." However varied in detail, syngamy is essentially nothing more than an intermingling of chromatin-substance derived from two distinct cell-individuals. Plus qa change, plus c'est la meme chose. The chromatin that undergoes syngamic union may be in the form either of chromidia or of nuclei ; in the former case the process is termed chromidiogamy, in the second karyogamy. Chromidiogamy , though probably the most primitive type, is known to occur only in a few Sarcodina (Difflugia, p. 230 ; Arcella, p. 148). In the vast SYNGAMY AND SEX IN THE PROTOZOA 127 majority of Protozoa, as in all known cases amongst Metazoa. and plants, syngamy takes the form of Jtaryogamy. The nuclei of the gametes are termed promiclei, and the nucleus that results from fusion of the pronuclei in the zygote is termed a synkaryon. In many Protozoa (e.g. Coccidium, Fig. 69) the fusibn of the two pronuclei is effected by means of a peculiar mechanism termed a " fertilization- spindle." When the two pronuclei are in contact, the female pronucleus first takes an elongated, fusiform shape, having its -chromatin-grains spread over an achromatinic framework. The chromatin of the male pronucleus is then spread over the same structure. This mechanism has nothing to do with nuclear division, but merely effects a complete intermingling of the chromatin of the pronucloi, after which the synkaryon assumes its normal appearance and rounded form. In Infusoria the two pronuclei fuse in the condition of the karyokinetic spindle in many cases. FIG. 69. — Fertilization of Coccidium schubergi. A, Pene- tration of the macrogamete by one of five micro- gametes ; the female pronucleus has an elongated form ; B, the favoured microgamete has passed into the interior of the macrogamete, which has secreted a membrane (oocyst) at the surface of the body, ex- cluding the other microgamctes ; C, the female pro- nucleus has assumed an elongated, spindle-like form, while the male pronucleus lies at one pole of the spindle in the form of a little mass of granules ; the excluded microgametes are degenerating ; D, the granules of the male pronucleus have spread themselves over the spindle-figure formed by the female pronucleus ; E, the fertilization-spindle seen in D has rounded itself off to form the synkaryon. imd fertilization is complete, c? , Microgametes ; ? , macrogamete ; n $ , male pronuclous ; n?( female pronucleus; /.«., fertilization-spindle; c, oocyst; no, synkaryon. After Schaudinn (99), magnified 2,250. True syngamy, as defined above, must be distinguished carefully from certain other phenomena which are likely to be confused with it ; it must not be assumed that every fusion of cells, or even of nuclei, is necessarily a case of syngamy. In some Protozoa tho mother- colls of tho gametes, the gametocytes, enter into a more or less close association prior to the formation of gametes, 'which are produced in due course and then perform the act of syngamy in the normal manner. An example of such association is seen in gregarines (p. 330), where association between adult gametooytes is the rule. Sometimes the two gametocytes associate in the earliest stages of their growth, as in Diplocystis (Fig. 70, A), and their bodies may then fuse completely into one ; but their nuclei remain distinct, as in Cystobia (Fig. 70, B), and give rise in due course to the pronuclei of distinct gametes. Forms in which precocious association of this kind occurs are described as being " neogamous " (Woodcock). In many cases, union of distinct individuals can bo observed which have nothing to do with syngamy, since no fusion takes place of nuclei, but only 128 THE PROTOZOA of cytoplasm. Such unions are distinguished as plastogauiy (or plasmogamy) from true syngamy. Plastogamic union may bo temporary or permanent ; in the latter case it loads to the formation of plasmodia, as in the Mycetozoa (p. 239). The significance of plastogamy is obscure in many cases, but in some it may perhaps be comparable to the association of gametes already described, and in this way may throw light on some cases of so-called " autogamy" (see p. 138, infra). A further case of unions which are not in any way sexual in nature is seen in the remarkable phenomena of agglomeration exhibited by some Protozoa — for example, trypanosomes. In this case the organisms adhere to each other by the posterior or aSagellar end of the body, apparently by means of a sticky secretion formed by the kinetonuclous, so that large clumps are formed composed of numerous individuals. The phenomena of agglomeration are associated with conditions unfavourable to the parasite, and appear to be due to the formation of special substances, agglutinins, in the Mood of the host. Similar phenomena are well known in bacteria as agglutination, since in this case the agglutinated individuals are unable to separate, whilo in FIG. 70. — Precocious association and neogamy of gametocytes in gregarines. A, Diplocyatis minor, parasite of the cricket:. m., common membrane uniting the two associates ; g., grains of albuminoid reserve-material. B, Cystobia holothurice, parasite of Holothuria tubulosa, showing the two nuclei in an undivided body. A after Cuenot, magnified about 120 diameters ; B after Minchin. the case of trypanosomes that are agglomerated it is possible for the indi- viduals to become free again if the conditions are ameliorated. In other Protozoa, also, phenomena of the nature of agglomeration are seen in de- generating forms (see p. 209, infra). Certain aspects of syngamy must now be discussed in more detail — namely, the relation of syngamy to the life-history as a whole ; its occurrence in the world of living beings ; its significance for the life-cycle ; and its effects on the species and the individual. 1. Syngamy in Relation to the Life-History of the Organism. — In any living organism the principal manifestation of vital activity is the power of assimilation, resulting in growth. As a general rule, however, the growth of an organism is not indefinite, but has a specific limit ; an individual of a given species does not exceed a certain size, which may be variable to a slight extent, but which is fairly constant for normal individuals of the species in question under similar environmental conditions. When the limit is SYNGAMY AND SEX IN THE PROTOZOA 129 reached the organism tends to reproduce itself. In Protista, as described in the last chapter, two principal types of reproduction occur — namely, simple or multiple fission. In either case the organism grows to its full specific size, and then divides into smaller individuals ; the greater the number of daughter-individuals pro- duced at each act of reproduction, the more minute those daughter- individuals. Following the act of reproduction comes a period of growth, during which the small forms grow up into full-sized individuals which reproduce themselves hi then1 turn. Thus the life-history of a Protist may be described as an alterna- tion of periods of growth and periods of reproduction. If, how- ever, the life-history consists of only these two events in alternating succession, it is an infinite series, not a cycle ; continuous, not recurrent. Possibly such a condition, varied only by states of repose interrupting the vital activity of the organism is found in Bacteria and allied forms of life, where true syngamy apparently does not occur. But it is probable that in all Protozoa, as in all Metazoa and plants, the life- history is a recurrent cycle, of which an act of syngamy may be taken as the starting-point ; this point will now be discussed. 2. The Occurrence of Syngamy in the Series of Living Beings. — With regard to this question, there are two possibilities ; first, that syngamy and sexuality constitute a fundamental vital phenomenon, common to all living things ; secondly, that it is an acquisition at some period or stage in the evolution of organisms, and not a primary characteristic of living beings. The sex-philosopher Weininger* has argued in favour of the first of these hypotheses, and goes so far as to regard all protoplasm as consisting primarily either of arrhenoplasm '(male) or thelyplasm (female), standing in fundamental antithesis to one another, and combined in varying proportions in a given cell or sample of the living substance. A view essentially similar has been put forward by Schaudinn, and is discussed below. It is beyond question that sexuality is a universal attribute of all living beings above the rank of the Protista, whether animals or plants. In Protista, however, syngamy has not been observed to occur with certainty in the Bacteria and organisms of a similar type of organization. It is true that certain rearrangements of the chromatin, observed in some larger organisms of the bacterial type at certain phases of their life-history, have been compared to sexual processes, but such an interpretation is, to say the least, highly doubtful. In Protozoa, syngamy. has been observed to occur in a vast number of forms, but by no means in all. In the * Weininger, O., " Sex and Character," chapter ii. London : W. Heinemann, 1906. 9 130 THE PROTOZOA case of those species in which syngamy has not been observed, there are three abstract possibilities : first, that it does occur, but has not yet been seen ; secondly, that it is secondarily in abeyance ; thirdly, that it is primarily absent — that is to say, that it has never occurred either in the form in question or in its ancestral lineage. On the whole, the first of these three possibilities is the most probable, though the second must, perhaps, also be taken into account, as will be shown later. So far as a generalization is possible or permissible in the present state of knowledge, it appears that sex and syngamy are phenomena of universal occurrence in all truly cellular organisms, but we have no certain knowledge that they exist in any organisms of the bacterial type of organization. With the passage from the bacterial to the cellular type of structure, syngamy became, apparently, a physiological necessity for the organism, and was probably acquired once and for all. 3. The Significance of Syngamy in the Life-Cycle. — In order to appreciate the part that syngamy plays in the life-histories of organisms generally, it is necessary to compare briefly and in general outline the life-cycles of Metazoa and Protozoa in typical cases. In the Metazoa the cycle starts from a single cell, the zygote or fertilized ovum, which multiplies by cell-division in the ordinary way. Thus is produced a multicellular individual, composed always of at least two classes of cells — tissue-cells (histocytes) and germ-cells. The histocytes are differentiated in various ways, related to various functions, to form tissues, and so build up the soma. The germ-cells are not differentiated for any functions but those of sex and reproduction, and occur primarily as a mass of undifferentiated cells constituting the germen ; they are lodged in the soma and dependent upon it — parasitic upon it, so to speak — but in a sense distinct from it ; they draw their sustenance from the soma, influence greatly its development and activities, but contribute nothing to the work of the cell-commonwealth. Of these two portions of the Metazoan individual, the soma is neces- sarily mortal, doomed inevitably to ultimate senility and decay. The cells of the germen, on the other hand, are potentially im- mortal, since under favourable conditions they can separate from the soma and give rise in their turn to a new individual of the species with soma and germen complete again This type of generation is always found in every species, though non-sexual methods of generation may also occur in many cases. In the life-cycle of the Metazoa, as sketched above in its most generalized form, two individualities must be clearly distinguished, the one represented by the soma together with the germen, con- SYNGAMY AND SEX IN THE PROTOZOA 131 stituting the complex body of a Metazoan individual; the other represented by the single cells of which both soma and germen alike are built up. The phrase " reproduction," whether sexual or non-sexual, as applied to the Metazoa, refers only to the complex multicellular body as a whole, and not to its constituent cells, which reproduce themselves uninterruptedly by fission during the whole life-cycle. In the comparison of a typical Protozoan life-cycle with that of the Metazoa, we may start in both cases alike from a single cell- individual which is the result of an act of syngamy. In Protozoa, also, the zygote multiplies, sooner or later, to produce numerous cell-individuals ; but in this case the cells remain separate from one another and independent, so that no multicellular body is produced, except in the colony-building species, nor is there any distinction of somatic and germinal cells, save in rare cases, such as Volvo? (p. 267). In Protozoa the phenomena of vital exhaustion, so-called " senility " (Maupas) or " depression " (Calkins, Hertwig), appear to be as inevitable as in the cells of the Metazoan body (see pp. 135 and 208, infra) ; but if the derangement of the bodily functions and the vital mechanism has not gone too far, the organism is able to recuperate itself by self-regulative processes, of which the most important and most natural are those involved in the normal process of syngamy. Consequently no cell - individuals among Protozoa are doomed necessarily and inevitably to decadence and death, but all possess equally potential immortality — that is to say, the capacity for infinite reproduction by fission under favour- able conditions. The Metazoan individual is represented in the Protozoa only by the entire life-cycle, from one act of syngamy to the next, and not by any living organic individual. In the life-cycle of a Protozoon, as there is only one individuality, so there is only one method of reproduction — that, namely, of the cell, by fission ; and it must be made clear that the reproduction of the cell-individual is not in any special relation to syngamy in Protozoa, any more than in Metazoa. It has been pointed out above that the life-history of a Protist organism consists of alternate periods of growth and reproduction. In those Protozoa in which syngamy has been observed, it is found to take place sometimes at the end of a period of growth and before a period of reproduction, sometimes at the end of a period of reproduction and before a period of growth, and sometimes there may be a difference between the two sexes of the same species in this respect. In the first case, syngamy takes place between full-grown individuals of the species, as in Actinophrys (Fig. 71) — so-called macrogamy, which is almost always isogamous. In the second case, syngamy is between the smallest individuals produced 132 THE PROTOZOA by fission or gemmation, as in Foraminifera (p. 235), Arcella (Fig. 80), etc. — so-called microgamy, which may be isogamous or slightly anisogemous. In the third case, syngamy is between two individuals showing the utmost disparity in size, a tiny micro- gamete and a bulky macrogamete, as in Coccidium (Figs. 69, 152) ; the result being anisogamy of the most pronounced type. From these facts, it is abundantly clear that syngamy in the Protista cannot be regarded as related specially to reproduction, but as a process affecting the life-cycle as a whole, related equally FIG. 71. — Copulation of Actinophrys sol. A, Two associated free-swimming individuals. B, The two individuals are beginning to encyst themselves ; their nuclei (N., N.) are preparing for karyokinesis ; an outer gelatinous envelope (g) is secreted round the two gametes, and also round each individual an inner cyst-envelope (c.), incomplete at the surface of contact. C, The nucleus of each gamete is dividing by karyokinesis (first polar spindle, p.sp.). D, Formation of the polar bodies or reduction-nuclei (r.n.) ; the reduced pronuclei (pn.) take a central position in the body of the gamete ; the bodies of the gametes are beginning to fuse. E, The pronuclei are fusing ; the reduction-nuclei have passed through the wall of the inner cyst. F, The synkaryon (sk.) is beginning to divide by karyokinesis ; the degenerating reduction-nuclei have passed out of the inner cyst. N., N., Nuclei of the gametes before reduction. After Schaudinn, magnified about 850. to all vital functions of the organism, and therefore only indirectly to reproduction — that is to say, only in so far as reproduction may result from renewed and invigorated vitality. This is equally true of the Metazoa, where, however, the life-cycle begins and ends with the production of a complex multicellular body, composed of soma and germen. Hence, in the Metazoa syngamy is brought into relation with the production of a higher individuality, the body, comparable to the whole Protozoan life-cycle, and it is in this sense that the phrase " sexual reproduction " must be under- SYNGAMY AND SEX IN THE PROTOZOA 133 stood ; as already pointed out, syngamy has no special relation in Metazoa to cell-multiplication. In Protozoa sexual reproduction means simply reproduction following the sexual act ; but sex and reproduction must be considered as two things entirely distinct. The comparison instituted above between the life-cycles of the Protozoa and Metazoa, according to which an entire Protozoan individual is the mor- phological equivalent of a single constituent cell of a Metazoan body, is that which I personally have always held and taught. It is, I believe, the pre- vailing view among zoologists, and has been enunciated clearly by Calkins (5). It has, however, been attacked vigorously by Dobell (110), who lays great stress on the physiological analogy between the single Protozoon, as a com- plete organism, and the entire Metazoan body. On this ground he expresses the view that " a protist is no more homologous with one cell in a metazoon than it is homologous with one organ (e.g., the brain or liver) of the latter " ; he considers it " incredible that anybody could advocate the view that the Metazoa have arisen from aggregated Protozoa," and he puts forward the view that, if the Metazoa have arisen from protist forms, "'it is far more natural to suppose that they did so by developing an internal cellular structure, and not by the aggregation of individuals to form a colony." Similar ideas have been put forward also by Awerinzew (890). From these and other considerations, Dobell draws the conclusion that the Protista are not to be regarded as unicellular, but as " non-cellular " organisms. So far as the word "cell ".is concerned, I have already expressed the opinion above that by the term should be understood a certain stage in the evolution of the Protista^ and that many protist organisms should not be termed " cells," but only those which have reached what may be considered as the truly cellular type of organization. I am not, therefore, concerned with DobelFs attack on his own conception of the cell-theory so far as it concerns Protists generally, but only in so far as it applies to the Protozoa. It is not possible here to discuss in detail the ontogenetic development of the Metazoa. It must suffice to state that in all primitive types of embryonic development among Metazoa the cells which build up the body originate by repeated binary fission of a single cell, the fertilized ovum ; and that the only cases in which the ovum breaks up into cells Ky the development of cell- limits internally are those in which the development is modified by the presence of yolk, or where there is good reason to believe that yolk was ancestrally present in the egg. For confirmation of these statements the reader must be referred to the qrdinary textbooks of embryology. I must content myself with a single instance, that, namely, with which I am best acquainted by personal study. In the development of a simple Ascon sponge, such as Clathrina blanca or other species (see chapter " Sponges" in Lankester's " Treatise on Zoology," part ii., p. 68), the ontogeny may be divided into four phases or periods, which indicate clearly, in my opinion, the general lines in the evolution of the Metazoa from Protozoan ancestors. 1. Starting with the fertilized ovum, strictly homologous with a Protozoan zygote, it divides by repeated binary fission into a number of cells (blasto- meres), each similar to the ovum in every respect except size ; the process is in every way comparable to the division of a Protozoan zygote into a number of individuals which remain connected to form a colony, as, for example, in many Phytomonadina. 2. Of the blastomeres thus formed, a certain number, variable in different species, but relatively few, retain their original characteristics, while the rest become differentiated into columnar flagellated cells forming the wall of a cavity (blastocoele). The undifferentiated blastomeres give rise to the archseocytes, from which ultimately the germ-cells and gametes arise. The flagellated cells are the ancestors of the tissue-dells (histocytes) in the future sponge. At this stage, in which the embryo is hatched out as a free-swimming 134 THE PROTOZOA larva, it is perfectly comparable to a colony of flagellates such as Volvox, in which the ordinary individuals have lost the power of becoming, or giving rise to, gametes, which can onty arise from certain special individuals. 3. The free-swimming larva, composed mainly of flagellated cells, with the archaeocytes either at the hinder pole or in the internal cavity, undergoes changes as it swims about, which consist in some of the flagellated cells losing their flagellum, becoming modified in structure, and migrating into the interior of the larva ; in this manner the two germ-layers are established, and the organism has then, so to speak, passed from the condition of a Protozoan colony to that of a true Metazoon. 4. When the germ-layers are established, the larva fixes itself, and of the subsequent development it is sufficient to state that the cells of the two germ-layers become differentiated into the tissues of the adult sponge, and that in the metamorphosis of the larva the cells undergo a complete rearrange- ment, which shows clearly that every cell has an individuality as distinct as that of any Protozoan individual, a conclusion fully borne out by the recent experiments of Wilson and Huxley (Phil. Trans., B., ccii., pp. 165- 189, pi. viii.) on the power of regeneration in sponges after complete separation of the cells from one another. I am unable, therefore, to accept the standpoint of Dobell with regard to tho relations of Protozoa and Metazoa, but consider that the comparison of a Protozoan individual to a single cell in a Metazoan body is fully justified both morphologically and physiologically, and is a reasonable phylogenetic deduction from the ontogenetic data. The objection that there are no animals known which correspond to the four-cell, eight-cell, and blastula stages in embryological development misses the point and is not strictly true ; the stage at which an embryo consists only of four or eight blastomeres is the homologue of a Protozoan colony, and in the Flagellata species are known in which the colony consists only of four, eight, sixteen, or thirty-two cell- individuals (p. 275). To the query, " Has anyone ever found a metazoon which is composed of nothing but coherent gametes T" it may be replied that in many Volvocineae the colony also consists only in part of gamete- producing individuals. The theory that the Metazoa arise by cleavage of a multinucleate plasmodium, equivalent to a single Protozoan individual, has often been put forward, but has never found support from a general con- sideration of the facts of Metazoan embryology. In Protozoa the plasmodial phase is always temporary, and ends sooner or later by breaking up into separate uninucleato individuals. 4. The Effects of Syngamy — (1) upon the Individual, (2) upon the Species. — 1. Of all Protozoa, the ciliate Infusoria are the group in which syngamy is most easily observed and studied — In the first place because in these organisms it is readily distinguished from simple fission, which is transverse, while in syngamy the two conjugants apply themselves laterally to one another ; in the second place, owing to the fact that the species of Ciliata are practically monomorphic (p. 440), and can be identified without difficulty. Hence in this group elaborate and exhaustive experi- mental studies upon syngamy and its relation to the life-cycle have been carried out by many investigators, more especially by Maupas, Hertwig, Calkins, and Woodruff. The results of these investigators is briefly as follows : After syngamy the fertilized individuals appear vigorous, feed actively and multiply actively. After many generations of reproduction by fission, however, the race, if kept in an unchanged environment, becomes less vigorous SYNGAMY AND SEX IN THE PROTOZOA 135 and shows signs of enfeeble ment and " senility " or " depression " — a condition which, with continued isolation, reaches such a pitch that the organism is unable to assimilate, grow, or reproduce, but dies off inevitably unless conjugation with another individual takes place. At a result of syngamy, the vigour of the race is renewed, and the organisms once more grow and reproduce them- selves actively until senility supervenes again. From these and many other facts it would appear as if syngamy produced a strengthening or re-organizing effect upon the organism, restoring vigour and activity to vital functions that have become, as it were, worn out and effete. One very important discovery has resulted from the experi- ments of Calkins and Woodruff — namely, that the necessity for syngamy can be greatly deferred by change of environment. A strain which has become senile and exhausted can be stimulated and revived by a change of food. Even this remedy appears to have its limits, however, a degree of exhaustion being reached sooner or later which nothing can restore to its pristine vigour. The animals may even reach a pitch of exhaustion so great that they are unable to conjugate, but die off in a helpless manner. Calkins explains such cases as due to the senility having affected not only the vegetative, but also the generative chromatin ; pro- ducing generative senility, which is incurable, instead of mere vege- tative senility, for which syngamy is a remedy. Nevertheless, the fact that the advent of senility and exhaustion can be deferred by the stimulation of changed conditions is a very important discovery. It must be remembered that the Ciliata are organisms of extremely complex organization, and it is not unreasonable to suppose that in such forms the work thrown upon the vegetative chromatin is much heavier, and therefore the tendency to exhaustion much greater, than it would be in an organism of simpler constitution ; in such a form the stimulus of change of environment might defer the advent of senility very greatly, perhaps even for an indefinite period (Woodruff, 141).* This suggestion applies particularly to parasitic forms, in which the organization is always greatly simpli- fied, and in which change of environment from generation to generation is inseparable from their mode of life. It would not be surprising, therefore, if syngamy were found to be completely in abeyance in a parasitic form of simple structure. It should be noted here that examples of syngamy being in abeyance are not wanting even in higher organisms. An instance * In his most recent work on Paramecium, Woodruff (142) expresses the view that " most, if not all, normal individuals have, under suitable environmental conditions, unlimited power of reproduction without conjugation or artificial Stimulation." Compare also Woodruff and Baitsell (143). 136 THE PROTOZOA is the banana-tree. In the wild-banana, seeds are produced from flowers of a normal type by fertilization, just as in any other flower- ing plant ; in the cultivated banana, however, the flowers ate sterile and incapable of fertilization, consequently the tree bears fruit which are entirely seedless. Hence the cultivated banana- tree is propagated entirely by a non-sexual method — namely, by the production of suckers growing up from the roots, and in no other way. Whether this complete abolition of sexuality will in time lead to exhaustion of the cultivated race of banana remains to be seen, but at present there seem to be no signs of loss of vigour under cultivation. If syngamy can be entirely dispensed with in an organism rela- tively so high in the stale of life as a flowering plant, it seems probable 'in the highest degree that the same may be true in many cases for unicellular organisms of simple structure, and especially for those parasitic forms which live, like cultivated plants; in a medium rich in nutritive substances, and in an environment which is changed at least once in each developmental cycle. Instances of this are perhaps furnished by the various species of pathogenic trypanosomes, strains of which have been brought to Europe and propagated for many years from one infected animal to another by artificial inoculation, without the natural agency of an inverte- brate host. If it be true, as is generally believed, that in trypano- somes syngamy takes place in the invertebrate host, then in the long-continued artificial propagation of pathogenic trypanosomes sexuality has been in abeyance for a vast number of generations without any apparent loss of vital powers. The case of the patho- genic trypanosomes cannot, however, be cited, in the present state of our knowledge, as an absolutely conclusive example of syngamy in abeyance, since it is by no means certain that this process does not take place in the vertebrate host, where its occurrence has frequently been affirmed (see p. 306, infra). But it is certain that in trypanosomes gonerally, whether pathogenic or non-pathogenic, syngamy is a rare phenomenon, since it has not yet been demonstrated satisfactorily in a single instance, either in the vertebrate or the invertebrate host, in all the many species that have been studied. It is possible that, in these and many other forms of life, sexual processes may intervene only at long intervals in the life-history, and by no means in every complete cycle of development or alternation of hosts. It then becomes necessary to distinguish a developmental cycle, consisting of a recurrent series of similar form-changes in regular succession, from a complete life-cycle marked by the occurrence of an act of syngamy. In such forms as the parasites of malaria, for example (p. 358), the life-cycle and the developmental cycle coincide — that is to say, SYNGAMY AND SEX IN THE PROTOZOA 137 syngamy occurs once for each complete cycle of development with alternation of hosts, though it must not be forgotten that the development in the vertebrate host comprises a vast and quite indefinite number of generations of the parasite. On the other hand, in such forms as trypanosomes, a complete life- cycte, from one act of syngamy to the next, may comprise, ap- parently, a great number of developmental cycles and alternations of hosts. From the foregoing considerations it is evident that syngamy, though usually a necessity for the continued existence of uni- cellular no less than of multicellular organisms, can be dispensed with for a very large number of generations, perhaps even indefinitely, in some in- stances or under special circumstances. Two other phenomena of apparently widespread occurrence point to the same con- clusion — namely, the phe- nomena of parthenogenesis and autogamy. Partheno- genesis is a mode of re- production so common in Metazoa of various classes Fro. 72. — Parthenogenesis of Plasmodium vivax. . A female ganietocyte, of which the nucleus is dividing into a darker portion (n1) and a lighter portion (n2); B, the separation of the two P*1*9 is comPlete '• c- the darker nucleus has divided into a number of portions ; D, a number of merozoites are formed from the darker nuclei ; the lighter nucleus is abandoned in the residual protoplasm (r.p.) containing the melanin-pigment. After Schaudinn (130) that it is unnecessary to J Cite instances of it here; it may be defined briefly . , . as the power to develop without Syngamy possessed 11 _!•«. by a sexually-differentiated gamete, which under nor- mal circumstances could do so only after syngamy with a gamete of the opposite sex. To this it must be added that the gamete which has this power is always the female ; but this limitation receives an explanation from the extreme reduction of the body of the, male gamete and its feeble trophic powers, rendering it quite unfitted for independent reproduction, rather than from any inherent difference between the two sexes in relation to reproductive activity. Parthenogenesis has been de- scribed by Schaudinn for the human malarial parasite (Fig. 72) and in Trypanosoma noctuce, and by Prowazek for Herpetomonas muscce-domesticce ; none of these cases, however, are entirely free 138 THE PROTOZOA from doubt, and in any case parthenogenesis seems to be of much rarer occurrence among Protozoa than among Metazoa.* Autogamy, on the other hand,- is a phenomenon which has been frequently observed in Protozoa, chiefly, though not exclusively, among parasitic forms ; it may be defined as syngamy in which the two gametes, or at least the two pronuclei, that undergo fusion are sister-individuals derived by fission of the same parent cell or nucleus. Hartmann (116) has brought together the many cases of autogamy known to occur among Protozoa and other- Protist organisms, and has classified them under a complex terminology. It is sufficient here to mention two typical cases, those, namely, of Actinosphcerium and Entamceba coli, made known by R. Hertwig (64) and Schaudinn (131) re- spectively. In Actinosvhcerium an ordinary indi- vidual (Fig. 3) be- comes encysted as a multinucleate ' mother - cyst , " which becomes di- vided up into a num- ber of uninucleate "primary cysts," after absorption of about 95 per cent, of the nuclei 'originally present. Each pri- mary cyst then di- vides completely into two distinct cells — " secondary cysts." Each secondary cyst then goes through a process of nuclear re- duction (see below), after which it is a gamete ; the two gametes then fuse completely, cell and nucleus, to form the zygote. * Prowazek (557) has described in Herpetomonas musccR-domesticce a process interpreted by him as parthenogenesis (" etheogenesis ") of m»le individuals, bat the correctness both of his observations and of his interpretations are open to the gravest doubt. According to Flu (536), the objects to which Prowazek gave this interpretation are in reality stages in the life-history of a quite distinct organism, named by Flu Octwnorea muscce-dome-iticce, and now referred to the Miorosporidia. It is greatly to be deprecated that interpretations of such un- certain validity should be used, as has been done, to support general theories in the discussion of the problem of syngamy. Fro. 73. — Autogamy in Entatncd>u colt. A, The amoeba at the beginning of encystation with a single nucleus ; B, the nucleus dividing ; 0, the two daughter-nuclei throwing off chromidia ; a space has appeared be- tween them ; D, each nucleus has formed two re- duction-nuclei, which are being absorbed ; E, a resistant cyst-membrane has been secreted ; the partial division in the protoplasm has disappeared, and the two reduced nuclei are each dividing into two ; F, each daughter-nucleus of the two divisions in the last stage has fused with one of the daughter- nuclei of the other division to form two synkarya. After Hartmann (116), drawn by him from the de- scription given by Schaudinn (131). SYNGAMY AND SEX IN THE PROTOZOA 139 Tn Entamceba coli (Fig. 73) the process starts in like manner from a uninucleate individual, the nucleus of which divides into two, but the cell divides incompletely and only temporarily. Each nucleus then breaks up completely into chromidia and disappears from view. Some of the chromidia are absorbed, while from others a secondary nucleus is formed on each side of the cell, so that two nuclei reappear again in the cyst, but smaller than before and staining feebly. Each secondary nucleus now divides twice to form three nuclei on each side, two of which degenerate as re- duction-nuclei, while the third in each caso persists as a gamete- nucleus. As soon as the process of reduction is complete, the incomplete separation of the two cells disappears, so that the two gamete-nuclei lie in a single cell, which at this stage forms a tough cyst. Now each gamete-nucleus divides into two pronuclei, those of the same pair being slightly different from those of the other, according to Schaudinn (133). Then a pronucleus of each pair fuses with a pronucleus of the other pair, so that two synkarya result. At a later stage each synkaryon divides twice, and eight amoebulffl are formed by division of the cell within the cyst. From these two examples, it is seen that autogamy is a process of extreme inbreeding as regards the gametes. In typical cases of syngamy the two gametes must be derived from two distinct strains, and those of the same strain will not conjugate ; Schaudinn (131), for example, observed that the gametes of PolystameUa crispa would only copulate when a couple came together in which each gamete was of distinct parentage. In a great number of Protozoa the differentiation of the gametes and their mode of formation makes it certain that the couple which join in syngamy are derived from different parents. On the other hand, in many cases of autogamy that have been described, it is equally certain that the conjugating gametes and pronuclei have a common parentage, and it is hardly possible to consider autogamy otherwise than as a degeneration of the sexual process, evolved in forms in which one feature of true syngamy — namely, the mixture of distinct strains — is, for some reason, no longer a necessity ; we shall return to this point when discussing the nature and origin of the syngamic process. It is possible, moreover, to recognize progressive stages of the degeneration, as shown by -the two examples selected. In the less advanced stage (Actinosphoerium) the parent cell divides into two complete cells, each of which, after a process of matura- tion, becomes a gamete. In the more advanced stage (Entamceba coli), the division of the parent-cell is checked, and only its nucleus divides, each daughter-nucleus becoming a pronucleus after reduction. The occurrence of autogamy has been asserted in a number of cases which are, to say the least, extremely doubtful, as for example, the Myxosporidia 140 THE PROTOZOA (p. 407) and allied organisms, where it iff far from certain that the two nuclei or cells, from which ultimately the pronuclei or gametes arise, have a common parentage. Autogamy has recently become very fashionable, and there is a tendency to regard as such, not only many cases which are probably truly heterogamous, but also nuclear fusions or appositions which are not in any way sexual (e.g., Schilling, 134). The essential point to consider, in cases of autogaiay, i whether there is a union of chromatin derived from distinct strains — amphimixis — or from a common parentage — automixis. Thus, it has been pointed out above that in gregarines two gametocytes may associate, and even fuse intorone body, but with the nuclei remaining distinct (Fig. 71, B). When gamete-formation takes place in a " neogamous " species of this type, the gametes of one sex derive their pronuclei from one gametocyte-nucleus, those of the opposite sex from the other, with subsequent syngamy 6f a truly heterogamous type. If the fusion of the gametocytes were to go farther, a plastogamic, non-sexual union of the two nuclei might result, producing a single nucleus containing , chromatin from two distinct sources ; in that case, when gamete-formation took place, the syngamy would be, to all intents and purposes, a typical case of autogamy, and would certainly be so described if it were not known that the single gametocyte-nucleus had arisen by fusion of two distinct nuclei. If, however, in each couple of copulating gametes, one pronucleus contained chromatin derived from one of the two original gametocyte-nuclei, the other pronucleus, similarly, chromatin derived from the other nucleus, the result would be a true amphimixis, just as in ordinary heterogamy. In Actinosphcerium, plastogamic fusions of the ordinary vegetative, multi- nucleate individuals are stated to be of common occurrence ; it is therefore possible that an individual which encysts may contain frequently nuclei from distinct sources. According to Brauer, fusion of nuclei takes place in the mother- cyst to form the nuclei of the primary cyst. There is therefore at least a possibility that the autogamy of Actinosphcerium may be, in some cases, combined with amphimixis. In other cases, however, such as Entamoeba coli arid Amoeba albida (Fig. 87), there seems little reason to doubt that the autogamy is a true automixis. Analogous cases of self -fertilization are well known in flowering plants, where they are sometimes the rule, sometimes an alternative to cross-fertilization. In free-living Ciliata, also, syngamy has been observed between cousins, the descendants of an ex-con jugant after but four divisions (Jennings, 121), which is not far removed from automictic autogamy. The conclusion put forward above, on experimental grounds, that syngamy has a strengthening or invigorating effect on the cell-organism, receives further support from the many instances in which it is observed to occur as a preliminary to the production of resistant stages destined to endure unfavourable conditions of Me. Thus, in free-living Protozoa syngamy occurs commonly in the autumn, previously to the assumption of a resting condition in which the organisms pass through the winter. In Difflugia, for instance, syngamy in the autumn is followed by encystment, and the cysts remain dormant until the spring. This is strictly comparable to the state of things known in many Metazoa, such as Rotifers, Daphnids, etc., where in the summer soft-shelled eggs are produced which develop parthenogenetically, but in the autumn hard-shelled winter-eggs are produced which require fertilization. In parasitic forms, such as Coccidia and Gregarines, syngamy is related to the formation of resistant cysts which pass out of the host SYNGAMY AND SEX IN THE PROTOZOA 141 and endure the vicissitudes of the outer world, until taken up by a new host in which the parasite is set free from its cyst and starts upon a fresh cycle of growth or multiplication without syngamy, under the most favourable conditions of nutrition. 2. As regards the effects of syngamy upon the species, it must be pointed out, in the first place, that a great difference exists between multicellular and unicellular organisms as regards the effects of external conditions of life upon the sexual process. In Metazoa the germ-cells, as already pointed out, are a race of cells apart, and are sheltered by their position In the body from the direct effects of external conditions — at least, to a very large extent. In Protozoa, on the other hand, there is no special race or strain of germ-cells, but any individual may become a gamete or the progenitor of gametes, and all alike are exposed to the direct action of the environment. If, now, Protist organisms placed under slightly different conditions of existence, tend to vary in their •characters as a direct consequence of environmental influences, syngamy would check any such tendency, and would, on the con- trary, tend to keep a given species constant and uniform in char- acter, within narrow limits. Were there no mtermingling of distinct strains, such as syngamy brings about, individuals of a species subject to different conditions of life would tend to give rise to divergent strains and races ; syngamy levels up such diver- gencies and keeps the tendency to variation within the specific limits (compare Eririques, 112 and 113 ; Pearl, 124). If this sup- position be correct, it would follow that no true species could exist until syngamy had been evolved ; and if it be true that no syngamy occurs in organisms of the bacterial type of organization, then such organisms must be regarded as having diverged under direct environmental influences into distinct races and strains, but not as constituting true species. The " species " of bacteria would then be comparable to the races of the domestic dog, rather than to the natural species of the genus Canis. Not until syngamy was acquired could true species exist amongst the Protista, a condition which was probably first attained after the cellular grade of organization had been evolved. The conclusions reached in the foregoing paragraphs may be summed up briefly as follows : Syjigamy_i§^a__process of inter- mingling, in a 8mgje^ll-individual,of chromatin derived from Two dmmclTlndividuals. gameteg7~"gEc^-m^y ^xhjtot dijfitrfti^Sit.ion into "jnale^_jndixiduals, ^characterized by preponderance of kinetic activity, and " female," in which trophic activities are more pronounced. Syngamy is probably of universal occurrence in organisms of the cellular type of organization, and from them has been inherited by the higher plants and animals, but apparently 142 THE PROTOZOA it does not occur amongst organisms of the bacterial grade. Syn- gamy is related to the life-cycle as a whole, and not specially to cell-reproduction. In its effects on the cell-individual, syngamy appears to have an invigorating effect, renewing vital powers that have become effete and exhausted ; but in species that live in very favourable conditions of nutrition, etc., whether such conditions are due to artificial culture or to natural causes, such as parasitism, syngamy may be deferred for a very long time, and may even be completely in abeyance, or may degenerate into parthenogenesis or autogamy. In its relation to the race, syngamy tends to level down individual variations, and so produce true species amongst the Protista. Before proceeding to discuss the nature and probable origin of the syngamic process, it is necessary to take into account a process which appears to be a universal concomitant of syngamy — namely, the process of nuclear reduction in the gametes. In all cases of syngamy that have been carefully studied, it has been found that the gametes differ from the ordinary cell-individuals of the species in having undergone a process of so-called " maturation " which con- sists essentially in nuclear reduction — that is to say, in a diminution of the normal quantity of the chromatin by so-called "reducing" divisions of the nucleus. Hence the pronuclei which undergo syngamic fusion differ in their constitution from the nuclei of cells not destined for this process, and do not multiply, as a rule, under normal conditions so long as they remain single. In some cases among plants, however, the cells that have undergone nuclear reduction may multiply by fission and produce a multicellular organism (gametophyte) from which gametes ultimately arise ; in this way is brought about the well-known alternation of genera- tions of the ferns and flowering plants. Since, moreover, in Metazoa, ova that have undergone nuclear reduction can be stimulated artificially to start their development without fertilization, it is clear that the nuclear reduction does not in itself inhibit further development or cell-multiplication. True nuclear reduction in gametes must be distinguished clearly from the process of elimination of effete or vegetative chromatin which precedes the formation of the gametes or their nuclei, probably in every case. As has been stated above (p. 72), vegetative and generative chromatin may be combined in the same nucleus, or may occur, the one in the form of a nucleus, the other in the form of chromidia, or may constitute two distinct nuclei. When the two are combined in one nucleus, a necessary preliminary to gamete- formation is the purification of the generative chromatin of all effete vegetative material. When the vegetative chromatin is already separate from the generative, the latter alone takes a SYNGAMY AND SEX IN THE PROTOZOA 143 share in syngamic processes, and the vegetative chromatin, whether as chromidia or a nucleus, disappears from the life-history. Nuclear reduction, in the strict sense, concerns simply the nuclei composed of generative chromatin, and is a process which results in the reduction of the chromatin to half the specific quantity, a deficiency made up again to the full amount by the union of the two pronuclei to form the synkaryon. It is therefore a process which is seen in its most characteristic form in those cases where it is possible to gauge the amount of chromatin in the nucleus more or less accurately by the number of chromosomes formed during division. In the Metazoa, where each species is characterized by possessing a number of chromosomes which is generally constant (the so-called " somatic number "), the process of reduction appears to be ex- tremely uniform in its essential details throughout the whole series, from the Sponges and Ccelenterates up to man, and admits of a description in general terms. The gametocyte (oocyte or sperma- tocyte), when at the full term of its growth, has a large nucleus which then goes through two maturative divisions in rapid succes- sion. When the gametocyte-nucleus prepares for division, it appears with half the somatic number of chromosomes ; but each chromosome is in reality bivalent, and produced by the fusion or close adherence of two separate somatic chromosomes. In the first reduction-mitosis, the two adherent chromosomes in each case separate from one another and travel to opposite poles of the spindle ; hence this division is in reality a reducing, though it simulates in some of its features an equating, division. Im- mediately or very soon after the two chromosomes of each pair have separated, they split longitudinally in preparation for the next mitosis, which follows hard upon the first, and in which the two sister-chromosomes of each pair go to opposite poles of the spindle. Consequently the second reduction-division is in reality an equating mitosis, though on account of the precocious splitting of the chromo- somes it may simulate a reducing division. Thus, to sum up the process briefly, the number of chromosomes in the germ-cells is reduced to half the somatic number by two successive mitoses, the first a reducing, the second an equating division. In the male sex, the spermatocyte divides into four gamete-cells of equal size, the spermatids, each of which becomes a spermatozoon. In the female sex the oocyte-di visions are very unequal, producing the ovum, ripe for fertilization, and three minute sister-cells of the ovum which, as the so-called " polar bodies," are cast off and die away. By syngamy between a ripe ovum and a spermatozoon, each containing half the somatic number of chromosomes, the full somatic number is restored. 144 THE PROTOZOA In Protozoa the chromosomes are seldom so sharply defined as in Metazoa, and consequently it is difficult or impossible to deter- mine their number. Many cases in wnich a fixed number of chromosomes is alleged to occur, as in Trypanosoma noctuce (Schau- dinn, 132), cannot be accepted without question in the present state of our knowledge. On the other hand, in all groups of the Protozoa, where the sexual processes have been carefully studied, the union of the gamete-nuclei has been found to be preceded in a great many cases by two successive divisions of each nucleus, with one or the other of the following results : either the successive formation of two reduction-nuclei,* which are cast out of the cell or absorbed without dividing further, while the third persists as the pronucleus of the gamete ; or the production of four nuclei, all of which, or only one of them, persist as pronuclei. These reducing divisions in Protozoa suggest forcibly a comparison with those of the Metazoa, and from this analogy it may be further inferred that in Protozoa also the chromatin of the conjugating pronuclei has undergone a reduction to half the specific quantity ; but it is seldom possible to confirm this inference by accurate enumeration of the chromosomes. In the case which has been the most care- fully studied of all others, that, namely, of Actinosphcerium, Hertwig (64) found the number of chromosomes in the first reduction- spindle to be between 120 and 150 ; in the second reduction-spindle the number was about the same, but the chromosomes were about half the size of those in the first reduction-spindle. Moreover, in both the reducing divisions of Actinosphcerium the chromosomes in the equatorial plate divide to form the daughter-plates, as in ordinary karyokinesis, whereas in the reducing divisions of Metazoa the individual chromosomes are not divided, but merely sorted out. Hence it would appear that in Actinosphcerium, and probably many other Protozoa, the reduction of the chromatin in the pronuclei is effected by more direct, though perhaps less exact, methods than in the highly-perfected process seen in the Metazoa. Nevertheless, a few cases are known among Protozoa in which the small number of chromosomes permits of their being accurately counted, and in which they are seen to be reduced to hah* the usual number in the maturation-divisions of the gametes. In Pefomyxa the first division reduces the chromosomes from eight to four ; the second division, however, is equating, and no further reduction takes place (p. 150). In some Infusoria it has been observed that * These reduction-nuclei are sometimes termed " polar bodies," by analogy with the maturative process of the Metazoan ovum, but the term is to be avoided in this connection, as it places upon these divisions an interpretation which is at least highly doubtful ; the polar bodies of Metazoa are sister-cells of the ovum ; the reduction-bodies in Protozoa are simply nuclei which are extruded or absorbed. It is certainly not justifiable in fact, and: probably no more so in theory, to regard their formation as abortive cell-division. SYNGAMY AND SEX IN THE PROTOZOA 145 the first division of the micronucleus is an equating division, the second reducing ; so in Opercvlaffia (Enriques, 112), Chilodon (Enriques, 113), Carche#ium (Popoif, 125), Didinium (Prandtl, 126), and Anoplophrya (Fig. 74) ; in the last named the second division of the micronucleus reduces the chromosomes from six to three, and union of the pronuclei brings the number up to six again. In Carchesium the number of chromosomes is reduced from sixteen to eight. A similar reduction-process has been described by Mulsow (123) in gregarines (p. 335). Hence in these cases the pronuclei Fio. 74. — Behaviour of the micronucleus during successive stages of the con- jugation of Anoplophrya (CoUinia) branchiarum. A, Micronuoleus of one conjugant preparing for division ; B, later stage, with six chromosomes dis- tinct ; 0, nuclear spindle, with an equatorial plate of six chromosomes ; D, diaster-stage, with six daughter-chromosomes at each pole of the spindle ; E, later stage, with the chromosomes at each pole fused into one mass ; F, O, H, reconstruction of the daughter-nuclei ; the remains of the spindle between them disappears gradually ; /, the two micronuclei preparing for division ; appearance of six chromosomes in each (one nucleus is seen in profile, the other from one pole) ; J, diaster-stages, showing three chromo- somes at each pole of the spindle (reducing division) ; K, later stage, the chromosomes fused into masses, of chromatin ; L, four granddaughter-mioro- •'nuclei ; M, one of them grows in size, the other three oegin to degenerate ; N, division of the persistent micronucleus to form the two pronuolei ; O, two pronuclei and three degenerating mioronuclei. After Collin (50), magnification about 2.000 diameters. have exactly half the amount of chromatin contained in the ordinary nuclei, just as in the Metazoa. Doflein (111) and Hartmann (116) consider that a process of reduction is absolutely essential to the conception and definition of syngamy, and regard reduction as a criterion whereby true syngamic union of gametes and pronuclei can be distinguished from plastogamic and nuclear fusions which have nothing to do with the sexual process. " No fertilization without reduction " (Hartmann). But it must be acknowledged that in a great many cases of gamete-formation in Protozoa a reduction of the chromatin- 10 146 THE PROTOZOA substance of the conjugating pronuclei cannot be deduced from observation, and could only be inferred from analogy. In the gamete-formation of Coccidium schubergi, so carefully studied by Schaudinn (99), a large number of male pronuclei are formed simultaneously by local condensations of chromidia thrown off from the nucleus of the gametocyte, which is left behind in the residual protoplasm, with its conspicuous karyosome (Fig. 50) ; in the female gamete, also, the process of reduction appears to consist of a simple elimination of the karyosome (Fig. 75), a process which could be interpreted more naturally as elimination of effete vegetative chromatin than as a process of true nuclear reduction. In the case of Coccidium, as in others that might be cited, it must either be assumed that reduction-processes, in the strict sense of FIG. 76. — Four- stages in the maturation of the female gametocyte of Coccidium schubergi. A, Full-grown macrogametocyte contained in the host-cell ; B, the macrogametocyte is beginning to round itself off and to expel the karyosome from its nucleus ; 0, the Karyosome expelled from the nucleus of the macrogametocyte has reached the surface of the body and broken up into a number of fragments, which lie scattered in the body of the host-cell or are extruded from it ; D, the macrogametocyto has now become a ripe macro- gamete, having rounded itself off, eliminated the karyosome from its nucleus, and divested itself entirely of the host-cell, n., Nucleus of the gametocyte ; k., its karyosome ; n.', nucleus of the host-cell ; k.', k.', fragments of extruded karyosome. After Schaudinn (99), magnified 1,000. the phrase, occur but have been overlooked, or that the method of reduction is one that can only be brought into line with the typical method by theoretical interpretation founded on analogy. It must therefore remain an open question, in the present state of our knowledge, whether a process of nuclear reduction strictly comparable to the process seen in Metazoa is essential to the definition of true syngamy, or whether such a process has not been evolved and perfected gradually as a consequence of the sexual process. It is quite conceivable that syngamy may have been at its first origin merely a process of intermingling of chromatin of distinct cell -individuals ; that in this crude and primitive form syngamy would tend to disturb the normal balance of nucleus and cytoplasm, since it would lead to quantitative excess of the SYNGAMY AND SEX IN THE PROTOZOA 147 nuclear substance; that, consequently, by a regulative process which may primitively have followed the syngamic union, the chromatin of the zygote was reduced to the normal quantity by elimination of half of its mass ; and that from this hypothetical primitive process of regulation of the nucleo-cytoplasmic balance a process of nuclear reduction preceding the syngamic act has been gradually evolved until it reaches its perfection in the form seen in the Metazoa. On this view, it is to be expected that in Protista a great diversity in the methods of nuclear reduction would occur, from those of the roughest type to others highly elaborated and perfected ; and this expectation certainly receives justification from the data of observation. Hertwig (119), on the other hand, compares the reducing divisions hi the maturation of the gametes to the so-called " hunger-divisions " in Infusoria, which exhibit a great disproportion in the relative mass of nucleus and cytoplasm as the result of starvation in artificial cultures ; in such forms the body is smaller than in forms from a normal culture, but the nucleus is not merely relatively, but absolutely, larger than that of a normal form. The disturbance in the nucleo-cytoplasmic ratio (see p. 70, supra) can, however, be regulated by reducing divisions of the nucleus. On the ground of this comparison, Hertwig considers that the maturative processes of the gametes are to be regarded as the necessary consequences of antecedent events* in the life-history — as processes which in their turn bring about syngamy, and not such as have the object of preparing the nuclei for fertilization. In order to give a more concrete idea of the processes of syngamy and reduction in Protozoa, a few typical examples will now be described, selected in order to illustrate the salient features of these processes. The most convenient method of classification of the examples chosen is to distinguish those cases in which cliro- midia are present in addition to nuclei from those in which nuclei alone are present. 1. Syngamy and Reduction wiih Nuclei and Chromidia. — In a great many Sarcodina, especially those belonging to the orders Amoebaea (p. 218) and Foraminifera (p. 231), chromidia may be present in the gamete-forming individuals as a permanent con- stituent of the body-structure. In such cases the chromidia represent, wholly or in part, the generative chromatin, and give rise, by formation of secondary nuclei, to the nuclei of the gametes. As an example Arcella may be taken, the life-cycle of which is described in a subsequent chapter. In this form two distinct forms of syngamy have been described. * It is, of course, hardly necessary to point out that starvation is by no means the only influence which can bring about a disturbance of the nucleo-cytoplasmic equilibrium ; over-nutrition, for example, may have the same effect. 148 THE PROTOZOA (a) Karyogamy. — The body of an Arcella gives rise by multiple gemination to a number of amoebulae, each containing a secondary nucleus derived from the chromidia, while the primary nuclei of the parent-form degenerate (Fig. 80). The number and size of the amoebulae vary, however, in different individuals. In one Arcella the number is less and the amoebulae are larger, eight or nine macramcebce being produced. In another the amoebulae are more numerous and smaller, about forty micramcebce being formed. In either case the amoebulae swarm out of the parent-shell and are the gametes. A micramoeba copulates with a macramoeba, the two fusing completely to form a zygote with a synkaryon. The amoeboid zygote thus produced is the starting-point in the growth and development of an Arcella (Fig. 80, A). In this example the karyogamy is a case of microgamy, which, like other such cases, precedes a period of growth and follows a period of active reproduction. It is possible that the syngamy of the gametes is preceded by reducing divisions of the nuclei of the amoebulae, but no such reduction has been observed in Arcetta. In Foraminifera (p. 235), in which the syngamy is perfectly isog- amous, each secondary nucleus formed from the generative chromidia divides twice to form the gamete-nuclei—divisions doubtless to be regarded as reducing divisions. In Centropyxis, according to Schaudinn (131), amoebulae, all of the same size, are produced as in Arcetta, by formation of secondary nuclei ; but in some broods each amoebula divides into four micramoebae (micro- gametes), while hi other broods the amoebulae remain undivided as macramoebae (macrogametes) ; copulation then takes place between two gametes of different size. (6) Chromidiogamy (Fig. 80, M — Q). — Two ordinary adult Arcettas come together and apply the mouths of their shells. The proto- plasm of one individual flows over almost entirely into the other shell, taking with it both chromidia and primary nuclei, only so much protoplasm being left in the one shell as suffices to hold the two shells together. The primary nuclei now degenerate, and the chromidia derived from each conjugant break up into a fine dust of chromatin-particles and become intimately commingled. When this process is complete, the protoplasm with the chromidia becomes again distributed between the two shells, and the two conjugants separate. Then hi each individual secondary nuclei are formed from the chromidia, and by a process of multiple gem- mation a number of uninucleate amoebulae are formed which swarm out of the shell, and, like the zygotes resulting from karyogamy, become the starting-point of a new Arcetta. Thus chromidiogamy is here a case of macrogamy which, like other similar cases, follows a period of growth and precedes a SYNGAMY AND SEX IN THE PROTOZOA 149 period of active reproduction. Chromidiogamy is a rare but very interesting form of syngamy which, from the standpoint of general notions with regard to the evolution of the nucleus, may be re- garded as the most primitive type. It is known to occur also in Difflugia (Zuelzer, 85), where also copulation of swarm-spores takes place as an alternative method (p. 230). A case must now be considered in which the chromidia represent vegetative, while the nuclei contain the generative, chromatin. An example of this state of things is furnished by Plasmodiophora brassicce, a well-known parasite of cabbages, turnips, etc., in which I FIG. 76. — Gamete-formation and syngamy in Plasmodiophora brassica. A, Normal vegetative nuclei of the myxamcebse ; B, G, extrusion of chromidia from the nuclei ; D, division of the nuclei by karyokinesis (first reducing division) ; E, nuclei after reduction ; F, formation of gametes which are fusing in pairs ; G, spore (zygote) containing two nuclei, one of which is going through a further reduction-division ; H, fusion of the two pronuclei within the spore ; 1, ripe spore with synkaryon and two centrioles. After Prowazek (127), magnified about 2,250 diameters. it produces a disease known as " fingers and toes " (Kohlhernie). According to the investigations of Prowazek (127) and others, Plasmodiophora goes through a development which may bo briefly summarized as follows : At the end of the " vegetative " period of growth and multiplication, there are found within the cells of the infected plant a numbey of " myxamcebae," amoeboid individuals (plasmodia) each with many nuclei containing distinct karyosomes (Fig. 76, A). From the nuclei chromidia are given off into the cell, and during this process the karyosomes disappear and centrosomes make their appearance (Fig. 76, B, C). The chromidia are ab- 150 THE PROTOZOA sorbed and disappear, and the nuclei divide twice by karyokinesis (Fig. 76, D), so that their number is quadrupled. The myxamoeba then undergoes multiple fission into as many cells as there are nuclei in the plasmodium (Fig. 76, F), and each of these cells is a gamete. The gametes now conjugate in pairs, and the zygotes become encysted to form the spores. Within the spores the nuclei of the gametes are stated to undergo a further process of reduction before they fuse to form the synkaryon (Fig. 76, O). The syngamy in Pla&modiophora is stated to be a case of autogamy, but this allegation assumes that the nuclei of the myxamoabae are sister- nuclei derived all from the division of one original nucleus ; they may equally well be nuclei of different origins brought together by plastogamic fusions. The two examples selected, Arce.Ua and Plaamstdiophora, show that the chromidia may represent generative chromatin in one case, vegetative in another. Goldschmidt (57) has proposed to distinguish these two conditions by a special terminology, retaining the name " chromidia " (trophochromidia, Mesnil, 74) for those which are purely vegetative, and coining a new term, sporetia (idio- chromidia, Mesnil) for those of generative nature. It is more convenient, however, to retain the term " chromidia " in its original significance, to denote simply excranuclear particles of chromatin, and to qualify the term by the adjectives " vegetative " and " generative " when required (see also Goldschmidt, 41, p. 130). The formation of vegetative chromidia, which are finally absorbed, is a common phenomenon in many Protozoa ; it may take place as a purely regulative process, as in Actinosphcerium during de- pression-periods (p. 208), when hypertrophy of the nuclear apparatus is corrected by the extrusion from the nuclei of chromidia, which ultimately degenerate and become converted into masses of pig- ment, and as such are eliminated from the protoplasm. The account given by Bott (103) of gamete formation in the common Pdomyxa (Amoebcea nuda, p. 227) describes a condition in which chromidia, extruded from the nuclei, are partly vegetative, partly generative ; secondary nuclei are formed from them, which later cast out a portion of their chromatin, then give rise to the gamete -nuclei. After the secondary nuclei have been purified in this way of their vegetative chromatin, the generative chromatin remaining in each of them forms a karyokinetic spindle with eight chromo- somes, and a reducing division follows by which each daughter- nucleus obtains four chromosomes. The " pronuclei of the first order, resulting from the first reducing division, divide again, forming a spindle with four chromosomes which split, so that the " pronuclei of the second order " have also four chro- mosomes. From the nuclei that have undergone reduction in this manner the nuclei of the gametes arise in a somewhat remarkable fashion : the pro- nuclei of the second order separate into two compact masses of chromatin ; a vacuole is formed near them ; and the chromatin of the two masses wanders, in the form of finely- divided granules, into the vacuole to form the definitive pronucleus of the gamete, which forms a membrane when the process is complete. When formed the gametes wander out as Heliozoon-like indi- SYNGAMY AND SEX IN THE PROTOZOA 151 viduals, which copulate in pairs, and the uninucleate zygote grows up into the multinucleate Pelomyxa. The conception of vegetative and generative chromidia has not been accepted universally or without criticism. Hartmann, as pointed out above, considers that many cases of generative chromidia are really the result of a disruption of a polyenergid nucleus ; Awerinzew (47) is of opinion that, while all Protozoa possess vegetative chromidia at some stage at least in the life- cycle, generative chromidia are to be considered as a new acquisition, a hasten- ing of the process of the formation of numerous gamete-nuclei ; Doboll (51) puts forward a similar view -with regard to generative chromidia. With regard to the latter criticism, it may be pointed out that nuclei may become resolved into chromidia in order to undergo simple binary fission. With regard to Hartmann's view, there is at present, at least, little evidence that it is an adequate explanation of the many cases of formation of secondary generative nuclei from chromidia known amongst the Sarcodina. The ques- tion is discussed further below (p. 255). 2. Syngamy and Reduction with Nuclei only. — A very simple example is furnished by the common Actinophrys sol (Fig. 71), as described by Schaudinn (129). Conjugation takes place between two adult forms (macrogamy), which come together and become enclosed in a common cyst. The nucleus of each individual then divides by karyokinesis, and one nucleus of the pair thus produced is expelled from the body and undergoes degeneration as a reduction nucleus. The persistent nucleus of each individual then repeats the process and forms a second reduction-nucleus. The nucleus now remaining in each cell is the definitive pronucleus. The two gametes now copulate, their pronuclei fusing to form the synkaryon, after which the synkaryon divides by karyokinesis and the zygote divides into two individuals which later escape from the cyst and resume the free-living vegetative life. The course of syngamy in Actinophrys is exactly similar to that performed by the two " secondary cysts " derived from division of a " primary cyst " in Actinosphcerium (see p. 138, supra). In both cases alike the nucleus of the conjugants may be supposed to contain both vegetative and generative chromatin mixed together. It is possible that the vegetative chromatin is extruded from the nucleus in the form of chromidia prior to the reducing divisions, but no elimination of vegetative substance has been described. The last example of syngamy in Protozoa that need be con- sidered specially at this point is that of the Infusoria, which have been the subject of numerous investigations. These organisms present the highest degree of specialization of the body-structure and elaboration of the nuclear apparatus found in any Protozoa. Their syngamic processes vary in detail to some extent in different cases (see p. 448), but the whole process is essentially as follows (Fig. 77) : Two individuals come together and adhere, placing themselves side by side. The two conjugants may be similar in visible constitution, or may differ to a greater or less extent, and 152 THE PROTOZOA . 77. — Diagram showing the successive stages of conjugation in Infusoria. A, The two conjugants attached, each with a macronucleus (N) and a micro- nucleus (n) ; B, G, the micronucleus of each conjugant dividing ; D, each conjugant has two micronuclei which are beginning to divide again ; E, each conjugant has four micronuclei ; the macronuclei are beginning to become irregular in form ; in later stages they degenerate, break up, and are absorbed ; F, three of the four micronuclei of each conjugant are degenerating and being absorbed ; the fourth is dividing ; G, one half of each dividing micro- nucleus of the preceding stage has travelled over into the other conjugant as the migratory pronucleus ; H, I, fusion of the stationary pronucleus of each conjugant with the migratory pronucleus derived from the other conjugant to form the synkaryon (S.) ; J ' , the two conjugants now separate ; in each ex-con jugant the synkaryon (S.) divides ; the old macronuclei are now almost completely absorbed ; K, L, the synkaryon has divided into two nuclei, one of which grows large and becomes the new macronucleus, the other remains small and becomes the new micronucleus, of each ex-conjugant. After Delage and Herouard. SYNGAMY AND SEX IN THE PROTOZOA 153 are sometimes markedly different in size (Doflein, 111). The greatest amount of differentiation is seen in the order Peritricha (p. 448), where microconjugants and macroconjugants can be dis- tinguished. Each conjugant has a micronucleus and a macro- nucleus. The macronucleus begins to degenerate, and finally dis- appears completely. The micronucleus, on the other hand, en- larges and divides by a simple form of karyokinesis (see p. 114, supra). The division of the micronucleus is repeated twice as a rule, but sometimes three times, and, as stated above, in one of these divisions the number of chromosomes is halved in a great many, possibly in all, cases. Of the four (or eight) micronuclei thus formed, all but one represent reduction-nuclei which are absorbed and disappear. The persistent micronucleus then divides by equating division into two pronuclei, which may be distinguished as migratory and stationary, respectively ; they sometimes exhibit distinct structural differentiation. At this juncture the cuticle of each conjugant is absorbed at the point of contact, and the migratory pronucleus of each conjugant passes over into the protoplasm of the other and fuses with its stationary pronucleus. The gap in the cuticle is now repaired and the two individuals separate, each " ex-conjugant " having a synkaryon constituted by a fusion of one-eighth (or one-sixteenth) of its own original micronucleus with the same fraction of the micronucleus of the other partner. The synkaryon grows and divides into two nuclei, one of which grows and becomes the macronucleus, while the other remains small and becomes the micronucleus, of the ex-conjugant, which thereby becomes indistinguishable from an ordinary in- dividual of the species, and proceeds to start on a course of vegeta- tive growth and reproduction in the usual manner, until the next act of syngamy initiates a fresh cycle. It has been observed that the two ex-conjugants sometimes differ markedly in their capacities, one of them multiplying much faster than the other. In the syngamy of Ciliata it is seen clearly that the macronucleus represents effete vegetative or " somatic " chromatin, which is eliminated bodily from the life-history of the organism, while the micronucleus represents reserve generative chromatin from which, after reduction, the entire nuclear apparatus is regenerated. The remarkable feature in the syngamy of Infusoria is the manner in which- the conjugants remain distinct, and merely exchange pronuclei (so - called " partial karyogamy "). Versluys (137), following Boveri, derives this from an ancestral condition of iso- gamic copulation — that is to say, a condition in which the two conjugants fused completely as gametes, both body and nucleus, after which the zygote divided into two individuals ; on this view che final division of the micronucleus which gives rise to the two 154 THE PROTOZOA pronuclei is to be regarded as the equivalent of the division of the synkaryon which took place ancestrally after syngamy. While, however, there is a general agreement that partial karyogamy (conjugation) is to be derived from total karyogamy (copulation), it is very doubtful if the two conjugants in Infusoria represent simple gametes ; it is more probable that the type of syngamy characteristic of Infusoria is derived from an ancestral condition in which each conjugant produced a number of minute gametes (swarm-spores) which copulated (compare especially Popoff, 125, and Hartmann, 116, and see p. 453, infra). On this view the divisions of the micronucleus represent a primitively much larger number of divisions which produced the numerous gametes, and the conjugants themselves are not to be regarded as true gametes, but rather as gametocytes or gamonts. Having now illustrated by typical examples the various forms which the syngamic process takes in Protozoa, we may conclude this chapter by a consideration, necessarily brief, of the problem of the significance and origin of syngamy and sex. This is a problem which has a vast literature, and it is only possible here to indicate in outline some of the theories that have been put forward, none of which can claim to be a complete solution of one of the profoundest mysteries of the living substance and its activities. Considering first the fertilization of the Metazoa, it is evident that the union of the spermatozoon with the ovum has two prin- cipal results. In the first place the spermatozoon brings with it a pronucleus, the equivalent of that contained in the ovum, but derived from a distinct individual, and therefore possessing different hereditary tendencies acquired from its own particular ancestral history. The union of the male and female pronuclei brings about, therefore, a process for which Weismann has coined the term amphi- mixis— that is to say, a mingling of different hereditary tendencies in one and the same individual. In the second place the spermato- zoon produces a result which may be termed briefly " developmental stimulus " (Entwicklungserregung) — that is to say, it produces a disturbance in the equilibrium of the protoplasmic body of the ovum which causes it to start on a course of cell-division oft-re- peated, a process of cleavage which converts the unicellular ovum into the mass of cells which supplies the material for the building up of the multicellular body. It is very probable that the develop- mental stimulus is s applied by the greatly -developed centrosome of the spermatozoon, that of the ovum having completely atrophied, apparently, after the completion of its maturative processes. The introduction of a male pronucleus — that is to say, the process of ampllimixis — can be effected only by the spermatozoon. But the researches of Loeb and others have demonstrated fully that the SYNGAMY AND SEX IN THE PROTOZOA 155 spermatozoon is not indispensable for supplying a developmental stimulus ; an unfertilized ovum can be induced by artificial stimuli of various kinds to start upon a course of development similar to that initiated, under natural circumstances, by fertilization with a spermatozoon. Hence, of the two results produced in the fertiliza- tion of Metazoa, amphimixis alone would appear to be that which is essential and peculiar in the process, and which only fertilization can bring about. From the above considerations, amphimixis is regarded by many thinkers as the essence of syngamy, a necessity for the evolution of living beings in that it supplies, by the intermingling of different hereditary tendencies, the conditions required for the production of " innate " variations in organisms in which the germinal substance is shielded from the direct influence of external conditions by its position within a multicellular body. Apart from the question, however, whether any such innate variations exist in the Protozoa, where all colls alike are exposed equally to the direct action of the environment, the criticism has often been made that amphimixis gives only a teleological explanation of the sexual process, and as such cannot be invoked as a causal explanation of its origin. The intermingling of distinct hereditary tendencies, however useful to the organism or important in the evolution of living beings generally, cannot be regarded as the incentive to syngamy at its first appear- ance in the Protista. In other words, amphimixis must be regarded as a secondary consequence not as a primary cause, of syngamy. It is necessary, therefore, to seek some explanation for the first origin of syngamy other than the benefits which it may confer through amphimixis, and it is undoubtedly among Protist organisms that the conditions under which syngamy first arose must be sought. It has been pointed out above that syngamy appears to have a strengthening or recuperating effect upon the cell-organism, and upon such grounds has been founded the theory of " rejuven- escence " (Verjiingung). According to this theory, connected chiefly with the name of Maupas, the cell-protoplasm, after many generations of reproduction by fission, tends to become effete and senile to an ever - increasing degree, a condition which, if not remedied, ends in the death of the organism ; the natural remedy is furnished, however, by the process of syngamy, .which has the effect of renewing the " youth " of the cell and starting it upon a fresh series of generations, until senility, once more supervening, necessitates syngamy again. The rejuvenescence-theory has been criticized by many critics who have themselves done little more, in some cases, than give a more precise meaning to the terms " youth " and " old age," terms that certainly stand in need of further explanation, since 156 THE PROTOZOA it can hardly be supposed that the time-factor alone can account for the exhaustion or depression of the vital faculties. It IB gener- ally admitted that unicellular organisms, such as the Protozoa, tend, after a greater or less number of generations, to exhibit a certain degree of exhaustion in their vital properties, or, it may be, of derangement in their organization and vital mechanisms. Hert- wig (164) is of opinion that " the conditions of death exist in the living substance from the beginning, and are a necessary conse- quence of its vital function " — a generalization which may be accepted for those Protista in which the body exhibits the degree of specialization and structural complication proper to a true cell (as the term is understood in this book — see p. 98) ; but it is very doubtful if it is true also for the simplest forms of life, such as the bacteria and allied organisms. If it be further admitted that syngamy is the natural remedy in unicellular organisms for a natural disease, the problem before us is to discover, if possible, the precise nature of the derangements, and of the method by which the remedy restores them to the normal functional condition. At the outset, attention must be drawn to a very constant and general preliminary to syngamy in Protozoa — namely, the elimina- tion of a large amount of chromatin which appears to have been regulating the vital activities during previous generations (vegeta- tive chromatin), and its replacement by chromatin which has been inactive and lying in reserve (generative chromatin). This process is seen in its most striking form in the Ciliata, where the macro- nucleus is entirely eliminated during the act of syngamy, and is replaced in subsequent generations by a new inacronucleus derived from the micronucleus formed by fusion of portions of the micro- nuclei of the partners in syngamy. Hence it might seem as if the chief result of syngamy was to replace effete vegetative chromatin by fresh generative substance which through inactivity has retained its powers unimpaired. But in the first place it must be pointed out that, to effect a replacement of this kind, the union of two individuals is not necessary ; it would be sufficient for a single individual to form a new nucleus from its store of generative chromatin, and to get rid of its old, effete vegetative chromatin. If we regard the chromidia of Arcella as composed of generative chromatin, the buds produced by formation of secondary nuclei from the chromidia would represent nuclear regeneration of this kind. Secondly, it is open to doubt how far the theory of vegeta- tive and generative chromatin can be applied throughout the whole series. In such forms as Arcella the chromidial mass, although it furnishes the gamete-nuclei, is a cell-element in a functional con- dition, and in the more primitive forms the distinction between vegetative and generative chromatin cannot be pressed so far as SYNGAMY AND SEX IN THE PROTOZOA 157 in highly- organized forms, such as the Ciliata. Hertwig (68) con- siders that the separation of two kinds of chromatin is an adaptation to particular conditions of life, evolved progressively, and attaining its greatest perfection in the Ciliata ; whereby chromatin which has become functionally effete is separated from that which has retained its constitution. According to the view put forward by Hertwig (118), syngamy remedies the effete condition of the cell chiefly by regulating the necessary quantitative balance between the nucleus and the cyto- plasm. Such regulation may be effected also by internal re- arrangements of the nucleai substance or by plastogomy, but is brought about most efficiently by syngamy, since the definite and necessary mutual relations between nucleus and cytoplasm are better maintained by " arrangements which prevent disturbance, than by arrangements which compensate for disturbances that have already set in." The obvious criticism of this theory is that it is difficult to understand why an internal regulative process of the cell should require the co-operation of two individuals, and the reason contained in the sentence just quoted from Hertwig scarcely seems an adequate explanation. The fact that two cells participate in syngamy indicates in itself that the necessity for syngamy depends on a loss of balance between two constituents or substances in the cell, and that the union of the two gametes restores equilibrium. As Hertwig (119) has pointed out, the quantitative relation of nucleus to cytoplasm is more altered in the gametes of Metazoa than in any other cells, and to opposite extremes in the two sexes ; in the ovum the quantity of cytoplasm is enormous in proportion to the nucleus, while in the spermatozoon the exact reverse is the case. The same argu- ment applies to a greater or less degree hi the case of anisogamous gametes of Protozoa. It would not, however, apply to the many cases of isogamy in Protozoa where the quantitative relations of nucleus and cytoplasm are the same in each gamete ; in such cases union of the gametes would leave the nucleo-cytoplasmic relation exactly what it was before. A theory of a different kind has been put forward by Schaudinn (133) and his followers Prowazek (128) and Hartmann (116), which is based on the notion that sex and sexual differentiation are primary characteristics of living matter. A normally function- ing cell is regarded as hermaphrodite, having male and female elements equally balanced. The differentiation which leads to the formation of gametes arises, as Butschli originally suggested, from inequalities hi the results of cell-division, which may be supposed to lead always to more or less imperfect partition of the qualities of the parent-cell between the daughter-cells. As a result 158 THE PROTOZOA of the defects in the process of cell division, some cells acquire more " male " properties, other more " female " ; the cells preponder- atingly male show greater kinetic and motile energy, those that have more female qualities show greater trophic activity. With con - tinued cell-division these opposite tendencies tend to accumulate in certain cells which in consequence become altogether one-sided in their vital activities. Thus a want of balance in the vital functions is brought about, which may reach such a pitch that the organism is unable to continue to assimilate and reproduce, and must die unless the balance is resorted by syngamy with an individual that has become specialized in the opposite direction. By the union of two gametes differentiated in this manner, equilibrium is restored and the vital functions are reinvigorated. No gametes, however, whatever their degree of specialisation, are to be considered as perfectly unisexual, but only relatively so ; a male gamete will always contain a certain amount of female substance, and a female gamete a certain amount of male substance, thus accounting for the possibility of partheno- genesis. Schaudinn's theory of sex is thus very similar to that developed by Weininger on purely psychological grounds. Schaudinn, whose work on Protozoa must secure full considera- tion for any statement of his observations, however inherently improbable the facts or the interpretations based upon thejn may seem, founded his theory chiefly on data alleged to have been observed by him in the development of Trypanosoma noctuae (Schau- dinn, 132). According to him, an " indifferent " ookinete might give rise either to male or female forms. In the formation of males, certain nuclear elements were separated out to become those of the daughter-cells, while certain other nuclear elements remained behind and degenerated together with a quantity of residual protoplasm. In the formation of females, the same two sets of nuclear structures were separated out, but those proper to the male sex degenerated, while those of the female sex, which were just those which degenerated in the formation of males, in this case persist and become the nucleus of the female gamete. Thus the indifferent ookinete was supposed to be really hermaphrodite, containing male and female elements mixed together, and giving rise to individuals of one or the other sex by persistence of one set of characters and atrophy of the other. It must be noted here that these observations of Schaudinn's are entirely unconfirmed, nothing similar having as yet been found by other investigators, either in trypanosomes or in any other Protozoa ; and further that, even if Schaudinn's observations be accepted as exact in every detail, they will not bear the interpretations which he places upon them — namely, that the small and large forms produced as he describes are males and females, since, as he himself admits, they do not, SYNGAMY AND SEX IN THE PROTOZOA 159 when developed, perform any act of syngamy. The alleged sexuality of the forms described by Schaudinn lacks the only de- cisive criterion of sexual differentiation — namely, sexual behaviour ; and the differentiation exhibited by the two forms of trypanosomes described by Schaudinn admits of an entirely different and far less forced interpretation (see p. 176, infra). There are two further criticisms that may be made of Schaudinn's theory. The first concerns the alleged universality of sexual differences in living matter It must be pointed out that, as stated above, at the present time we have no evidence whatever of the occurrence of true syngamy in any organisms of the bacterial grade. The processes that have been interpreted by Schaudinn as autogamy in certain bacteria may be much more easily regarded as processes of internal regulation of the chromatin-substance. Nowhere yet has the union of two distinct gametes been observed in any bacterial organisms. The theory that sex is a universal characteristic, and syngamy an elementary function, of living things, does not rest at the present time on any basis of established fact. The second criticism is that the terms " male " and " female " require definition and explanation, without which they remair meaningless, connoting merely unknown, mystic properties, nc further analyzable, of the living substance. The characteristic feature exhibited by male cells is the preponderance of kinetic activity, and by female cells, of trophic functions, as Schaudinn and many others have pointed out. Before Schaudinn, the same idea was expressed in different language by Geddes and Thomson (114), who regarded the male sex as characterized by katabolic, the female sex by anabolic activities. It we suppose that these two manifestations of physiological activity have each a distinct material basis in the living cell, then it can easily be imagined that the imperfections of cell-division may lead to the production of cells in which one or the other substance predominates. This is the view that Doflein (7) has developed in his very interesting critical summary of the views that have been put forward upon the sexual problem. He supposes, further, that these two different physio- logical qualities depend upon substances which have intense mutual interactions and attract each other strongly, and that a certain equilibrium between them is necessary for the normal life of the cell. When, therefore, one or the other substance preponderates greatly in a cell, a functional derangement results ; but since cells differentiated in opposite directions attract each other strongly, they tend to unite, and by their union to restore equilibrium. The question of the sexual differentiation of the gametes is one that will be discussed at greater length in the next chapter. It is only necessary to point out here that a clear distinction must be 160 THE PROTOZOA drawn between intrinsic differences, not necessarily visible, and structural or other differences which are more or less obvious. The fact that gametes and pronuclei tend to unite proves that in all cases there must be intrinsic differences between them which stimulate them to do so ; in this sense, at least, we may endorse fully the dictum of Hertwig, that " fertilization depends on a fusion of sexually-differentiated cell-nuclei." On the other hand, gametes of opposite sexes exhibit every possible condition from complete similarity in structure and appearance to the greatest possible contrast in every feature of their organization. There can be no doubt that visible differentiation of the gametes is largely, if not entirely, an adaptation to the functions that they have to perform ; and this conclusion is by no means weakened by the fact that there are many cases of isogamy which are un- doubtedly secondary, in which a more primitive and phylogeneti- cally older structural differentiation has gradually become annulled, under circumstances in which adaptive differences in the gametes are no longer necessary — as, for example, in gregarines (p. 173). In Metazoa it is generally recognized that the two pronuclei that undergo fusion are perfectly equivalent,* and that the dif- ferences seen between them in the gametes are temporary and, in the case of the spermatozoon, an adaptation to circumstances ; here the real differentiation of the gametes affects only cytoplasmic characters. In Protozoa, on the other hand, the conjugating pronuclei often exhibit differences of structure when the cells themselves appear perfectly similar. In the Infusoria, for instance, differences have been noted between the migratory and stationary pronuclei ; how far these differences may be correlated directly with the differences in their activities must remain an open question. In the foregoing paragraphs we have set forth and discussed some of tne attempts that have been made to solve the problem of sex. It cannot be said that a perfectly satisfactory solution has been attained, but at least certain conditions of the problem may be laid down. In the first place, no theory of sex is satis- factory which daes &ot explain why the union of two cells should be necessary in syngamy. In .the second place a teleological inter- pretation, such, as amphimixis, can only state a secondary con- sequence, not a primary cause, of sexual union ; but such a consequence may suffice to explain the retention and persistence of sexual phenomena after the conditions have ceased to exist under which they came- into existence. In the simplest Protista of the bacterial grade, it may be supposed, either that the living matter is not differentiated into localized substances having distinct physiological qualities, or that in such * Apart, that is to say, from the much -discussed question of the supernumerary chromosome. SYNGAMY AND SEX IN THE PROTOZOA 161 minute bodies reproduction by fission does not produce differentia- tion in the fission-products. With increased size such differences may arise, at first to a minor extent, and capable of being adjusted by internal rearrangements of the living substance such as have been described in the larger Bacteria. Not until the process of natural evolution had gone so far as to produce the full complica- tion of structure seen in a true cell would localized differences in the living substance be brought about to a sufficient extent to lead to differences between the daughter-cells produced by fission, as a consequence of the imperfections of the process of cell-division. The differences produced in this way might be changes in the nucleo-cytoplasmic balance, as Hertwig supposes, or in the relative proportions of substances exerting different physiological activities, as suggested by Butschli, Geddes and Thomson, Schaudinn and Doflein, or possibly of all these and other changes yet unknown. In any case it is reasonable to suppose that the imperfect character of the primitive types of cell -division, described in the last chapter, might produce accumulated material or structural inequalities in the daughter-cells, such as could only be rectified by the union of two cells differentiated in opposite directions, thus making syngamy a necessity for the continued existence of the species. This theory explains the necessity for syngamy recurring with greater frequency in forms having a high degree of structural differentiation than in forms of a primitive and simple type of organization. With increasing perfection in the process of the division of the cell, and especially of the nucleus, the primary cause of, or necessity for, syngamy might be expected to disappear ; but at this stage in evolution other benefits to the species consequent on the process of amphimixis might be a sufficient cause for the retention of a process already well established. This conclusion appears to receive some support from the fact that intensive culture, whether artificial, or natural as in parasitism, seems to diminish the necessity for syngamy. It can hardly be supposed that intensive culture can diminish consequences arising from defective cell-division ; but it might conceivably produce a strengthening effect equal to, and capable of supplanting, the benefits derived from amphimixis. Enriques (113) has stated that in Infusoria ex-conjugants may proceed to conjugation again, so that between one act of syngamy and the next there may not be a single cell-division intervening. In this case neither cell-division nor any consequences of cell- division can be the factor bringing about sexual union, but some other explanation must be sought. Enriques considers that the function of syngamy in Infusoria is to maintain the fixity of the species. Bibliography. — For references see p. 479. CHAPTER IX POLYMORPHISM AND LIFE-CYCLES OF THE PROTOZOA A. POLYMORPHISM. ONE of the most striking peculiarities of living beings is the infinite variety of form, structure, and appearance, which they present. There is, perhaps, no living individual of any kind which is exactly similar, in all respects, to any other. Nevertheless, the most uncultured intellect cannot fail to recognize that, in the case of all ordinary, familiar plants and animals there is a pronounced tendency to segregation into distinct kinds or species — that is to say, natural groups of individuals which, though they may vary greatly amongst themselves, yet resemble one another far more than they do the individuals of another species. It is not necessary to point out that species are not to be regarded as permanent or immutable entities. It is certain that a species may in course of time become modified so as to acquire characters different from those it originally possessed, thus giving rise to a new species, or that a single parent-species may become split up into a number of groups which, by a similar process of modification, became so many daughter-species differing from one another and from the parent- species to a greater or less degree. The problem of the origin of species is one that it is not necessary to discuss here ; it is sufficient to point out that the mutability of species often makes it very difficult to define or delimit a given species exactly, of which a striking example is seen in the pathogenic trypanosomes of the brucii - group, probably to be regarded, as pointed out above (p. 27), as instances of species in an incipient or nascent condition. Some species are sharply marked off from others, some are much less so, and some are of questionable rank, regarded by one naturalist as distinct, by another as mere races or varieties — a state of things perfectly intelligible if existing species are regarded as having arisen by descent, with modification, from pre-existing species. In the Protozoa the existence of distinct species is just as marked as in the higher plants and animals, and is universally recognized. As has been pointed out in the previous chapter, it is probably syngamy which is responsible for the segregation of individuals 162 POLYMORPHISM AND LIFE-CYCLES 163 into species, by blending the divergent characters that may be supposed to arise from the influence of different conditions or circumstances of life. Thus, syngamy in unicellular organisms appears to have an effect which is the opposite, to a large extent, to that which it produces in multicellular organisms, in which there are special germ-cells, sheltered to a greater or less degree from the direct influence of the environment, and in which amphimixis appears rather to be a means by which variations arise. The conception of a species is by no means incompatible with the occurrence of a number of distinct forms in its life-history. Taking well-known instances from the Metazoa, there may be, in the first place, ontogenetic or developmental differences ; not only may the individuals of the same species differ in size at different periods in the development, but they may differ so greatly in appearance and structure that only a knowledge of the life-history enables us to assert that they belong to the same species — as, for example, a caterpillar and a butterfly, or a hydroid and a medusa. Secondly, the adult individuals may differ to an enormous extent in the two sexes. Thirdly, there may be in many cases differences between individuals of a species related to differences in the functions which they perform, not merely at successive phases in the life-history, as in some cases of ontogenetic differentiation already mentioned, but even at corresponding phases of the lif e-history — a phenomenon best seen in social or colony-forming organisms, as in the case of ants and termites, or in the colonies of Hydrozoa. In Protozoa, similarly, a given species may show distinct phases or forms at different or corresponding periods of its life-history to a greater or less extent. In some species the form-changes are very slight, and the individuals occur always under a similar form and aspect, at least during the active state, and are therefore recog- nizable without difficulty as regards their specific identity ; such forms may be termed monomorphic, and as examples the species of ciliate Infusoria can be cited. Other Protozoa, on the other hand, are extremely polymorphic — that is to say, they occur under a variety of widely-differing forms at different stages in the life-cycle or in response to variations in the conditions of life. Hence it is often difficult or impossible to refer a given form to its proper^ species without tracing out its life-history and following its develop- ment step by step. The unravelling of the complicated life-cycles of Protozoa is attended by far greater difficulties than in Metazoa, since one important criterion fails us altogether in the Protozoa, that, namely, of sexual maturity. A naturalist has no hesitation in pronouncing a trochophore to be a larval form, and a rotifer to be an adult organism, from the fact that the former is sexually immature, while the latter produces ripe generative cells. In the 164 THE PROTOZOA Protozoa, however, there is no visible criterion of any similar state of maturity or the opposite which might be a guide in estimating the significance of a particular form. It is certain that with in- creasing knowledge many species of Protozoa now regarded as distinct will prove to be developmental stages of others, as has happened so frequently in the case of Metazoa. The polymorphism of the Protozoa may be related directly or indirectly to a variety of causes, which may be grouped generally under three headings — life-conditions, growth and development of the individual, and sex, 1. Polymorphism in Relation to the Conditions of Life. — Under this heading are included all those cases where the individual is forced to adapt itself to inevitable changes in the environment, or else succumb to their effects ; hence this type of polymorphism may be termed briefly adaptive. The animal may adapt itself to such changes in one or the other of two ways : passively, by passing into a resting state, in which vital activities are temporarily sus- pended ; or actively, by changes of form, structure, and function, adapted to the changed conditions. Methods of passive adaptation to unfavourable conditions occur probably in all Protozoa — perhaps it might be said in all Protista, so that no species can be said to be absolutely monomorphic. The commonest form of such adaptation is the process of encystment, whereby the organism protects itself by secreting a firm, resistant envelope, or cyst, round its body. The first preliminary to encystment in Protozoa is usually a rounding off of the body-form. In the case of naked amoeboid forms such a change of form follows naturally, as pointed out above, from cessation of the locomotor activity. It is, however, also observed that a similar change takes place in corticate forms, a phenomenon which indicates that the cuticle or cortex must be absorbed, or softened, and that any internal form-giving elements must be dissolved, so that the protoplasm is free to conform to the natural physical tendencies of a fluid body. In the great majority of cases, an individual in process of encystment becomes perfectly spherical, whatever may have been the form of its body in the active state, but in some cases the spherical form is not fully attained, and the body becomes ovoid or pear-shaped. During the process of rounding off, any food-particles or foreign bodies contained in the cytoplasm are rejected or absorbed, as a rule ; the contractile vacuoles, if there be any, cease to be formed and vanish ; and all locomotor organs, such as cilia, flagella, and of course pseudo- podia, are absorbed or cast off. At the same time the protoplasm of the organism becomes less fluid and more opaque, and usually diminishes appreciably in bulk, probably through loss of water ; it POLYMORPHISM AND LIFE-CYCLES 165 thereby becomes denser in consistence, but of less specific gravity Lastly, the cyst-membrane itself appears round the body, if it has not already done so ; it generally stands off distinctly from the surface of the body, and may vary in nature in different cases, from a soft, slimy or gelatinous coat to a firm membrane of variable thickness, often exceedingly tough and impervious. In the encysted state, Protozoa are able to withstand the many vicissitudes to which they are naturally subject. They can then be dried up, frozen, or sun-baked ; and since the protoplasm becomes much lighter, they can be transported great distances by winds, a fact which accounts for the appearance of Protozoa in infusions exposed to the air in any situation — a peculiarity from which the name Infusoria is derived. In general the function of encystment is to protect the organism against unfavourable conditions or violent changes in the environment — for instance, in freshwater forms, against drought and climate, the cold of winter or the heat of a tropical summer. In parasitic forms it is an adaptation commonly connected with a change from one host to another. In parasites two types of cysts can be distinguished. In the first place, full-grown forms may produce relatively large, resistant cysts (Dauerzysten) of the ordinary type, almost invariably spherical or ovoid in form. In the second place, the smallest forms in the developmental cycle, the products of multiple fission or " sporulation," may secrete round themselves tough, resistant envelopes, within which they may multiply further ; in this case the envelope is termed a sporocyst, and the entire body a spore* * The word " sporo " has come to be used in two distinct senses, as applied to Protozoa, thereby producing a regrettable confusion and ambiguity. The word itself is derived from the Greek nts represent the multiplicative, the sporonts the propagative, phase, and that in the latter resistant cysts are pro- duced as a protection against the vicissitudes of the outer world, to which the parasite must expose itself during this phase of its life-history. An example of a parasite which infects two distinct species of hosts in the course of its life-history is furnished by the malarial parasites (p. 360, Fig. 156). In this case there are first of all schizonts which, like those of Coccidium, reproduce themselves by multiple fission, this part of the life-cycle being passed in the blood of a vertebrate host. Later, sporonts are generated which under normal circumstances are incapable of multiplication in the verte- brate host, or, indeed, of any further development, unless taken up by another host, in this case a mosquito, which takes them from the vertebrate host by sucking its blood. In the stomach of the new host the sporonts behave in a similar manner to those of Coccidium — that is to say, they give rise as gametocytes to gametes, which by syngamy produce zygotes. The zygotes grow and repro- duce themselves by multiple fission, forming an enormous number of minute germs or sporozoites, which do not develop further unless they pass from the mosquito back into the blood of a suitable vertebrate host, in which they start a fresh developmental cycle. The life-cycle of the malarial parasites shows that a given phase of a parasite is only to be regarded as multiplicative or propagative in relation to a particular host. In the vertebrate blood the schizont is the multiplicative, the sporont the propagative, phase. As soon, however, as the sporont passes into the mosquito, it becomes there the multiplicative phase which gives rise ultimately to the sporozoites, representing the propagative phase in the mosquito. The sporozoites in their turn, when they reach the blood of the vertebrate, develop there into schizonts. Thus one and the same stage in the life-cycle represents one phase in one host and another in another, according to circumstances. It should be noted further that in the life-cycle of the malarial parasites resistant cysts are unnecessary, since the parasite never comes out into the open, but passes the whole of its existence in one or the other of its two hosts ; consequently such cysts are not formed at any stage of the life- cycle in these forms. 168 THE PROTOZOA Another example of a parasite with alternation of hosts, in which the course of events is different from that of the malarial parasites, is furnished by the species of the genus Aggregate (p. 353). Here the schizonts are parasitic in crabs, and reproduce themselves by multiple fission without encystment to form naked germs, mero- zoites, which grow up into schizonts, and multiply again in the same way. If, however, the crab is devoured by a Cephalopod, the merozoites adapt themselves to their new surroundings and become sporonts, which produce gametes. The zygotes form resistant cysts in which they multiply to form spores enclosed in tough sporocysts. The resistant phases pass out of the Cephalopod in its faeces, and to develop further they must be devoured by a crab, in which they become schizonts again. In this case there is no special differentiation of propagative phases in the crab, but the same stage can serve both functions ; on the other hand, in the Cephalopod there is no multiplicative phase, but only a propagative phase with resistant cysts. 2. Polymorphism in Relation to Growth and Development of the Individual. — In Protozoa which multiply only by equal binary fission, as, for example, many Infusoria, there is practically no difference between young and old forms beyond a slight variation in size. An individual feeds, and in consequence grows slightly beyond the size characteristic of the species to which it belongs. It then divides by equal binary fission into two individuals each slightly below the specific size, and they in their turn feed and grow and reproduce themselves by fission in due course. In other cases, however, young and adult forms of a species can be clearly distinguished, and may differ in structure as well as in size. Beginning with reproduction by binary fission, the simplest case is where the adult individual divides into two unequal portions, so that parent and daughter can be distinguished, the former not appreciably smaller than ordinary full - grown individuals, the latter, however, very much smaller ; it may be relatively minute. Examples of this type of reproduction are furnished by trypano- somes, a group in which all gradations may be found between equal and very unequal fission (Fig. 127). Still greater differences between parent and young individuals are seen in cases of gemma- tion— that is to say, where the offspring is set free in an undifferen- tiated condition, and acquires after separation from the parent the characters of the adult, as in Acinetaria. The greatest differences between young and old forms are seen, as might have been expected, in cases of reproduction by multiple fission or gemmation. In such cases the young forms produced often differ from the adult in structure and appearance, as well as in size. An example of multiple fission is furnished by the common POLYMORPHISM AND LIFE-CYCLES 169 Trypanosoma leivisi of rats, in which two types of such fission are seen : either the multiplication of a small individual by repeated binary fission to form a " rosette " composed of several daughter- individuals (Fig. 127, J, K), or the separation of several small daughter-individuals from a large one (Fig. 127, F, G, H). In both cases the multiple fission is simply rapid and repeated binary fission. The young individuals resulting from the fission are sometimes crithidial in type (p. 294), and grow into the adult trypanosome- form. In multiple gemmation (sporulation) the parent body breaks up into a number, sometimes very large, of small or even very minute individuals, buds, or germs, usually given off from a more or less considerable mass of residual protoplasm, which degenerates and dies off. The buds when set free may become active at once, or they may pass first into a resting state to which an active state succeeds at a later period. In the latter case they may form sporocysts, and become the spores already described. Within the sporocyst the minute germ may multiply further water under a cover-slip on a slide, they tend to gather round the drop. As the drop diffuses in the surrounding water, the Paramecia arrange themselves in a ring in the region of optimum concentration. If, however, the drop of fluid employed is of a strength which represents the optimum of chemotaxis for the species, Fid. 83. — Diagram showing the course taken by a Paramecium which has entered a drop of fluid to which it is positively chemotactic. The forward movements of the Paramecium are indicated by arrows ; its backward movements by dotted lines ; the outline of the drop of fluid by a circle. Each time the Paramecium, in its forward movement, reaches the confines of the drop, it conies into contact with fluid which is less positively chemotactic than the drop into which it has entered ; it then shrinks backward (avoiding reaction), after which it moves forward again with the same result every time it reaches the edge of the drop. After Liang (10). the Paramecia gather within it, and in such a case the position taken up by each Paramecium depends on the avoiding reaction made by it when it comes in contact with a less attractive medium. Thus, if a Paramecium, swimming in a straight line, enters a drop of fluid which is positively chemotactic to it, when it has crossed the drop to its opposite boundary it comes to the region where it meets with fluid which is less chemotactic to it ; it then shrinks back with an avoiding movement ; after a time it again moves forward, and comes again into the negatively chemotactic region, with the same result as before. Thus its movements are as if caught in a trap (Fig. 83), in which it is held by the automatic movements called forth by the difference between the more and the less chemotactic fluids, unt'l 204 THE PROTOZOA the differences slowly disappear by the diffusion of the one liquid into the other. Chemotaxis is a phenomenon which is obviously of the greatest importance in the natural life of the organism. It comes into play in the search for food and in sexual attraction, for example. It has long been known that certain Protozoa are attracted towards food- substances, especially those species which feed more or less exclusively upon certain particular foods. Plasmodia of Mycetozoa, for example, "scent" their food from a considerable distance, and move towards it. Rhumbler (34, 204) has studied the ingestion of food by amoebae, and has made a number of experiments on the manner in which drops of fluid take up or cast out solid particles. Thus, » drop of chloroform suspended in water draws into its interior a glass splinter coated with shellac when brought into contact with it ; after a time the coating of shellac is dissolved in the chloro- form, and the glass splinter is then ejected from the drop. This experiment furnishes data for a mechanical explanation of the ingestion of food and ejection of faecal master ; and it might be expected that amoebae in Nature would ingest mechanically, and as it were helplessly, many substances of a useless kind with which they are brought into contact. This may occur experimentally when amoebae are brought into contact with substances of no nutritive value ; Rhumbler observed an amoeba which ingested carmine- particles until it died. In Nature, however, there can be no doubt that amoebae exercise a certain choice or selection in the food they ingest, doubtless as the effect of rhemotactic reactions (compare Jennings, 168). In the Ciliata, however, tiiere appears to be no selection of the food- particles wafted down the oesophagus except as regards their size (compare Greenwood, 162). Purely mechanical reactions, on the other hand, may possibly explain the apparent selection which many Protozoa exhibit in building up houses of cer- tain special materials (p. 34). Chemotactic reactions to particular substances must play a large part in determining the migrations of certain parasitic Protozoa towards particular organs of the body in which they are parasitic, in so far as such migrations are not purely passive on the part of the parasite, or determined to some extent by rheotaxis (see below). The attraction of gametes to one another can hardly be effected by any- thing but chemotaxis. It is well known that the antherozoids of the fern- prothallus are positively chemotactic to malic acid, which is secreted by the oogonium. In Coccidium achuJberfi, Schaudinn (99) observed that the macro- gamete, as. soon as it had expelled its karyosome, but not before, became attractive to the microgamete. The effects of drugs and reagents on the activities of the Protozoa is a field of investigation which cannot be dealt with in detail here. Some reagents have a quickening effect on the movements, others the contrary. Narcotics, on the other hand, such as alcohol, ether, etc,, may at first have a stimulating, later a deleterious, action on the vital activity. Minute doses of alcohol, according to Woodruff (216), diminish the rate of division at one period, augment it at another, of the life-cycle, but in the latter case the rate is not continuous, but decreases again ; increase in the amount of alcohol will, however, again cause a more rapid cell-division for a limited period Thyroid extract is stated to have an attractive effect on Paramectum, and also increases its capacity for reproduction (Nowikoff, 183). For the effects of other drugs and poisons, see Giemsa and Prowazek (159), and Prowazek (191, 192, and 195). In the same culture different individuals often exhibit different powers of resistance to the effects of reagents. THE GENERAL PHYSIOLOGY OF THE PROTOZOA 205 (6) Phototaxis and Effects of Light and Other Bays. — Many Pro- tozoa appear quite indiff erent to light — at least of ordinary intensity ; others show a very decided reaction, as already mentioned, either negative or positive. Thus many amoebae, Pdomyxa, etc., are negatively phototactic, and pass at once into a condition of rest and inactivity when exposed to light. According to Mast (176), a sudden increase in the intensity of the illumination inhibits move- ment in Amoeba proteus ; but if the illumination remains constant, movement begins again in a few moments. If the illumination is very gradually in- creased, it produces no response. In strong light Amceba proteiis orientates itself, producing pseudopodia only on the less illuminated side. Many flagellates, on the other hand, especially the holophytic forms such as Dinoflagellates, Phytomastigina, Eugle- noids, etc., show the opposite reaction, moving towards the light or becoming active when exposed to it,, and passing into a resting state in the dark. The positive phototaxis of the holophytic Protozoa has an obvious bionomical sig- nificance, since the holophytic nutrition can only proceed in the presence of light. In the majority of holophytic flagellates the phototactic reaction is associated with the possession of a special organ, the etigma or "eye-spot." The stigma of Euglena consists of a protoplasmic ground-substance forming a fine network, in which is embedded pigment in the form of drop-like bodies. The pigment granules are brightly refractile, with a distinct outline, and form a single layer. In some cases the granules are spherical and all of the same size ; in others they are more irregular in form and of different sizes The pigment appears to be a derivative of chlorophyll. The stigma is in close contact with a well-marked thickening on one of the two branches into which the flagellum bifurcates at its base. Wager (213) suggests that this thickening (blepharoplast ?) is a specialized sensitive organ which is stimulated by the light-absorbing pigment-spot, the stigma, and that in this way the reaction of Euglena to light is determined. Euglena swims towards a moderate light, but away from strong sunlight. If kept in bright sunlight it comes to rest, rounds itself off, and ultimately becomes encysted. The blue and violet parts of the .spectrum exert the strongest stimulus on flagellates. In the case of Amoeba proteus, Mast (176) found the blue rays nearly as efficient as white light in causing reactions, but violet, green, yellow, and red, to be but slightly active. Paramecium and some other Protozoa are stated to react only to the ultra-violet rays. The effect of radium-rays upon various Protozoa has been investigated by Zuelzer (221). Some species are more affected by them than others; a. 84. — A, Anterior end of Eiujlena viridis. fl., Flagel- lum ; ces., oesophagus ; bl., thickening (blepharoplast ?) on one of the two roots of the nagellum ; at., stigma ; rh, the two roots of the nagellum passing through the reservoir ( R) of the contractile vacuoles, two to be attached to its opposite side. B, Stigma in surface view, highly magni- fied, showing the pigment- grains imbedded in a proto- plasmic basis. After Wager (213). 206 THE PROTOZOA Amoeba Umax, for example, was very resistant to the rays, while other Protozoa were very soon injured by them. In all cases long exposure to the rays was fatal. The first effect of the rays was generally to quicken the movements; the next was an injurious action. The rays appear to act more particularly upon the nucleus in the first instance, with subsequent gradual deleterious effects upon the cytoplasm. In experiments on the effect of Rontgen-rays on Paramecium and Volvox (Joseph and Prowazek, 169), these forms wore found to exhibit a negative taxis, collecting in ten to fifteen minutes in a part not exposed to the rays. Exposure of Paramecium to the rays caused the pulse of the contractile vacuoles to become slower to a marked degree as a rule, but individual variations were observed in this reaction, the effect being inconsiderable in some cases ; and the animals gradually regain the normal pulse. Intro, vitam staining of the nucleus of Paramecium exposed to the rays gave a result similar to that obtained by staining Paramecia fatigued by being shaken evenly and continuously for two hours. Long- continued action of the rays killed the organisms. (c) Thermotaxis end Effect* of Temperature. — For a given species of the Protozoa there is an optimum temperature at which its vital activity is at its highest pitch, and above which the activity is diminished until ii reaches a point at which the vitality is impaired and the animal is finally killed. A temperature, however, at which the animal succumbs sooner or later may at first have a quickening effect upon the "ital functions. Thus, many experiments have shown that a rise of temperature increases greatly the rapidity and frequency of the pulsations of the contractile vacuoles ; and in the case of Glaucoma colpidium Degen (154) found that, although the animal was killed by a temperature above 30° C., the maximum frequency of the pulsations was produced temporarily by a tempera- ture of 34° C., above which the frequency was rapidly diminished (compare also Khainsky, 170-5). The optimum temperature may, however, be different at different stages in the life-cycle, as in parasitic Protozoa which infest a warm- blooded and a cold-blooded host alternately ; in such cases a change of temperature may perhaps be a factor in bringing about develop- mental changes. In free-living Protozoa the phases of the life-cycle are often related to seasonal changes, and are probably induced largely by conditions of temperature. Experimentally it has been shown that Protozoa tend to move towards regions of more favourable temperature, and away from those less favourable. Khainsky (170'5) found that rise of temperature produced a quickening of the digestive processes in Paramecium, very marked at 24° C. or above. At 30° C. and above Paramecium takes up scarcely any more food ; the contents of the food-vacuoles, which continue to be formed, then consist almost entirely of water. The effects of temperature on the development in cultures are very marked. Popoff studied the growth of Frontonia leucas in cultures kept at 14° C. and 25° C. respectively ; at the lower temperature the animals divide once in about eighty or ninety hours, in the warmer culture once in about seventeen hours ; in the cold both the nucleus and the body grow to a size absolutely larger than in the warmth, but in the former case the nucleus is about -<*j, in the latter about -J4, the bulk of the whole body (Hertwig, 92). In the case THE GENERAL PHYSIOLOGY OF THE PROTOZOA 207 of Actinosptuerium, the experiments of Smith (207), Mackinnon (174), and Boissevain (151), show that increased temperature hastens on encystment, and causes fewer and larger cysts to be formed in which the nuclei are larger but poorer in chromatin ; while at lower temperatures the encystment is retarded, and finally inhibited altogether, and the cysts produced are smaller and more numerous, with nuclei smallerthan the normal but rich in chromatin. (d) Barotaxis and Effects of Mechanical Stimuli. — This category includes Geotaxis, or reactions to gravity ; Thigmotaxis, or reactions to the mechanical contacts of hard surfaces ; and Rheotaxis, or reactions to the pressure of currents in the surrounding medium. The influence of gravity is seen in the manner in which many Protozoa, when placed in a vessel, seek of their own accord the bottom in some cases, the surface-film in others. The plasmodia of Mycetozoa exhibit often a well- marked rheotaxis, and move in the opposite direction to currents of water. It has been suggested that a similar rheotaxis may explain the passage of blood-parasites from the invertebrate to the vertebrate host during the act of blood- sucking ; but it is probable that such migrations are purely passive, so far as the parasites are concerned. Contact-stimuli acting from one side often have a marked effect on the movements of Protozoa. An amoeba tends to adhere to, and spread itself over, a firm surface with which it comes in contact. The movements of Ciliata often cease when they come in contact with a firm substance, and the animal remains still ; Putter (197) has shown that the contact-stimulus may be sufficient to prevent a Paramecium from reacting to thermal or electric stimuli, which would otherwise produce a marked effect upon its movements. Under effects of mechanical stimuli must be included those brought about by changes in the tonicity of the surrounding medium. Such effects have already been discussed above as regards their action on the contractile vacuoles. For the remarkable experiments of Verworn on the change in body-form and in the nature of the pseudopodia exhibited by amoebae under the action of different media, see p. 217, infra. Free-living Protozoa are probably seldom if ever subject to such changes, though they might well occur in the environment of marine forms living near the upper limit of the tide-marks, in rock-pools, or other places where the tonicity of the medium might be lowered temporarily by influx of fresh water, as the result of rain or other natural causes. On the other hand, parasitic forms, and especially those which pass from one host to the other, may be subject to rapid changes of tonicity in their environment. In this connection special interest attaches to the experiments of Robertson (503) on nsh-trypanosomes ; it was found that in undiluted blood or in blood diluted with isotonic solutions the trypanosomes underwent no change in vitro, but that when the blood was diluted with water the trypanosomes multiplied by division, and went through changes similar to the first stages of the natural development in the leech. It was concluded, therefore, that the principal stimulus which initiates the developmental changes in the organism was a lowering of the osmotic tension, with consequent absorption of water by the protoplasm. Neumann (677) also found that the " exflagellation " of the Profeoscmia-parasite of birds was greatly furthered by addition to the blood of not more than one-fifth of its volume of water. 208 THE PROTOZOA (e) Galvanotaxis and Effects of Electrical Stimuli. — Protozoa placed in an electric field — that is to say, in a drop of water between the two poles of a battery under a cover-glass on a slide — are affected to a marked degree, but with opposite results in different species. 0-palina places itself parallel to the direction of the current, with its anterior end towards the anode. With a current of moderate intensity it swims towards the anode ; but with a stronger current the speed at which the animal moves is diminished, and with still more increased strength of current it is carried passively towards the kathode, with its hinder end forward, as the result of kataphoric action (Wallengren, 215). Chilomonas behaves in a similar manner. Paramecium and Colpidium, on the other hand, move towards the kathode. Spirostomum with a moderate current also moves towards the kathode, but with stronger currents it first contracts its myonemes spasmodically, and then takes up a position transverse to the direction of the current, and remains still. According to Wallengren (215), the apparently different galvano- tactic phenomena exhibited by different ciliates admits of a uniform explanation, by a combination of two effects. In the first place, in the half of the body turned towards the kathode the expansion - phase of the ciliary movement is stimulated ; in the anodic half of the body, the contraction-phase is stimulated. In the second place, the turning movements of the ciliates are determined mechanically (compare the " avoiding reactions " mentioned above), and may be effected either by the expansion or by the contraction of certain cilia. Consequently, if the turning movements are effecte-1 by beats of expansion, the animal places itself automatically in a posi- tion in which it moves towards the anode ; if beats of contraction are effective in the turning movement, it moves towards the kathode. According to Statkewitsch (209), the galvanotactic re- action is one which overcomes «hemo tactic stimuli, and leads the animals irresistibly into toxic media in which they are killed. 6. Degeneration and Regeneration. — The fact that under certain conditions Protozoa undergo a process of physiological degenera- tion, which may end in death, has been observed frequently by all those who have kept cultures of Protozoa under observation for a long time. It has been pointed out in a previous chapter (p. 135) that the life-cycles of Protozoa exhibit depression-periods (Calkins) which are characterized chiefly by cessation of feeding, metabolism, growth, and reproduction, together with increase in the size of the nucleus, and tendency to deposition of grains of fat or other sub- stances in the protoplasm, giving the body a characteristic dark- grey appearance. Such periods jecur regularly and apparently normally in the life-cycles both of Protozoan and Metazoan cells THE GENERAL PHYSIOLOGY OF THE PROTOZOA 209 (Popoff, 184) ; they may also be induced artificially in various ways by unfavourable conditions, such as overfeeding or starvation, changes of temperature, or treatment with reagents (compare Smith, 207 ; Popoff, 186 ; Boissevain, 151). A state of depression may be regulated naturally by conjuga- tion, or by restoration of the nucleo-cytoplasmic balance through a process of self-regulation on the part of the organism. The regulative processes consist of absorption of a large part of the superfluous chromatin, so as to restore the normal quantitative relation of the nucleus and cytoplasm. On the other hand, the depression may lead to complete degeneration of the organism without possibility of recovery, and death ensues by a process of disruption of the protoplasm into granules — so-called " granular disruption " (korniger Zerfall). Some examples are given below : Actinosphcerium can be brought into a condition of depression either by starvation or overfeeding (Hertwig, 164). In the depressed state a great quantity of chromatin is extruded from the nuclei in the form of chromidia which degenerate into pigment, so that the animal during a depression-period has a characteristic brownish tint, more or less pronounced in proportion to the degree of depression. In extreme cases the protoplasm is bereft of its nuclei, and becomes incapable of continuing to live. The nuclei may become entirely resolved into chromidia ; or some of the nuclei grow to a relatively gigantic size and are cast out, while other nuclei break up ; or the entire medullary layer surrounding the enlarged nuclei may be thrown off. The pseudopodia may disappear altogether or become deformed in various ways, the difference between cortical and medullary substance may be annulled or abnormally increased, and the metabolism may be modified, all these changes being in relation to nuclear alterations. In Opalina, according to Dobell (155), physiological degeneration can be induced by starvation of its host, the frog. The degenerating Opalines lose their cilia and become irregular in form j peculiar refringent eosinophile globules appear in the cytoplasm ; the nuclei undergo increase in size and modification in structure, give off chromatin, and undergo irregular fusions ; and the body divides irregularly, sometimes producing buds which contain no nucleus. Ultimately the Opalines disintegrate. Prandtl (187) has described the degeneration of Amoeba proteus. The nucleus increases in size and becomes hyperchromatinic. Chromidia are extruded into the cytoplasm, and may there degenerate, with formation of numerous small crystals. The chromatin in the nucleus also degenerates to form a mass of brown pigment, which is extruded en bloc into the cyto- plasm, or forms a ring of fine granules round the nucleus. The pigment may also spread through the whole cytoplasm, giving it a brownish tinge. Finally the nucleus breaks up and disappears altogether. Degenerating amoebae are subject to the attacks of parasites. A noteworthy feature is the tendency of the degenerating amoebae to associate in clumps, and plastogamic fusion of two amoebae was observed by Prandtl. The tendency to fusion may be compared with the agglomeration of trypanosomes, etc. (p. 128), which is common also in degenerating forms or under unfavourable conditions.* It is not improbable that many of the plastogamic unions of Sarcodina often * The " conjugations " observed by Putter (201, p. 582) in Opalines kept without oxgyen must have been also phenomena of the nature of agglomeration, since in Opalina syngamy takes places between special gametes, and not in the form of conjugation of adult forms as in other Giliata (p. 453). 14 210 THE PROTOZOA described may be phenomena of agglomeration associated with a similar condition. In Radiolaria, Borgert (152) describes fatty degeneration affecting the nucleus as well as the protoplasm, both endoplasm and ectoplasm. The nucleus becomes converted entirely into a vesicle filled with a mass of fat- globules, or into a number of such vesicles. In Tocophrya quadripartite subjected to starvation, after the refringent bodies (p. 458) have been absorbed, the nucleus becomes modified in structure, the tentacles are retracted, active budding takes place, and with the last bud formed the nucleus disappears and the remaining protoplasm dies away. From a consideration of the various examples of degeneration from different causes, it appears that the first part to be affected is always the nucleus, and that the other derangements of the structure and functions of the body are secondary consequences of an abnormal condition of the nucleus. The regeneration of lost parts of the cell-body of Protozoa has been the subject of experiment by a great number of investigators. The methods employed have consisted mainly in mutilating the body or cutting it up hi o a number of pieces, in order to find out to what extent the fragments possess the power of regenerating the lost parts. The experiments have led to one very definite result, which can be expressed briefly : no separate part of the body is capable of continuing its vital activities indefinitely, or of regenera- ting any of the deficiencies in the structure of the body, if it does not contain the nucleus or a portion of the nucleus. Non-nucleated fragments may continue to live for a certain time ; in the case of amoeba such fragments may emit pseudopodia, the contractile vacuole continues to pulsate, and acts of ingestion^ or digestion of food that have begun may continue ; but the power of initiating the capture and digestion of food ceases, consequently, all growth is at an end, and sooner or later all non-nucleate fragments or enucleated bodies die off. A Polystomella which possesses a nucleus can repair breakages to the shell ; an individual deprived of its nucleus cannot do so (Verworn). On the other hand, an isolated nucleus, deprived of all protoplasm, dies off ; but a small quantity of protoplasm containing the nucleus or a part of it is able in some cases to regenerate the whole body, and to produce a complete individual of small size. In experiments on regeneration the Ciliata are the objects of choice ; their complicated structure permits the regeneration that has taken place to be estimated accurately ; their size renders the mutilation more easy to perform ; and the large size and frequently extended form of the nucleus makes it possible to divide up this body also. In recent experiments Lewin (171) has succeeded in dividing Paramecium into a number of fragments (" mero- zoa "), containing each a portion of the macronucleus. Only one of the merozoa obtained in this manner contains the micrpnucleus, which is too minute to be divided by a mechanical operation. Except when the Paramecium was in process of division, only one merozoon recovered the normal body- form and proceeded to divide ; and the interesting result was obtained that THE GENERAL PHYSIOLOGY OF THE PROTOZOA 211 the merozoon which survived was not necessarily the one which contained the micronucleus. Regenerated individuals multiplied for a number of genera- tions, producing a culture of " amicronucleate " Paramecia. If, on the other hand, a Paramccium in process of division was halved, each half regenerated the entire body and was capable of division. These experiments indicate that Paramecium contains a division-centre independent of the nuclei, and that its presence is necessary for regeneration of the body. Prowazek (189) observed occasionally a certain power of regeneration hi non-nucleated fragments of Stentor, but considered it possible that extra- nuclear chromatin might have been present. The same author (190) ob- served abnormal regeneration, leading to monstrosities with three hinder ends, in a culture of Stylonychia mytilus during a depression-period which led finally to the extinction of the culture. The recent experiments of Lewin (172) on Stylonychia mytilus show that, in the regeneration which follows artificial mutilation, multiplication of micronuclei may occur, with the result that the regenerated individual may have more micronuclei.than the number typical of the species or race. Bibliography. — For references see p. 481. CHAPTER XI SYSTEMATIC REVIEW OF THE PROTOZOA : THE SARCODINA As stated in Chapter I., the Protozoa are commonly divided into four principal classes. Of these, two — namely, the Sarcodina and Mastigophora — may be regarded as the more primitive groups, comprising* the main stock of less specialized and typical forms from which the other two classes have been evolved. The Sporozoa are an assemblage of exclusively endoparasitic forms exhibiting clearly the modifications and adaptations induced by, or necessary for, their particular mode of life ; and it is practically certain that the Sporozoa are not a .homogeneous class showing mutual affinities based upon a common ancestry, but that one section of the group is a specialized offshoot of the Mastigophora, the other of the Sarcodina, and that the two sections are united only by characters of convergence due to the influence of a similar mode of life. The Infusoria, on the other hand, are a specialized group in which great complexity of organization has been attained ; they are the highest class of the Protozoa, and furnish examples of the most extreme degree of structural differentiation of which a unicellular organism is capable. While there is but little difficulty, as a rule, in defining the classes Sporozoa and Infusoria, or in assigning members of these groups to their proper systematic position, the case is different, very often, when we have to deal with the other two classes. The verbal distinction between them is based chiefly on the use of the word " adult ": Sarcodina are Protozoa which have no permanent organs of locomotion in the adult condition, but move by means of pseudo- podia extruded from the naked protoplasmic body ; Mastigophora, on the other hand, bear organs of locomotion in the form of flagella in the adult condition, whether the protoplasmic body is naked and amoeboid or corticate and of definite form. In both classes the youngest stages may be flagellate ; if, in an amoeboid form, the flagella are retained in the adult, the organism is classed in the Mastigophora ; if lost in the Sarcodina. The word " adult " when applied to the Metazoa has a meaning which can be defined clearly, as a rule, by the criterion of sexual 212 THE SARCODINA 213 maturity. In the Protozoa no such criterion is available, and the distinction between young and adult is based on differences in size and growth, or on phases of the life-cycle selected in an arbitrary manner. In many cases the distinction presents no difficulty ; it is perfectly easy to distinguish young from adult stages in such forms as the Foraminifera and Radiolaria among Sarcodina, or the genus. Noctiluca among Mastigophora. But in other cases it is purely a matter of opinion which phase in the lif e-cycle is to be regarded as adult. Such a form as Pseudospora has a flagellated and an amoeboid phase (Robertson), and can be placed in either the Sarcodina or the Mastigophora with perfect propriety. The amoeba-like genus Mastigamceba is placed in the Mastigophora because the flagellum is retained ; but if any species of this genus were to lose its flagellum when adult, rigid adherence to verbal definitions would necessitate its being classed in the Sarcodina. The difficulty of separating and defining the stems of the Sarco- dina and Mastigophora at their root is only to be expected on the theory of evolution. The two classes are undoubtedly descended from a common ancestral type, which has become modified in two divergent directions, giving rise to two vast groups of organisms which may differ from one another very slightly or very greatly in selected examples. The systematist may meet with many obstacles when it is required to lay down verbal distinctions between the two classes, but it is easy to recognize, in a general way, two principal morphological types, round which each class is centred, and which may be realized to a greater or less extent in given cases. 1. Sarcodine Type. — Protozoa which grow to a relatively large size ; in the so-called " adult phase " permanent organs of loco- motion are wanting, and the naked protoplasmic body moves or captures food by means of pseudopodia ; the young stages may be flagellate or amoeboid. 2. Mastigophoran Type. — Protozoa usually of minute size, seldom with a large adult phase (as, for example, Nocti Hca) ; flagella retained throughout active life, only lost in resting phases ; body, amoeboid or corticate. THE SARCODINA. The name Rhizopoda is sometimes used for this class but this name is only applicable, strictly speaking, to the first four orders recognized below, in which the pseudopodia are more or less root- like, and not to the orders Heliozoa and Radiolaria, characterized by stiff radiating pseudopodia. General Characteristics. — As stated above, the Sarcodina are Pro- tozoa for the most part of relatively large size. Many Sarcodina 214 THE PROTOZOA are visible to the naked eye, and some of the Radiolaria, Foramin- ifera, and Mycetozoa, attain to a size that must be considered gigantic for Protozoa. The more primitive forms, on the other hand, are often very minute. The body-form is of two principal types, related to-distinct habits of life — namely, the amoeboid type, characteristic of forms that creep on a firm substratum ; and the radiate type, seen in floating forms. Amoeboid forms are found aquatic, semiterrestrial, and parasiti§ ; radiate forms are for the most part pelagic, living floating or suspended in large masses of water, marine or fresh-water. The protoplasmic body is in many cases distinctly differentiated into clear motile ectoplasm and granular trophic endoplasm. The surface of the protoplasm is naked, or may be covered in rare in- stances (Amceba verrucosa, A. terricola, etc.) by a very thin pellicle which modifies, but does not restrain, the amoeboid movements. A resistant cuticle or cell-membrane investing the body is not formed, but an external shell or internal supporting skeleton is frequently present. The locomotor organs in the adult are always pseudopodia, which may be of various types — lobose, filose, or reticulose (Chapter V., p. 46) ; they may lie in one plane, as in creeping forms, or may be given off on all sides, as in pelagic forms. The youngest forms (swarm-spores) /nay be flagellate or amoeboid. In some cases the pseudopodia of the young forms may differ markedly in character from those of the adult ; for example, the adult Amoeba protects has fluid protoplasm with thick lobose pseudopodia, but the young amoebula produced from the cyst of this species has viscid proto- plasm with sharp, spiky pseudopodia (Scheel). The free-living Sarcodina are almost without exception holozoic, capturing other organisms by means of their pseudopodia, and devouring them; but the remarkable genus Chlamydomyxa (p. 243) has chromatophores, and can live in either a holozoic or holophytic manner, like some flagellates ; and the genus Paulinella, allied to Euglypha, also possesses chromatophores. and is capable of holo- phytic nutrition (Lauterborn). The nuclear apparatus consists of one or more nuclei, in addition to which chromidia may be present. A single nucleus is charac- teristic of the majority of species, even of many which grow to very large size, such as many Radiolaria, in which the nucleus also attains to proportions relatively gigantic. In other cases increase in the size of the body is accompanied by multiplication of the nuclei ; there may be two nuclei constantly, as in Amceba binucleata (Schaudinn), or several, as in Difflugia urceolata, or many hundreds, as in Actinosphcerium and Pelomyxa, or even thousands, as in the Mycetozoa. In such forms the adult is a plasmodium, but the SARCODINA 215 numerous nuclei show no differentiation amongst themselves, and appear to be perfectly equivalent both in structure and function. Chromidia may be present as a permanent cell-constituent in many Amoebaea, such as Arcella, Difflugia, and the Foraminifera ; in other cases they are formed temporarily, as extrusions from the nucleus, during certain phases of the life-cycle, either as a preliminary to reproduction or as a regulative process under certain physiological conditions. The reproduction of the Sarcodina is effected either by binary or multiple fission. Binary fission may be absent in some of the larger, more specialized forms, as in many Foraminifera and Radio- laria, but in most cases it is the ordinary " vegetative " method of reproduction during the active trophic life of the organism. In plasmodial forms it takes the form of plasmotomy (p. 100). Mul- tiple fission or gemmation (sporulation) is in some cases the sole method of reproduction ; in other case it is combined with binary fission, and occurs only at certain crises in the life-cycle, in relation to seasonal changes, or as a preliminary to syngamy. In this type of reproduction the organism, breaking up rapidly into a large — often an immense — number of minute individuals, is necessarily put hors de combat as soon as the reproduction begins ; hence it is not uncommon for the sporulation to take place within a cyst, when a shell or protective envelope is not present, as in Amoeba proteus (Scheel). The minute germs produced by sporulation may be set free at once as swarm-spores ; or they may form a pro- tective envelope or sporocyst, and be liberated as resistant spores which are disseminated passively, and germinate when conditions are favourable, as in parasitic forms and in the semi-terrestrial Mycetozoa. The swarm-spores, whether produced directly by sporulation of an adult or indirectly by germination of a spore, may be either flagellulae or amoebulao. In many forms two types of sporulation occur — schizogony producing agametes, and sporogony producing gametes. The agametes may be structurally or morphologically distinguishable from the gametes. Thus in Foraminifera the agametes are amoabulaBj the gametes. are flagellulse. In Radio laria both alike are flagellulae, but the agametes produced in schizogony — the "isospores" — are distinguishable from the gametes produced in sporogony — " anisospores." In this class syngamy takes place rarely between adult indi- viduals ; but examples of this are seen in Actinophrys, where it takes the form of karyogamy within a cyst (Fig. 71), and in Arcella (p. 148) and Difflugia, where it takes the form of chromidiogamy between free individuals, followed in Difflugia by encystment. In the great majority of Sarcodina the syngamy is microgamous, and takes place 216 THE PROTOZOA between swarm-spores, either amoebulse or flagellulse. The microg- amy is isogamous or slightly anisogamous ; macrogamy, as in other cases, is perfectly isogamous. Microgamy occurs, as has been seen (p. 148), in Arcdla in addition to chromidiogamy ; and, according to a recent note of Zuelzer (86, p. 191, footnote), syngamy between free swarm-spores occurs in Difflugia also. As regards the life-cycle of the Sarcodina, there remains still so much to be discovered that to generalize is both difficult and dangerous. Even in the commonest forms, such as Ama&a proteus, the complete life-cycle has not been yet worked out. In some FIG. 85. — Changes in the form of an amoeba under the influence of differences in the [surrounding medium. A — G, In its natural medium (water) : A, contracted ; B, beginning to throw out pseudopodia ; 0, Umax-form. D — F, Forms assumed after addition of potash-solution : I), contracted, beginning to throw out pseudopodia ; E, F, radiosa-fov&a. After Verworn. cases the life-cycle appears to be of comparative^ simple type, and the species is monomorphic or nearly so, as in Actinosphcerium ; in other cases there is a well-marked alternation of generations, with dimorphism in the adult condition, as in Trichosphcerium (p. 182), the Foraminifera, etc. Classification. — The Sarcodina are subdivided into a number of orders, the distinctions between which are based principally on the characters of the pseudopodia and of the skeleton, when present ; in more highly differentiated forms, such as Radiolaria, the internal structure of the body is also taken into account. In the primitive THE SARCODINA 217 forms of simple structure, however, in which no skeleton is present, the subdivisions are defined entirely by the characters of the proto- plasmic body and the pseudopodia, which furnish distinctions of very doubtful validity. Not only may the characters of the pseudopodia vary in different phases of the life cycle, as already stated in the case of Amoeba proteus, but even in the same phase under the influence of different media. Thus, no two forms of amoeba could appear more distinct at first sight than the Umax and radiosa forms, originally regarded as distinct species. In the Umax-form the whole body flows forward as a single pseudopodium, gliding along like a slug ; in the rarftosa-fonn the spherical body becomes star-like, sending out sharp-pointed pseudopodia OB ..all sides. Nevertheless Verworn showed that the one form could be changed into the other by differences in the medium (Fig. 85). Doflein (238) obtained similar form-changes in Amoeba? vespertilio; and showed that the body-form and character of the pseudopodia were quite inadequate features for distinguishing the species of amoeba, depending as they do upon the conditions of the environ- ment and the nature of the medium. Compare also Gruber (246) on form-varieties of Amoeba proteu*. In view of the protean nature of these organisms, it is not sur- prising that much diversity of opinion prevails as to the arrangement of the groups and the exact position of some of their members. It is usual to put a number of primitive organisms together in a group termed Proteomyxa, the members of which probably have more affinities with various members of other groups than with one another. On the other hand, the more highly organized Sarcodina .are classified without difficulty into well-characterized orders ; such are the Foraminifera, Mycetozoa, Radiolaria, and Heliozoa, though even in these groups there are forms near the border-line and of doubt ul position. The classification adopted here is mainly that of Butschli (2), with the addition of some forms not included in his great work as follows : A. SUBCLASS RHIZOPODA. — Typically creeping forms with branched, root-like pseudopodia. I. Order Amozbom. — Amoeboid forms of simple structure ; skeleton lacking or in the form of a simple shell. 1. Suborder Reticulosa (Proteomyxa). — With filose or reticulose pseudopodia, without shell. 2. Suborder Lobosa. — With lobose pseudopodia. (a) Section Nuda, without shell or skeleton. (6) Section Testacea, with shells. II. Order Foraminifera. — With reticulose pseudopodia and shells. 218 THE PROTOZOA III. Order Xenophyophora. — With skeleton of foreign bodies and a peculiar internal structure. IV. Order Mycetozoa. — Semi-terrestrial forms with repro- duction by resistant spores and formation of plas- modia. B SUBCLASS ACTINOPODA (Calkins). — Typically floating forms with radiating, unbranched pseudopodia. V. Order Heliozoa. — Principally fresh-water, without a " central capsule." VI. Order Radiolaria. — Exclusively marine, with a central capsule. I. AMfEB^BA. 1. Reticulosa. — In this suborder are comprised a number of forms of doubtful affinities, sometimes ranked as a distinct order, Proteomyxa. The only positive character which they have in common is the possession of filose or retioulose pseudopodia, with which is combined the absence of a shell and skeleton. Hence it is not surprising that the position of many forms referred to this suborder is extremely dubious, and some of them are referred to distinct orders by many authorities. In general two types of organisms are referred to this suborder : (a) Large marine plasmodial forms ; an example is Poniomyxa ftava, described by Topsent from the Mediterranean and British Channel. Pontomyxa is a multinncleate plasmodium of yellow colour. It sends out branching root-like pseudopodia, which may spread out and form a network extending over two or three inches in length. Nothing is known of its development or life-cycle. (6) Small forms with a single nucleus, marine or fresh-water, which reproduce by process of multiple fission forming swarm- spores. These forms have been subdivided into two families, according to the type of swarm-spore found — Zoosporidce, pro- ducing flagellulae ; and Azoosporida, producing amoebulae. An example of the Zoosporidce is furnished by the genus Pseudospora, which preys upon algae, diatoms, Volvocineae, etc. The adult phase is amcsboid, flagellate, or even Heliozoon-like. It feeds on the cell- substance and chlorophyll of the prey, and multiplies by binary fission. It can also break up by multiple fission into flagellate swarm-spores, with or without previous encystment. Robertson has observed syngamy between flagellulae thus formed, which are therefore gametes ; in other cases the flagellulae are perhaps agametes. As already pointed out above, the position of this form amongst the Sarcodina is doubtful ; by many authorities it is classified in the Mastigophora. An example of the Azoosporidce is furnished by Vampyrella, a THE SARCODINA 219 small amoeboid form which, like Pseudospora, preys upon algae (Fig. 86), devouring the contents of the cell, and multiplying in the free state by binary fission. It also encysts and breaks up within the cyst by multiple fission to form a number of amoebulae, which creep out and grow up into the adult form. A large number of other genera are referred to the Reticulosa, for the most part so little investigated as regards their develop- ment and life-history that it is impossible to deal with them com- prehensively in a brief space. For an account of them see Delage and Herouard (6, p. 66), Hickson (248), and Rhumbler (288). 2. Lobosa. — This suborder comprises a great number of organisms, which it is convenient to subdivide into — (a) Nuda (Gymnamoebae), FIG. 86. — Vampyretta lateritia: various forms. A, Fjec Heliozoon-like phase ; B, creeping amoeboid phase ; C, amoeboid form attached to a Core/erm- fila- ment ; D, a similar form ; it has broken the algal filament at a joint, and has emptied one cell of its contents. A and B after Hoogenraad ; C and D after Cash and Hopkinson. with no shell ; and (6) Testacea (Thecamoebae, Thalamophora), with a shell or house. General Characters. — Familiar examples of the Lobosa Nuda are furnished by the species of the genus Amceba and allied forms. A very large number of free-living amoebae have been described and named, but it is very doubtful how far they are true species ; some of them, with pronounced and constant characteristics, such as Amoeba proteus (Fig. 2) and A. verrucosa (Fig. 23), are probably " good " species ; others, such as A. Umax and A. radiosa, are probably forms that may occur as phases in the development of other species of amoebae or of other organisms, such as Mycetozoa. 220 THE PROTOZOA At the present time the life-history has been worked out satisfac- torily in but few free-living amoebae, but in such protean organisms it is quite- xinsafe to attempt to characterize or define a species without a knowledge of the whole life-cycle. As regards the familiar Amoeba proteus, for example, practically all that is known of its life-cycle is that it encysts and multiplies within the cyst to form a great number of small amoebulae, very different in appear- ance from the parent-organism ; the amoebulae creep out of the cyst, and probably grow up into the adult form (Scheel). Calkins adduces arguments in favour of the occurrence of a sexual cycle, which remains at present, however, purely conjectural. The majority of free-living amoebae are aquatic in habitat. A certain number, however, are semi-terrestrial, inhabiting damp earth, moss, etc. Such is Amoeba terricola (vide Grosse-Allermann). The " earth-amoebae," like other terricolous Protozoa, probably play a great part in keeping down the numbers of the bacteria and other organisms in the soil and thereby lessening its fertility from an agricultural standpoint (compare Russell and Hutchinson, 24 ; Goodey, 16). A great many species of amoebae are found living within the bodies of animals of all kinds, for the most part in the digestive tract. The entozoic amoebae are commonly placed in a distinct genus, Entamceba, distinguished from the free-living forms by little, however, except their habitat and the general (but not invariable) absence of a contractile vacuole. A common example is Entamosba blattos, from the intestine of the common cockroach ; others are E^ ranarum of the frog (Do bell, 236, 237) ; E. muris of the mouse (Wenyon) ; the species parasitic in the human intestine, presently to be mentioned ; E. buccalis (Prowazek), from the human mouth ; and many others. Chatton has described a species, Amoeba mu- cicola, ectoparasitic on the gills of Labridos, and extremely patho- genic to its host. Life-History. — So far as it is possible to generalize from the scanty data available at present, the development of many free-living species of amoebae appears to be of a type very similar to that of Arcetta, described in a previous chapter (p. 179). In the free state the organisms reproduce themselves in two ways : first, " vegeta- tively,'* by simple binary fission, preceded by a division of the nucleus, which varies 'n different cases from a promitosis (p. 109) of the simplest type to very perfect mitosis ; secondly, by forma- tion of chromidia and subsequently of secondary nuclei, round which the cytoplasm becomes concentrated to form a number of internal buds, destined to be set free as amoebulae, agametes, which grow up into the adult form. In addition to these two methods of reproduction in the free state, the animal may become encysted, THE SARCODINA 221 and produce within the cyst a number of gametes in the same manner as the agametes already described, but with the following differ- ences of detail : the principal nucleus degenerates as soon as the chromidia are formed ; the number of secondary nuclei produced is much larger, and the gametes are much smaller than the agametes ; and the cytoplasm of the parent is entirely used up in their forma- tion. The gametes are ultimately set free from the cyst as amoe- bulae, and pair ; the zygote grows into the adult form of the amoeba. Such a cycle has recently been described by Popoff (264) for a species named by him Amoeba minuta ; the gametes in this species are isogametes, without any sexual differentiation as in Arcetta. This type of life-cycle is probably very common hi many amoebae, FlQ. 87. — Amoeba albida : autogamy in the encysted condition ; drawn in outline, with nuclear details only. A, Encysted amoeba ; B, the nucleus of the amoeba divides unequally into a larger vegetative and a smaller generative nucleus ; the vegetative nucleus, as seen in the subsequent figures, travels to the surface of the cyst, degenerates, and disappears ; the generative nucleus gives rise to the gamete-nuclei ; G, incomplete division of the generative nucleus ; D, one half of the generative nucleus is budding off two reduction-nuclei (on the right) ; E, four reduction-nuclei have been budded off, two from each pole of the incompletely divided generative nucleus ; F, the reduced generative nucleus completes its division ; the four reduction-nuclei are degenerating ; O, the two pronuclei far apart ; H, the two pronuclei coming together ; /, the pronuclei fusing. After Nagler (95). with specific differences of detail in different cases, of which the most important are, that in some cases, probably, the nucleus divides to form the gamete-nuclei, instead of becoming resolved into chromidia, and that autogamy within the cyst may occur, instead of free gametes being formed, as A. albida (Fig. 87). According to Nagler (95), autogamy of this type is characteristic of all amoebae of the Umax-group ; in such cases only two gamete- nuclei are formed in the cyst, which after going through reducing divisions fuse to form a synkaryon. The zygote then leaves the cyst and begins a fresh vegetative cycle. A different type of life-cycle is exemplified by that which Schepo- tieff has described in the case of a marine amoeba identified by him as A. flava. In this case also the ordinary vegetative form is a 222 THE PROTOZOA uninucleate amoeba, which reproduces itself by binary fission of the ordinary type ; but large multinucleate forms occur which become encysted. Within the cyst the nuclei break up into chromidia, from which a great number of secondary nuclei are formed. The protoplasm becomes concentrated round the secondary nuclei to form a number of small cells, which acquire flagella and are set free from the cyst as flagellulae, believed to be gametes and to copulate ; the zygote is at first encysted, but becomes free from the cyst, and develops into the uninucleate amoeba. The life-cycle of A. proteus is possibly of this type, since in this species also multinucleate amoebae are commonly observed (see especially Stole ; compare also Paramceba (Fig. 49). Fio. 88. — Amoeba diploidea. A, The amoeba in the vegetative condition, with its two nuclei ; B — F, the sexual processes within the cyst, drawn in outline on a reduced scale ; B, two amoeba;, each with its two nuclei, encysted together, the nuclei beginning to give off chromidia ; C, the two nuclei of each amoeba fused, numerous vegetative chromidia in the cytoplasm ; D, the bodies of the amoebae fused, each synkaryon beginning its reduction-process ; E, the synkarya giving off reduction-nuclei which are degenerating ; F, the reduction- process complete ; the cyst contains a single amoeba with two nuclei (syn- karya), ready to emerge and begin its vegetative free life. After Nagler (95). Metcalf (257) describes " gemmulcs " budded from small free amoebae of the proteus-type, each gemmule becoming detached and developing into a flagellated gamete of a cercomonad type. The flagellulse were observed frequently to lose their flagella and become amceboid. Copulation of two flagellulse took place to form an amceboid zygote. Metcalf's observations upon the syngamy in this case recall strongly the observations of Jahn (294) on the sexual processes of Mycetozoa (p. 242). It is possible that the syngamy observed by him did not form a part of the life-cycle of the amoeba, but of some other organism. The sexual process described by Nagler (95) in Amoeba diploidea ia of a remarkable kind (Fig. 88). In the ordinary vegetative condition the amoeba possesses normally two nuclei, which divide simultaneously each time the THE SARCODINA 223 animal reproduces itself by fission. The sexual process begins by two such amoebae coming together and surrounding themselves with a cyst in common. Within the cyst their nuclei first give off vegetative chromidia, which are absorbed, after which the two nuclei in each separate amoeba fuse together tD form a single nucleus, a synkaryon. The protoplasmic bodies of the two amoebae now fuse completely into one, after which each synkaryon goes through two reducing divisions, producing each two reduction-nuclei, of which the first may divide again, so that there may be in the cyst six reduction- nuclei altogether, which are gradually absorbed. The two persistent synkarya, after undergoing this process of reduction, approach each other, but remain separate, and the amoeba is hatched out of the cyst to begin its vegetative life with two nuclei representing gamete-nuclei that have undergone reduction — that is to say, pronuclei — which remain separate and multiply by fission throughout the vegetative life, and do not undergo syngamic fusion until the — -end of it. In Amoeba binudeata, described by Schaudinn, the vegetative phase also contains two similar nuclei which multiply simultaneously by division each time the animal divides ; but in this case the complete life-cycle is not known. Owing to the practical importance of the entozoic amoebae, and the attention that has been directed to them in consequence, their life-cycles have been more studied and are better known than those of the free-living species. According to Mercier, Entamoeba blmttos multiplies by binary fission in the gut of its host, and later becomes encysted, passing out of the body of its host in this condition. Within the protective cyst it breaks up by multiple fission, follow- ing repeated division of the nucleus, into a number of amoebulse, which are set free from the cyst when it is devoured accidentally by a new host. The amoebulse are gametes which copulate after being set free, and the zygote grows into the ordinary vegetative form of the amoeba. E. blattos thus furnishes a very characteristic and primitive type of the life-cycle of an entozoic amoeba, and one which differs only in points of specific difference from that of Amoeba minuta, described above. *- The question of the human entozoic amoebae is at present in a somewhat confused state. The occurrence of amoebae in the hinder region of the human digestive tract, especially the colon, has long been known, and the name Amoeba coli was given by Loach to such organisms (synonym, Entamosba hominis, Casagrandi and Barba- gallo). It is, however, certain that more than one species of amoeba occurs in the human bowel, and Loach's name must therefore be restricted to one of these. An epoch in the study of human entozoic amoebae was marked by the researches of Schaudinn (131), who distinguished two species. The first, to which he restricted the name Entamoeba coli, occurs commonly in Europe and elsewhere as a harmless inhabitant of the intestine — that is to say, like E. blattce and many others, it is not, under normal circumstances at least, a parasite in any sense of the word, but a simple scavenger, feeding on bacterial and other organisms, detritus, etc., in the colon and rectum. The second species, to which Schaudinn gave the name E. histolytica,* is, on the contrary, * Liihc has proposed to place E. histolytica in a separate genus, Poneramceba n. g. (Schr. Physik. Oes. Kiinigsberg, vol. xlix., p. 421). 224 THE PROTOZOA a parasite of a dangerous kind, which occurs in tropical and subtropical regions, and is the pathogenic agent of amoebic dysentery and liver-abscess ; it attacks and devours the tissues of the host, destroying the wall of the intestine, whence it penetrates into the blood-vessels and is carried to the liver, where it establishes itself and gives rise to liver-abscesses. These two species of amoebae are distinguishable by structural characters. E. coli has a relatively fluid body, with ectoplasm feebly developed and with a fairly large spherical nucleus (or nuclei) lodged in the endoplasm. R. hiatolytica, FIG. 89. — Entamoeba coli. A and B, Living amoebae showing changes of form and vacuolation in the endoplasm ; C, D, E, amoebae showing different conditions of the nucleus (n.) ; F , a specimen with two nuclei preparing for fission ; G, a specimen with eight nuclei preparing for multiple fission ; //, an encysted amoeba containing eight nuclei ; /, a cyst from which young amoebae (al) are escaping ; J, K, young amoebae free. After Casagrandi and Barbagallo. on the contrary, has a relatively viscid body with greatly-developed ecto- plasm, as is seen clearly in the formation of pseudopodia, which may consist entirely of ectoplasm ; it is smaller than E. coli, and its nucleus has a com- pressed form, stains feebly, and is lodged in, or immediately below, the superficial eotoplasmic layer. The life-cycles of these two species are also very different, as described by Schaudinn. E. coli, in the amoeboid multiplicative phase, reproduces itself by binary THE SARCODINA 225 fission of the ordinary type, and also by a process of multiple fission is which the nucleus divides until there are eight nuclei in the body ; the characteristic 8-nucleate plasmodium then divides up into eight small amoebae, each of which grows into an ordinary adult form. Hence it in characteristic of E. coli to occur in various sizes, from very small to full-grown amoebae. The propagative phase of E. coli is initiated by the formation of a gelatinous envelope round a full-sized amoeba possessing a single nucleus. The nucleus then divides into two, and the process of maturation and autogamy takes place that has been described on p. 139, supra (Fig. 73). When it is complete, a tough resistant cyst is formed within the soft gelatinous envelope, and each of the two synkarya divides twice to produce four nuclei. Thus is formed the 8-nucleate resistant cyst which is characteristic, perhaps diagnostic, of this species. Within the cyst no further changes take place until it is swallowed by a new host ; then it is believed that the contents of the cyst divide up into eight uninucleate amoebulae, which are set free in the colon and are the starting-point of a new infection. Schaudinn was able to infect himself by swallowing the 8-nucleate cysts of the amoeba. Prowazek (A.P.K., xxii., p. 345) has described a variety of E. coli under the name E. williamsi. E. histolytica reproduces itself in the amoeboid phase by binary fission and by a process of gemmation in which the nucleus multiplies by division, and then small amoebulae, each with a single nucleus, are budded off from the surface of the body. In the process of gemma- tion, however, the number of nuclei in the body is irregular, and not definitely eight, as in E. coli. In its propagative phase E. histolytica does not form a cyst round the whole body, but its nucleus becomes resolved into chromidia, which collect in patches near the surface of the body. Little buds are then formed as outgrowths of the body, each bud containing a clump of chromidia. Round each bud a sporocyst is formed of so tough and impervious a character that no further cytological study of the bud is possible. The resistant spores formed in this way separate from the body, of which the greater part remains as residual protoplasm and dies off. The minute spores are the means of infecting a new hose, aa shown by Schaudinn in experiments on cats, which are particularly susceptible to the attacks of this amoeba. Schaudinn' s investigations, of which a brief summary has been given in •the foregoing paragraphs, first introduced clear ideas into the problem of the human entozoio amoebae. Many of the works of subsequent investigators have tended, however, rather to confuse and perplex the question, for various reasons. In the first place, in cultures made from human faeces, free-living, non- parasitic species of amoebae make their appearance, which have passed through the digestive tract in an encysted condition, and emerge from their 15 FIQ. 90. — Entamceba histolytica. A, Young specimen; B, an older specimen crammed with ingested blood-corpuscles; 0, D, E, three figures of a living amoeba which contains a nucleus and three blood- corpuscles, to show the changes of form and the ectoplasmic pseudo podia : n., nucleus ; b.c., blood-corpuscles. After Jurgens. 226 THE PROTOZOA cysts in the cultures ;* such amoebae, for the most part of the Umax-type, have been confused with the true entozoic amoeba, and have given rise to erroneous ideas. Secondly, it is certain now that the two species of amoebae recognized by Schaudinn does not exhaust the list of human entozoic amoebae. Thirdly, it is possible that Schaudinn did not see the entire life-cycle of the forms studied by him, or that in some cases he confused stages of different species in the same life-cycle (compare Hartmann, 247). It is still doubtful how many species of entozoic amoebae occur in man. Hartmann recognizes two dysenteric amoebae, in addition to the harmless E. coli : E. histolytica, Schaudinn, and E. tetragena, Viereck (synonym, E. africana, Hartmann). E. tetragena has been described from various parts of the tropics ; it differs from E. histolytica in its characters, and more nearly resembles E. coli, but is distinguished by the formation of resistant cysts containing four nuclei. In addition to these species, many others have been described by various investigators — for example, E. minuta, Elmassian, which, according to Hartmann, is merely a variety of E. coli. A summary of the various amoebae described from the human intestine is given by Doflein (7) and Fantham (241). In Cochin China, Noc obtained from liver- abscesses and dysenteric stools a small amoeba (not named) which in the multiplicative phase reproduces in two ways : by binary fission of the ordinary type ; and by budding off small amcebulae containing secondary nuclei formed from chromidia. In the propagative phase Noc's amoeba encysts and breaks up into amoebulae. Greig and Wells, in Bombay, obtained results very similar to those of Noc. In cultiires from liver-abscesses from. Bombay, Liston found two distinct forms of amoebae — a larger form containing a single nucleus and numerous chromidia, and a smaller form containing a nucleus only. The larger amoeba multiplies either by binary fission, with karyo- kinesis of the nucleus and partition of the chromidia ; or by the formation of endogenous buds containing chromidia from which a secondary nucleus is formed, the bud being finally set free as a small amoeba with a nucleus and a number of chromidia. The small amoeba multiples only by binary fission, preceded by amitotic division of the nucleus. Both large and small amoebae form resting cysts, in which, however, they remain unchanged, and from which they emerge when circumstances are favourable. It is evident that much of the life-cycle of these liver-abscess amoebae remains to be worked out. Prom, the foregoing it is clear that, with regard to the human pathogenic amoebae, many important problems remain to be investigated, especially as regards their specific distinctions, distribution, and life-history. Much recent work has been carried on by culture-methods, with valuable results, which, however, should be interpreted with caution, since it remains to be ascertained whether the forms and phases assumed by these organisms in cultures are identical in character with those which they exhibit under natural conditions ; and until this point has been cleared up it is not safe to describe the characters of a species of an amoeba, any more than of a trypanosome, from cultural forms alone. With regard to the lif e-cycle of the pathogenic amoebae, it is most important to discover what are the phases of development or conditions of life under which they occur outside the human body ; whether they exist only in an encysted, resting condition, or in an active state also ; and, in the latter case, whether as free-living organisms or within some other host. On general grounds it is un- likely that an organism adapted to an entozoic life should be capable also of living free in Nature, and it is more probable that the pathogenic amoebae out- side the human body occur only in the condition of resting cysts or spores, which produce infection through being accidentally swallowed with food or water (compare Walker, 276'5). In that case.unfiltered water, uncooked vegetables * Whether this also applies to cultures made from the pua of liver-abscesses, as asserted by Whitmore (279) and Hartmann (247), may well be doubted ; it is not easy to understand how an encysted amoeba Sould be transported passively from the intestine into a liver-abscess. THE SARCODINA 227 and herbs, or fruit that grows near the ground, are likely sources of infection by becoming contaminated with the resting stage of the amoebae scattered on the ground or in manure. In this connection the further question arises whether the human entozoic amoebae are specific parasites of man or not, and conse- quently whether their infective stages would be derived only from human faeces, or irom the excreta of other animals also. From general considerations of parasitism in Protozoa, it seems probable that the harmless E. coli is a specific parasite of man, but that the pathogenic forms are parasites of other animals also, and perhaps only occasionally find their way into the human body ; in which case garden-manure might be a fruitful source of contamination, through the medium of vegetables habitually eaten uncooked, such as lettuce, celery, etc. None of these questions can be answered decisively at present, however, and there is a wide field of in- vestigation open. Greig and Wells found that in Bombay amoebic infection shows a marked seasonal variation, closely associated with variations in humidity, but not corresponding with those of temperature, and reaching its maximum in August. In addition to the various species of Amoeba and of allied genera and subgenera, a number of other genera are included in the section under consideration, for an account of which the reader must be referred to the larger treatises ; but two deserve special mention — namely, the genera Pdvmyxa and Paramceba. The species of Pelomyxa (Fig. 91) are fresh- water amoebae of large size and " sapropelic " habit of life (p. 14). The body, which may be several millimetres in diameter, is a plasmodium in the adult condition, containing some hundreds of nuclei ; it is generally very opaque, owing to the animal having the habit of loading its cytoplasm with sand and debris of all kinds, in addition to food in the form chiefly of diatoms. The pseudopodia are of the lobose type, blunt and rounded, but the animal may also form slender reticulose pseudopodia under certain conditions (Veley). The cytoplasm is very vaeuolated, and contains a number of peculiar refringent bodies (" Glanzkorper ") of spherical form, with an envelope in which bacterial organisms (Cladothrix pelomyxae, Veley) occur constantly. The bacteria multiply by fission in a linear series in the form of jointed rods, which may branch ; as a rule they have five or six joints, or less, but at least two. The refringent bodies are of albu- minous nature (Veley). According to Gold- schmidt (57), the refringent bodies arise from the nuclei when they give off chromidia ; in this process the chromatin is given off into the cytoplasm, and the plastin-basis of the karyosome is left as a spherical mass which becomes the refringent body. At first the plastin-sphere is surrounded by the remains of the nuclear membrane, which disappears, and the refringent body grows in size. Re- fringent bodies, with their bacteria, are seen frequently to be ejected by the animal during life. Bott (103), on the other hand, states that the ivfringent bodies are reserve food-stuff, their contents of the nature of glycogen, and Fro. 91. — Pelomyxa palus- tris : a specimen in which the body is transparent owing to the absence of food-particles and foreign bodies, showing the vaeuolated cytoplasm and the numerous nuclei and refringent bodies (tho refringent bodies are for the most part larger than the nuclei) in the living condition. After Greeff, magnified 60. 228 THE PROTOZOA that they arise in the cytoplasm independently of the nuclei ; but their rejection by the animal is more in favour of the view that they are waste- products of the metabolism (Veley). It is not clear what is the role of the bacteria, whether they are parasites or symbionts. Petomyxa reproduces itself by simple fission or by formation of gametes. The sexual process, according to Bott, begins with extrusion of chromatin from the nuclei into the cytoplasm to form chiomidia, which may take place so actively that sometimes the nuclei break up altogether. A similar extrusion of chromidia may take place as a purely regulative process under certain conditions, such as starvation ; -but the vegetative chromidia formed in 'this way, and absorbed ultimately in the cytoplasm, must be distinguished from generative chromidia produced as a preliminary to gamete- formation. From the genera- tive chromidia secondary nuclei of vesicular structure arise, which, after elimination of chromatin followed by reduc- tion (see p. 150, supra), become the gamete-nuclei. The gametes arise as spherical in- ternal buds, each with a single nucleus, to the number of 100 or more, and are extruded when fully formed, causing the parent-individual to break up completely. Each free gamete is Heliozoon-like, with slender, radiating pseudopodia ; they copulate in pairs, and the zygote grows into a young Pdomyxa, either directly or after a resting period in an encysted condition. The genus Paramceba (Fig. 49) was founded by Schaudinn (81) for the species P. eilhardi discovered by him in a marine aquarium in Berlin.* In the adult stage the animal occurs as an amoeba, from 10 to 90 /* in diameter, of rather flattened form and with lobose pseudo- FIG. 92. — Portion of a section through the body of Pdomyxa. N., Nucleus ; r.b., refringent bodies ; 6., bacteria on th6 refringent bodies ; s., sand and debris in the protoplasm. After Gould. podia. It contains a single nucleus, and near it a peculiar body, the " Nebenkern " of Schaudinn (see p. 95). In this phase the amoeba multiplies by binary fission accompanied by division both of nucleus and Nebenkern. It also becomes encysted and goes through a process of multiple fission, which shows three stages ; in the first the Nebenkern multiplies by repeated division, the nucleus remaining unchanged ; in the second the nucleus divides repeatedly to form as many small nuclei as there are Nebenkerne present, and each nucleus attaches itself to a Nebenkern ; in the third the protoplasmic body undergoes radial super- ficial cleavage into a number of cells, each containing a nucleus and a Neben- kern. Each of the cells thus formed becomes a swarm-spore with two flagella. * The amoeba from the human intestine described by Craig under the name Paramceba hominia certainly does not belong to this genus. See Doflein (7), pp. 602, 603. THE SARCODINA 229 The swarm-spores are liberated from the cyst and live freely, feeding and multiplying by binary fission, in whioh the nucleus divides by mitosis and the Nebenkern acts like a centrosome. After a time, however, the swarm-spores lose their flagella, and become amoebulae which develop into the adult phase. Syngamy was not observed, but probably takes place between the flagellulse. Two new parasitic species of Paramoeba have been described recently by Janicki (7T5) ; see p. 95. To the order Amoebjea should be referred, probably, the parasite of the Malpighian tubules of the rat-flea (Ceratophyttus fasciatus), described by Minchin under the name Malpighietta refringens, and the parasite of Ptychodera minuta> described by Sur under the name Protoentospora ptychoderce. The section Lobosa Testacea or Thecamoebae contains a number of free-living forms familiar to every microscopist, such as the genera Dijflugia (Fig. 16), Centropyxis, Arcella (Fig. 32), etc. The majority of these forms inhabit fresh water, but Trichosphcerium (Fig. 81) is marine. Their common distinctive feature, in addition to the possession of lobose pseudopodia, is the formation of a shell or house into which they can be withdrawn entirely. The shell may be secreted by the animal, and then is chitinous (Arcella) or gelat- inous (Trichosphcerium), or may be made up of various foreign bodies cemented together (Difflvgia). Typically the house has the form of a chamber with a single large opening, through which the pseudopodia are extruded at one pole. When the animal multiplies by fission, the protoplasm streams out through the aperture, and forms a daughter-shell external to the old one, after which division of the nucleus takes place and the two sister-individuals separate. In TrichosphcBrium, however, the house has the form of a gelatinous investment to the body, with several apertures through which pseudopodia protrude, and when the animal divides the investing envelop divides with it. The protoplasmic body contains typically one nucleus — some- times more than one — surrounded by a ring of chromidia. In Arcella vulgaris there are constantly two primary nuclei ; in Difjlugia urceolata, from ten to thirty. Trichosphcerium possesses many nuclei, but no chromidia. The life-cycle, so far as is known, is of various types ; those of Arcella and TrichosphcBrium are described above (p. 177, Fig. 80, and p. 181, Fig. 81). The latter, with an alternation of generations combined with dimorphism in the adult condition, approaches that of the Foraminifera in character. In the testaceous amoebae the method of division varies in accordance with the nature of the shell. In those in which the shell is soft and yielding, as, for example, Cochliopodium and Cryptodifflugia, the division is longitudinal — i.e., in a plane which includes the axis passing through the mouth and apex of the shell (Doflein, 239) ; in Cryptodifflugia rapid division of this kind may lead to colony-formation. In forms with a rigid shell, on the other hand, such as Difflugia, Arcella,, Centropyxis, etc., the shell sets a limit to the growth of the animal, which, when it has filled the shell, ceases to grow for a while 230 THE PROTOZOA and stores up reserve-material. Prior to division a sudden and rapid growth takes place at the expense of the reserve-material and by absorption of water ; as a result the protoplasm grows out of the shell-mouth, a daughter-shell is formed, and the animal divides transversely (Pig. 50). In Difflugia urceolaia. Zuelzer (85) has described a process of chromidiogamy. Two animals come together with the mouths of the shells in contact, and the entire contents of one shell flow over into the other, the empt'y shell being oast off. The chromidia of the two animals fuse into a single mass; the nuclei, however, remain separate. Copulation of this kind is a preliminary to encystment, which takes place in Nature at the end of October or the beginning of November. Prior to encystment the pseudopodia are retracted, «11 foreign bodies, food-remains, excreta, etc., are cast out, and the proto- plasmic body rounds itself off in the shell, and diminishes to about a quarter of its former volume, becoming denser and more retractile. The cyst- membrane is then secreted at .the surface of the body. The old nuclei are gradually absorbed, and new nuclei are formed from the chro'midial mass. The reconstitution of the nuclear apparatus takes place from January to April ; in the spring the cyst is dissolved, and the rejuvenated Difflugia begins to feed and to enter upon a summer course of vegetative growth and repro- duction. In a recent note (86, p. 191, footnote) Zuelzer states that conjuga- tion between free gametes also OCCUIB in this species; In Cenfropyxis acvleata, according to Schaudinn (131 ), the ordinary vegeta- tive reproduction is by fission, the new shell that is formed being larger than the old one, until the m«.-rininm size is reached. Sexual processes are initiated by degeneration of the primary nucleus, which is single in this species. Then the protoplasm with the chromidia creeps out 6f the shell, and divides into a number of amoebulae, each containing chromidia Vhioh condense into a single nucleus. Some amoebulae form a shell at once ; others before doing so divide into four smaller amcebulse, and then form a shell. The larger are macrogametes, the smaller the microgametes ; they copulate and abandon their shells. The zygote forms a new shell, chromidia appear, and a fresh vegetative cycle is started. In a species of the genus Cryptodifllugia (" AUogromia") a remarkable type of life-cycle has been described by Prandtl (265) ; see also Doflein (7.), p. 310, Fig. 283. In this form also the organism, at the time of gamete- formation, quits its shell and penetrates into some other Protozoan organism, such as Amoeba proteus, in the body of which it becomes parasitic and goes through the process of gamete-formation. The nucleus breaks up into chromidia, from which secondary -nuclei are formed, producing a multinucleate plasmodium which multiplies by plasmotomy until the host is full of them. Ultimately the plasmodia break up into uninucleate cells, the gametes, which are set free and copulate. The zygote becomes a flagellated .Bodo-like organism, with two flagella, one directed forward, the other backward as a trailing flagellum (p. 270, infra) ; it feeds and multiplies in this form for •several generations in the free state, but ultimately it loses its flagella, becomes amoeboid, forms a shell, and develops into an adult Cryptodifflugia. Note- worthy in this development are the alternation of generations between the flagellated and the amoeboid phase, as in Pseudospora (p. 218), and the para- sitism in the gamete-forming phases; if, however, the Cryptodifflugia does not succeed in finding a suitable host, the gamete-formation may take place in the free state. From the life-cycles and sexual processes of ArceKa, Difflugia, Centropyxis, etc., it is seen that the primary nuclei of all these forms are vegetative in nature, while the chromidia give rise to the gamete-nuclei, and consist of, or at least contain, the generative chromatin. The- marine Trichosphcerium, however, stands apart from the fresh- water genera in regard to its structure, sexual processes, and life-cycle, in all of which it shows more similarity to the Foraminifera. THE SARCODINA 231 II. FORAMINIFERA. General Characters — Shett-Structure. — The characteristic features of this group are the possession of reticulose pseudopodia and of a shell or test. The Foraminifera are typically creeping forms, moving slowly, and using their net-like pseudopodia chiefly for food- capture. Certain genera, however, such as Globigerina, have taken secondarily to a pelagic existence, and float on the surface of the ocean, spreading their nets in all directions around them. On the other hand, some forms have adopted a sedentary life, attaching themselves firmly to some object. An example is seen in the genus Haliphysema (Fig. 17), once believed to be a sponge, and in the remarkable genus Polytrema and allied forms, recently monographed by Hickson (282) — organisms which in many cases have a striking and deceptive resemblance to corals. The test may be secreted by the animal itself, and then is usually either chitinous or calcareous, rarely siliceous or gelatinous (Myxo- theca) ; or it may be made up of foreign bodies cemented together, as in HaHphysema (Fig. 17), and is termed generally " arenaceous,'* but the materials used may be of various kinds, and the organism sometimes exhibits a remarkable power of selection (see p. 34, supra). The typical form of the shell, as in the Amoebaea Testacea, is a chamber with, a wide aperture — sometimes more than one — through which the pseudopodia are extruded, as hi Gromia (Fig. 21). In addition to the principal aperture, the wall of the shell may be perforated by numerous fine pores, through which also the protoplasm can stream out to the exterior. Hence the shells of Foraminifera are distinguished primarily as perforate and imperforate, the former with, the latter without, fine pores in addition to the principal opening. Whether perforate or imperforate, the 'shell remains a single chamber in the simple forms, as in the Amoebaea Testacea.. In some cases, when the animal reproduces itself by binary fission, the proto- plasm streams out through the principal aperture to give rise to the body of the daughter-individual, which forms a shell for itself, and, when the division is complete, separates completely from the mother, which retains the old shell. Division of this type is seen in Euglypha (Fig. 59). But in many species, when the animal out- grows its original single-chambered shell, -the protoplasm flows out and forms another chamber, which, however, is not separated off as a distinct individual, but remains continuous with the old shell, so that the animal, instead of reproducing itself by fission, remains a single individual with a two-chambered shell. By further growth , third, fourth, . . . nth chambers are formed successively, each newly- formed chamber being, as a rule, slightly larger than that formed 232 THE PROTOZOA just before. Hence a distinction must be drawn between mono- thalamous or single-chambered shells and polythalamous shells, made up of many chambers formed successively. In the latter type the new chambers may be joined in various ways to the old, 2.Lagena 4.Frondicularia O.CIobigenna O.Planorbulino 10 n.Nummulites Fia. 93. — Shells of various genera of Foraminifera. In 3, 4, and 5, a shows the surface-view, and b a section ; Sa is a diagram of a coiled shell without supple- mental skeleton ; 85, of a similar form with supplemental skeleton (.i.tk. ) ; 10, of a form with overlapping whorls ; in lla half the shell is shown in hori- zontal section ; b is a vertical section. In all the figures a marks the aperture of the shell ; 1 to 15, the successive chambers, 1 being always the oldest or initial chamber. From Parker and Haswell. producing usually either a linear or a spiral series, and the utmost variety of shape and pattern results in different species (Fig. 93). Some polythalamous species exhibit a peculiar dimorphism (Fig. 94) THE SARCODINA 233 in some individuals, hence termed microspheric, the initial chamber of the shell is smaller than in others, which are known as megalospheric. This point will be discussed further under the reproduction. It may be noted that if, in this order, a species were to form no shell, whether from having secondarily lost the habit or as a primi- tive form which had never acquired it, then such a species would be classed in the order Amcebaea Reticulosa. It is very probable that many of the large marine " Proteomyxa " are allied to the true FIG. 94. — Biloculina depressa : transverse sections of (a) the megalospheric form, magnified 50 diameters, and (b) the microspheric form, magnified 90 diameters. After Schlumberger, from Lister. Foraminifera, as forms either primitively or secondarily without a test ; and Rhumbler unites the Foraminifera proper with the naked forms in the section Reticulosa. The body-protoplasm exhibits no marked distinction of ecto- plasm and endoplasm. Contractile vacuoles are present in some of the fresh-water genera, but are not found in marine forms. The protoplasm contains metaplastic bodies of various kinds, and may become loaded with fseoal matter in the form of masses of brown granules, termed by Schaudinn the " stercome " (compare also Awerinzew, 281). Periodically a process of defaecation takes place, whereby the protoplasm is cleared of these accumulations, often as a prelude to the formation of a new chamber (Winter, 28). The 234 THE PROTOZOA nuclear apparatus varies in different forms, even in the same specie's, as will be seen in the description of the reproductive processes. The marine Foraminifera, so far as they have been investigated, show a well-marked alternation of generations in their life-history, St. FIG. 95. — Polystometta crispa : decalcified specimens to s^ow the structure of the two forms. A, The megalospheric type ; B, the microspherio type : 6, the central chambers of the latter more highly magnified ; r., retral processes ; st, communications between the chambers. From Lister. combined with dimorphism in the adult condition. An example it, Polystometta, which has been investigated by Lister (285) and TIIE SARCODINA 236 Schaudinn (131) ; their results have been confirmed in the case of Peneroplis by Winter, who gives a useful combined diagram of the life -history (28, p. 16, text -fig. A). The microspheric form (Fig. 95, B.) has many nuclei, which multiply by fission as the afiimal grows, and which also give off chromidia into the body- protoplasm. When reproduction begins, the nuclei become resolved entirely into chromidia, and the protoplasm streams out of the shell, which is abandoned altogether. Secondary nuclei are formed from the chromidia, and the protoplasmic mass divides up into a swarm of about 200 amoebulae (Eig. 96). Each amcebula contains a nucleus and chromidia, and secretes a single-chambered shell, which is the initial chamber of a megalospheric individual. The amoebulse separate, ar d each one feeds, grows, forms new chambers successively, and becomes a megalospheric adult. Thus the micro- spheric form is seen to be an agamont or schizont, which gives rise by a process of schizogony or multiple fission to agametes (amoe- bulae). The megaiospheric form, when full grown, has a single large nucleus and numerous chromidia (Fig. 95, A). The nucleus is that of the amoebula which was the initial stage in the develop- ment of this form ; as it grows the nucleus passes from chamber to chamber, and at the same time gives off chromidia into the cyto- plasm. Finally the primary nucleus is resolved entirely into chromidia, from which a great number of secondary nuclei are formed. Round each such nucleus the protoplasm becomes con- centrated to form a small cell, which may be termed a gameto- cyte. By two divisions of the nucleus and cell - body of the gametocyte four gametes are formed, each of which acquires two flagella, and is set free as a biflagellate swarm-spore. In Peneroplia, however, the gametes have a single flagellum, and in AUogromia ovoidea the gametes are amoebulae (Swarczewsky). Gametes pro- duced by different individuals copulate, losing their flagella in the process, and the zygote secretes a minute single-chambered shell, and thus becomes the starting-point of the growth of a micro- spheric individual. From the foregoing it is seen that the megalospheric form is the gamoat , which by multiple fission produces the gametocytes, and ultimately the gametes. Thus, if m. represents the microspheric form and M . the megalo- spheric, am. the amoebulae (agametes), and fl. the flagellulae (gametes), the life-cycle may be represented thus : m. — am. — M. — (fl. + fl.) — m. — am. . . . In some cases, however, the life-cycle does not present a regular alternation of sexual and non-sexual generations, but a number of non-sexual generations may take place before a sexual generation intervenes ; that is to say, the megalospheric forms may produce agametes and other megalospheric forms again for several generations, before gametes are produced and the sexual processes occur. Then the life-cycle may be represented thus : m. — am. — M. — am. — M. — am. . . . M. — (fl.+fl.) — m. — am. — M. . . . I ' t,"- ! , I FIG. 96. — Stages in the reproduction of the microapheric form of Poly- atomella crispa. In a the protoplasm is streaming out of the shell ; in 6 and c it is becoming divided up into amoebulse ; in d the amoebulae, having each formed a single- chambered shell, are dispersing in all directions, abandoning the empty shell of the parent. From Lister, drawn from photographs of ono specimen attached to the walls of a glass vessel. /t THE SARCODINA 237 Hence the dimorphism of the adults is due to their parentage, and is not necessarily related to the manner in which they reproduce. A microspheric form is produced sexually, and is always an agamont ; a megalospheric form is produced non-sexually, and may be either a gamont or an agamont. Very little is known of the life-cycle of the non-marine genera. The only form of which the cycle is known with any approach to completeness is CMamydophrys stercorea, the only entoeoic member of the order, which is found in the faeces of various vertebrates ; a second species, C. schaudinni, is distinguished by Schiissler (A.P.K., xxii., p. 366). The adult form has a chitinous single-chambered shell, and its protoplasm contains a single nucleus and a ring of chromidia. It reproduces itself vegetatively by binary fission, and also by multiple fission producing gamet«s. In the gamete -format ion, according to Schaudinn .(131), the nucleus is ejected from the shell together with all foreign bodies, food-particles, etc. In the shell is left a small quantity of protoplasm containing the chromidia, from which about eight secondary nuclei are formed, and then the protoplasm concentrates round each nucleus and divides tip into as many cells, the gametes, each of which becomes a biflagellate swarm-spore, and is sot free. The gametes copulate and the zygote encysts. In order to develop further, the cyst must be swallowed by a suitable host and pass- through its digestive tract. If this happens, the cyst germinates in the hind-gut, setting^ free an amoebula which forms a shell and becomes a young CMamydophrys, living as a harmless inhabitant of the hind- gut, and feeding on various organisms or waste products occurring there ; but according to Schaudinn it may, under circumstances not yet defined or explained, pass from the digestive tract into the peritoneal cavity, and multiply there as an amoeboid form without a shell, thus giving rise to the organism described by Leyden and Schaudinn, from ascites-fluid, under the name Leydenia gemmipara. The Foraminifera as a group comprise a vast number of genera and species, both recent and fossil, for an account of which the reader must be referred to the larger works. They are classified by Lister (286) into ten orders (suborders ?), containing in all thirty- two families ; Rhumbler (288) recognizes ten families in all. The vast majority are marine, but some of the simpler forms, such as Euglypha, are found in fresh water, and car scarcely be separated from the Lobosa except by the characters of their pseudopodia, a feature upon which great weight cannot be laid as an indication of affinity. Until the life-histories of these simpler forms have been studied, their true systematic position must be considered as some- what uncertain. But the affinities of such genera as Euglypha and Chlamydophrys would seem to be with the Lobosa Testacea rather than with the Foraminifera. III. XENOPHYOPHORA. This group was founded by F. E. Schulze (290) for a number of curious organisms of deep-sea habitat, the zoological position of which was a matter of dispute. By Haeckel they were believed to be sponges allied to Keratosa; such as Spongdhidce, horny sponges which load the spongin-fibres of the skeleton with foreign bodies of various kinds. Schulze established definitely thtir relationship to 238 THE PROTOZOA the Rhizopoda by showing that the soft body was a plasmodium containing numerous nuclei and chromidia, and forming a pseudopodial network, but with no cell-differentiation or tissue- formation. The body consists principally of a network of hollow tubes in which the plasmodium is contained. The wall of the tubes con- sists of. a hyaline organic substance resembling spongin. In the interspaces between the tubes great numbers of foreign bodies (" xenophya," Haecke)) are deposited, such as sand-grains, sponge- spicules, Radiolarian skeletons, and so forth. In one family (Stannomidce) the xenophya are held together by a system of threads, " linellae," in the form of smooth, refringent filaments, approxi- mately cylindrical, which pass from one foreign body to another, and are attached to them by trumpet-like expansions of their ends. The substance of the linellae is doubly ref ractile and allied to spongin in its chemical nature. Schulze compares them to the capillitium of the Mycetozoa (see p. 241, infra). The protoplasmic body within the tubes contains, in addition to nuclei and chromidia, enclosures of various kinds. Many tubes, distinguished by the darker colour of their walls, contain quantities of brown masses, apparently of faecal nature, and comparable to the stercome of the Foraminifera (p. 233). In other tubes, lighter in colour, there are found small, oval, strongly-refractile granules, or " granellae," which consist chiefly of barium sulphate. Schulze terms the system of stercome-containing tubes the " stercomarium," and those that contain granellae the " granellasiurn." The tubes of each system are distinguishable by their mode of branching, as well as by their colour and contents. In the tubes of the granel- larium the protoplasmic bodies are often found to contain isolated cells or groups of cells, each with a single nucleus, which are prob- ably stages in the formation of swarm-spores. Hence the sterco- marium probably represents the purely vegetative part of the body, in which the waste products of metabolism are deposited, while the granellarium is a differentiated region of the plasmodium in which the reproductive elements are produced. Nothing is known of the actual life-cycle of these organisms, but from the appearances already described, seen in preserved speci- mens, Schulze conjectures that they reproduce by formation of swarm-spores, much as is known to take place in the Foram- inifera. The affinities of the Xenophyophora .are seen to be with the Foraminifera. In their habit of forming a skeleton of foreign bodies they resemble the arenaceous Foraminifera, in which, how- ever, the foreign bodies build up the house which directly encloses the soft body, while in the Xenophyophora the soft body is en- THE SARCODINA 239 closed actually within the system of tubes. Nothing similar to the 1'nellae is known in any Foraminifera. For the classification of the Xenophyophora and their genera see Schulze (290). IV. MYCETOZOA. The Mycetozoa are a group of semi-terrestrial Rhizopods occur- ring in various situations, especially on dead wood or decaying vegetable matter of various kinds. Their most characteristic features are the formation of plasmodia, which represent the adult, vegetative phase of the life-history, and their method of repro- duction, consisting in the formation of resistant spores very similar to those of fungi. The Mycetozoa were originally classified amongst the Fungi as a group under the name Myxomycetes, but the in- vestigations of de Bary first made clear their Rhizopod affinities. The life-history of a typical member of this group exhibits a succession of phases, the description of which may conveniently begin with the spore. Each spore is a spherical cell with a single nucleus, enclosed in a tough protective envelope which enables it to resist desiccation. It may be dormant for a considerable period, and germinates when placed in water. The envelope bursts, and the contained cell creeps out as an amoebula with a single nucleus (Fig. 97), the so-called " myxamoeba." After a tune the amcebula develops a fiagellum, and becomes a flagellula or zoospore (" myxoflagellate"), which feeds and multiplies by fission. The flagellula (Fig. 98) retains its amoeboid form, and sometimes also the amoeboid method of locomotion, the fiagellum appearing to act as a tactile organ. It captures bacteria and other organisms by means of its pseudopodia, nourishing itself in a holozoic, perhaps also in a saprophytic, manner. It also may become temporarily encysted. The flagellate phase is succeeded by a second amoeboid stage, the flagellum being lost. The amoebulae of this stage tend to con- gregate together in certain spots, and the groups thus formed fuse together (their nuclei, however, remaining separate) to form the plasmodium, fche dominant vegetative stage, which feeds and grows, its nuclei multiplying as it does so, until from the small mass of protoplasm formed originally by the amoebulae, with relatively few nuclei, it becomes a sheet or network of protoplasm, which may FIG. 97. — The hatching of a spore of Fvligo septica. a, Spore ; b, c, contents emerging and under- going amoeboid movements prior to the assumption of the flagel- lula-stage ; d, flagellula. c.v., Contractile vacuole. After Lister, magnified 1,100. 240 THE PROTOZOA be several inches across and contain many thousands of nuclei. The plasmodium moves about in various directions, showing exquisite streaming movements of the proto- plasmic body (Fig. 99). The nature of the food varies in different species ; the majority feed on dead vegetable matter, but some attack and devour living fungi. The mode of nutrition is generally holozoic, but in some cases perhaps saprophytic. Contractile vacuoles are present in large numbers in the protoplasm, in addition to the innumer- able nuclei, which are all similar and not FIG. 98. — Flagellula of differentiated in any way. The plasmodia Stemonitia fusca, show- are often brightly coloured. From their mode of life, the plasmodia ing successive stages in the capture of a b&cillus. an anterior vacuole. In a it is captured by are naturally liable to desiccation, and when aTthl htdHndTin * *his occurs the plasmodium passes into the it is enclosed in a diges- sclerotial condition, in which the proto- plasm breaks up into numerous cysts, each containing ten to twenty nuclei. When From Lister, magnified moistened, the cysts germinate, the con- tained masses of protoplasm fuse together, and so reconstitute the active plasmodium again. The plasmodium represents the trophic, vegetative phase, which is succeeded by the reproductive phase, apparently in response to external conditions, such as drought, but more es- pecially scarcity of food. The reproduction begins by the plasmodium be- coming concentrated at one or more spots, where the protoplasm aggre- gates and grows up into a lobe or eminence, the beginning of the sporangium (Fig. 100), the capsule in which the spores are found. The sporangium is modelled, as it were, on the soft FlQ- "•— Part of * , pfcwnodium of Badhamia . . ' vtricularis expanded over a slide. Irom protoplasmic body, and Lister, magnified 8 diameters. takes the form of a rounded capsule, attached to the substratum by a disc-like attachment known as the hypothallus. Between the sporangium THE SARCODINA 241 proper and the hypothallus the body may be drawn out into a stalk. The first events in the reproductive process are the formation of the protective and supporting elements of the sporangium. Over the surface of the lobe a membrane or envelope is secreted, the " peridium," and in the interior of the protoplasmic mass a network, or rather feltwork, of filaments, the " capillitium," is produced, of similar nature to the peridium, and in continuity with it ; peridium and capillitium contain cellulose or allied substances and the former may contain carbonate of lime in some species. During the formation of the pro- tective peridium and the supporting capillitium the protoplasmic mass remains in the plasmodial condition, but when the accessory structures are Fio. 100. — Badhamia utricvlaris. completely formed the actual spore- formation begins. According to recent investigations, spore-formation is initi- ated by the degeneration of a certain number of the nuclei ; the nuclei that persist then divide by karyokinesis simultaneously throughout the whole plasmodium. The protoplasm then becomes divided up, directly or indirectly, into as many masses as there are nuclei. The cells thus produced, lying in the interstices of the capillitium, become surrounded each with a tough membrane, and are the spores (Fig. 101). They are liberated by bursting of the peridium, and the hygroscopic properties of the capillitium are the cause of movements in it which assist in scattering the spores. With the formation of the spores the life-cycle has been brought round to the starting-point that was selected. The spores are scattered in all directions by the wind, and germinate in favourable localities. a, Group of sporangia, magni- fied 12 ; b, a cluster of spores ; c, a single spore ; d, part of the capillitium containing lime- granules : b and d magnified 170. From Lister. Fiu. 101. — Trichia varia : part of a section through a sporan- gium after the spores are formed ; threads of the capil- litium are seen in longitudinal and transverse section. From Lister, magnified 650 dia- meters. The account given above may be taken as describing the typical series of events in the life-history, which is liable to considerable variations in particular 16 242 THE PROTOZOA types. In the subdivision termed the Sorophora or Acrasise there is no fiagellula-stage in the life-history, and the amcebuloc which are produced from the spores aggregate together, but form only a pseudo-plasmodium, in which the constituent amoebulae remain distinct, without fusion of their protoplasmic bodies, each amoebula multiplying independently. The details of the reproductive process also vary greatly. In the division known as the Exosporeae, represented by the genus Ceratiomyxa, no sporangium is formed, but the plasmodium grows up into antler-like processes, sporophorea, over the surface of which the plasmodium divides up into a mosaic of cells, each containing a single nucleus of the plasmodium. Each cell becomes a spore, which is produced on the free surface of the sporophore, and drops off when ripe. In the Sorophora the amoebae associated in the pseudo- plasmodium are not all destined to become reproductive individuals ; some of them join together to^ secrete a stalk, and develop no further; others form clusters (" sori ") of naked spores on the stalk. The cytological details of the life-history of the Mycetozoa have been the subject of a series of studies by Jahn, who, however, in his latest investigations, has come to conclusions different from those at which he arrived in his earlier works. According to the earlier accounts given by Jahn and Kranzlin, the spore -formation was preceded by a fusion of nuclei in pairs throughout the sporangium, a process which was regarded as the true sexual karyogamy, and was followed by reducing divisions. According to Jahn's latest investiga- tions (294), however, the nuclear fusions observed in the sporangium take place only between degenerating nuclei, and are to be interpreted as purely vegetative phenomena which have- nothing to do with the true sexual process, which is stated to be as follows : The nuclear division which immediately precedes spore-formation is a reducing division, whereby the number of chromosomes is reduced from sixteen to eight. Consequently the nuclei of the spores, and also the swarm-spores produced from them, both flagellulae and amoebulae, have half the full number of chromosomes. In Physarum didermoides the amoebulae multiply by fission, with mitoses showing eight chromosomes. After a certain number of such divisions, the amoebulae copulate in pairs as gametes. The zygotes thus formed are the foundation of the plasmodJa ; when one zygote meets another it fuses with it, the nuclei remaining separate, and by repeated fusions of this kind the plasmodia are formed. When, on the other hand, a young plasmodium or a zygote meets an amoebula (gamete), it devours and digests it. The nuclei of the plasmodia ^multiply by mitoses which show sixteen chromosomes. In Ceratiomyxa the reduction-division preceding spore-formation is followed by degeneration of one of the two daughter-nuclei ; the other becomes the nucleus of the spore. Within the spore the nucleus divides twice, forming four nuclei, and as soon as the spore germinates the contents divide into four amoebulae, which adhere in the form of a tetrahedron. Each amoebula has eight chromosomes in its nucleus, and divides into two amcebulse, also with eight chromosomes. Each of the amcebulae develops a flagellum and swims off. Possibly in this genus the syngamy takes place between flagellulec. From the investigations of Jahn, it is clear that the swarm-spores of Mycetozoa, like those of other Sarcodina, are the gametes ; their nuclei have undergone a process of reduction, and represent pronuclei, which after a certain number of divisions give rise by syngamy to synkarya, from which the nuclei of the vegetative phase, the plasmodium, takes origin. The Mycetozoa are classified by Lister (297) as follows : SUBORDER I. : EUPLASMODIDA (Myxogastres, Myxomycetes sens, strict.). — Mycetozoa with a flagellula- stage and a true plasmodium formed by plasto- gamic fusion of amoebulae. This suborder comprises forms with the full life- cycle described above. Sectiqn 1. Endosporece. — Spore -formation within a sporangium. Examples : Badhamia, Fuligo (Mthalium), etc. Section 2. Ectosporece. — Spores formed on the exposed surface of sporo- phores. Example : Ceratiomyxa. THE SARCODINA 243 SUBORDER II. : SOEOPHOBA (Acrasiae, Pscudoplasmodida). — With no flagellate stage in the life-history ; the amcebulae do not fuse completely to form a true plasmodium ; the spores are formed in clusters (" sori "). Here belong various genera, for the most part found in dung, such as Dictyostdium and Copromyxa. Acrasis occurs in beer-yeast. In addition to the typical Mycetozoa belonging to these two suborders, there are a number of forms on the border-line, referred by some authorities to the Mycetozoa, by others to other orders, such as the Proteomyxa. It is only possible to refer very briefly to these genera here. In the first place, there are a number of parasitic forms, placed together by DoHein in the suborder Phytomyxinoe, Schroter. In this suborder no sporangium is formed, the process of spore-formation being simplified, probably, in correlation with the parasitic mode of life. The typical members of this group are parasites of plants, but some recently-described parasites of insects have been assigned to Phytomyxinoe. The best known example of the group is the common Plasmodiophora brassicce, which attacks the roots of cabbages and other Cruciferae, producing a disease known as " Fingers and Toes " (" Kohlhernie "), characterized by knotty swellings on the roots. Other genera parasitic on plants are Tetramyxa and Sorosphcera. In Plasmodiophora the spores germinate to produce flagellulae, which are liberated in water or damp earth, and which in some way penetrate into the cells of the plant, and there appear as the myxamoebae after loss of the flagel- lum. The youngest myxamoebae seen have two nuclei. They grow in the cell-contents with multiplication of their nuclei, and fuse with one another to form plasmodial masses which fill the cell after absorption of its contents, In a diseased plant a number of cells are attacked by the parasite, and it is not certain whether the myxamcebae can pass from one cell to another, and so spread the infection, or whether all the infected cells are derived from the multiplication of the first cell infected. The second view, maintained by Nawaschin, is supported by Prowazek, and also by Blomfield and Schwartz, with regard to the allied genus Sorosphcera. When the host-cell is exhausted, the reproductive phase begins, according to Prowazek (127), by the nuclei of the plasmodium throwing out numerous chromidia, and becoming in consequence very indistinct. In Sorosphcera at this stage (Blomfield and Schwartz) the nuclei disappear altogether, being entirely resolved into chromidia from which secondary nuclei are formed. Spore -formation, preceded by sexual processes, takes place in the manner described above (p. 149, Fig. 76). In Sorosphcera, Blomfield and Schwartz found that, after reconstitution of the generative nuclei, the plasmodium divides up into uninucleate cells, each of which divides twice by karyokinesis ; after these divisions the cells become arranged as a hollow sphere, the " soro- sphere," and each cell becomes a spore. No cell-fusions or syngamic processes were observed. As stated above, certain parasites of insects are referred to this order by Leger. Such are the genera Sporomyxa, Leger (295), Mycetosporidium, Leger and Hesse, and Pdtomyces, Leger (C.R.A.S., cxlix., p. 239). Zoomyxa legeri, Elmassian (637), parasite of the tench, is perhaps also to be referred to the Mycetozoa. The position of these forms must, however, be considered somewhat doubtful at present. Chatton has thrown out the suggestion that the affinities of Pdtomyces are rather with the Cnidosporidia (p. 409), through the genus Paramyxo recently found by him (761). Lastly, mention must bo made of the remarkable genera Chlamydomyxa, Archer, and Labyrinthula, Cienkowski, the affinities of which are still obscure. By Lankester (11) they were ranked as an independent order of the Sarcodina under the name Labyrinthulidea ; by Delage and Herouard (6) and others they are placed as a suborder, Filoplasmodida, of the Mycetozoa. Chlamydomyxa is a fresh-water genus occurring either free or encysted. Its most remarkable feature is the possession of chromatophores which enable it to live in a holophytic manner, and consequently to assimilate and grow when encysted. On the other hand, when free it forms a network of long, 244 THE PROTOZOA filamentous pseudopodia, by means of whioh it is able to digest food in the ordinary holozoic manner. The body is a plasmodium containing, in addition to numerous nuclei, chromatophores, and peculiar "oat-shaped bodies," "spindles," or "physodes," stated to consist of phloroglucin. The cyst- cnvtilopo consists of cellulose, and has a stratified structure. In addition to reproduction by fission (plasmotomy), Chlamydomyxa appears to form flagel- late swarm-spores, possibly gametes. Labyrinthula occurs in marine and fresh water. In the active state it has the form of a network of filaments, 1 millimetre or so in extent, over which travel a great number of " units," each a nucleate cell or amcebula, sometimes brightly coloured. When dried, each unit encysts and hatches out again separately. The units multiply by fission. They were formally compared erroneously with the " spindles " of Chlamydomyxa. Lister (298) regards Labyrinthula as a colonial organism of which the units remain in connection by their pseudopodia. He considers these two genera as related in one direction to certain members of the Foraminifera (Gromiidce), in other drections to the Heliozoa and the Proteomyxa. V. HELIOZOA. The Heliozoa are characterized, as a group, by their spherical form and stiff, radiating pseudopodia, whence their popular name of " sun-animalcules." As in the case of the Radiolaria, these peculiarities of form are generally correlated with a floating habit of life, though in a few cases the animal is sedentary and attached to a firm support. In contrast with the Radiolaria, a " central capsule" (p. 250) is absent from the body-structure. A skeleton may be present or absent. The majority of species inhabit fresh water, but a few are marine. General Characters. — As in other orders of Sarcodina, a concise statement of the characteristic features of the group is rendered difficult by the occurrence of border-line forms, of which the exact position is doubtful. It is best, therefore, to consider first typical forms of which the position is incontrovertible, and then those which link the Heliozoa to other groups of Protozoa. The body-protoplasm exhibits commonly a vacuolated, frothy structure, with distinct cortical and medullary regions. The cor- tical zone, distinguished by vacuoles of larger size, disposed in a radiating manner, is regarded as ectoplasm ; the medulla/ry region, with smaller vacuoles irregular in arrangement, as endoplasm ; but it is open to doubt if these two regions correspond truly to the ectoplasm and endoplasm of an amoeba. The cortex contains the contractile vacuoles, and gives off the pseudopodia, which are typically stiff, straight, and filamentous, ending in a sharp point and supported by an axial organic rod (p. 48) ; but in some genera the supporting axis is wanting. In the medulla are lodged the nuclear apparatus, the food- vacuoles, and frequently also symbiotic organisms, which are probably in most cases vegetative, non- flagellate phases of holophytic flagellates (Chlamydo monads). As regards the nuclear apparatus, there are two types of arrange- THE SARCODINA 245 ment (compare p. 90). In the first or Actinophrys-type (Fig. 46) the nucleus is central, and the pseudopodia are centred on it. Actinosphcerium (Fig. 3) can be derived from this type by multi- plication of the nucleus, originally single, until there may be some hundreds present in large specimens. The marine form Campto- nema nutans, Schaudinn, is perhaps also to be referred to this type of structure ; it has as many pseudopodia as there are nuclei present, each pseudopodium arising directly from a nucleus (p. 91, Fig. 47). In the second or Acanthocystis-type (Figs. 18, 64) the centre of the spherical body is occupied by a "central grain" (p. 91), on which the axial rays of the pseudopodia are centred. The nucleus, on the other hand, occupies an excentric position in the body. In this type there is a tendency to a sessile habit of life, the animal being attached by the surface of the body, which may grow out into a stalk, as in Clathrulina (Fig. 19). In the interesting marine genus Wagneretta (Fig. 48), the surface of attachment has become drawn out in such a way that the body is divided into three parts — basal plate, stalk, and head. The nucleus is situated in the basal plate. The head contains the central grain, from which the pseudo- podia radiate. Thus, in this genus the excentric position of the nucleus is carried to an extreme ; it may be regarded as having grown out from the body in a lobe or prolongation which forms the basal plate and stalk, while the original body remains as the head with the central grain and pseudopodia. The skeleton, when present, may take various forms. It may be a simple gelatinous investment, or may contain mineral (sili- ceous) substance either in the form of loose, radiating spicules, as in Acanthocystis, or of a continuous lattice-like investment, as in Clalhrulina. In Wagner ella the basal plate and stalk are protected by a tough yellowish organic membrane, replaced in the head by a colourless gelatinous layer, and both head and stalk are further protected by siliceous spicules, which are formed in the protoplasm and transported by protoplasmic currents (Zuelzer, 86). Life - History. — Reproduction in the free vegetative phase is effected by binary fission or gemmation. Imperfect binary fission may lead to colony -formation, as in Rhaphidiophrys. The sexual phases are only known accurately in a few cases. In Actinophrys, Schaudinn described copulation within a cyst (p. 132, Fig. 71), with subse- quent division of the zygote and liberation of two individuals from the cyst. In Actinosphcerium (Hertwig), encystment of a large multinucleate individual is followed by degeneration of about 95 per cent, of the nuclei ; the remainder appear to fuse in pairs, and the body then divides into as many cells as there are nuclei. Round each cell a separate " prinfary " cyst is secreted within the gelatinous " mother-cyst " originally formed round the whole mass. 246 THE PROTOZOA Each primary cyst then divides into two secondary cysts, which after nuclear .reduction become the gametes and copulate. The zygote develops into a young Actinosphcerium with several nuclei, which emerges from the cyst and begins a vegetative life, but appears to divide frequently at the start into uninucTeate, Actinopkrys-like forms. In other genera, on the other hand, and especially in those of the Acant hocystis - type (Acanthocystis, Clathrulina, and Wagnerella), flagellate swarm - spores are formed, which probably represent gametes, as in many other Sarcodina. The life-history of Wagnerella has recently been studied in detail by Zuelzer (86) ; her investigations reveal a diversity in its modes of reproduction almost as great as that seen in Arcella, and indicate that there is much yet to be discovered with regard to the life-cycles of other forms. Wagnerella exhibits, according to Zuelzer, dimorphism correlated with alternation of generations. In June and July stout forms are observed, which are believed to arise from the conjugation of gametes ; they reproduce by binary fission, and by a process of schizogony giving rjse to amoabulse (agametes). The more usual form, on the other hand, is smaller and more slender, and multiplies by binary fission, gemmation, and formation of flagellate swarm-spores. Hence this peculiar form reproduces in a variety of ways. In the process of binary fission the nucleus migrates from "the. base up the stalk into the head, and places itself beside the central grain, which divides, its two halves passing to opposite sides of the nucleus ; then the nucleus follows suit and divides also. Divisions of tfie central grains, and subsequently of the corresponding nuclei, may be repeated until eight to ten nuclei and as many central grains are present. Each nuclear division is followed by division of the head, at first incomplete, so that a condition results resembling the colonial form Rhaphidiophrys, a number of daughter- individuals united together, and each sending out pseudopodia (Fig. 102, D). After a time the colony breaks up, the daughter-individuals separate,, and each one fixes itself and grows into the adult Wagner 'etta-lorm. Bud-formation in Wagnerella (Fig. 102, A — C) is initiated by division of the karyosome within the nucleus, which retains its position in the base. The process is repeated until the nucleus contains a number of karyosomes, each with a centriole. The nucleus then buds off one or more small daughter- nuclei, each containing a single karyosome. Sometimes the nucleus breaks up entirely into as many daughter- nuclei as there are karyosomes, in which case the parent-individual dies off, in a manner similar to Arcella (p. 180), after liberation of the buds. Each daughter-nucleus migrates up the stalk into the head, where it becomes surrounded by a layer of protoplasm to form the bud, which is set free at first as an amoeboid body. Before or after being set free, the bud may multiply by binary fission with mitosis, in which the centriole in the karyosome acts as a controsome. Finally each amoeboid body develops into a Wagnerella, and in the process the centriole passes out of the nucleus and becomes the central grain, while the nucleus becomes displaeed from the centre. In the process of gemmation the central grain of the parent-individual takes no share whatever. In the formation of the swarm-spores, minute secondary nuclei arise from chromidia near the principal nucleus in the base. Each secondary nucleus forms a centriole and divides by mitosis ; the division is repeated until the whole body, stalk and head as well as base, is filled with small nuclei, while the primary nucleus degenerates. The body then divides up into as many cells as there are secondary nuclei, each cell becoming a bifiagellate swarm- spore which is set free, while the parent-individual degenerates. The destiny of the swarm -spores is uncertain, but they are believed to be gametes. THE SARCODINA 247 In the " schizogony " of the stout forms the nucleus breaks up into a number of daughter-nuclei, as in gemmation ; each daughter-nucleus grows, its karyo- some multiplies by fission, and it breaks up in its turn into granddaughter- nuclei. Continued multiplication of the nuclei in this manner proceeds until the body is filled with vesicular nuclei ; it then breaks up into as many amo3bulae, which are set free, leaving a residual body with the central grain, which degenerates. F(O. 102. — Wagnerella borealia, showing budding and fission. A, Specimen with a single bud (b) : e.g., central grain ; B, specimen with four buds (b) ; C, en- larged view of the head of a speoimen containing two buds (6) in process of extrusion ; D, speoimen in which the head has multiplied by fission to produce a Phaphidiaphrys-like colony ; six individuals are seen, five of them each with nucleus and central grain, the sixth in process of fission, with two nuclei and two central grains. After Zuelzer (86). The Heliozoa are classified into four suborders : SUBORDER I. : APHROTHORACA. — Body naked in the active state ; envelopes, sometimes with siliceous spicules, only formed during 248 THE PROTOZOA encystment. Examples : Actinophrys (Fig. 46), Actinosphcerium (Fig. 3), Camptonema (Fig. 47), etc. SUBORDER II. : CHLAMYDOPHORA. — Body protected by a soft gela- tinous envelope, but without solid skeletal elements. Example : Astrodisculus. SUBORDER III. : CHALAROTHORACA. — Body invested by a soft envelope containing isolated spicules, usually siliceous, sometimes chitinous. Examples : Acanthocystis (Figs. 18, 64, 68), Wagneretta (Figs. 48, 102), Heierophrys (Fig. 103). SUBORDER IV. : DESMOTHORACA. — Body invested by a continuous, lattice-like skeleton. Example : Clathrulina (Fig. 19). ^o. 103. — Helerophrya fockei, Archer, c., c., Contractile vacuoles ; «., radial ohiti- nous spines surrounding the envelope. A nucleus is present in the body, but is not shown ; the bodies in the protoplasm represent zooxanthella?. From Weldon and Hickson, after Hertwig and Lesser. A certain number of genera must be mentioned which are of doubtful position, referred by some authorities to the Heliozoa, by others to other orders. Some of these genera perhaps do not represent independent, " adult " forms, but may be only developmental phases of other genera. Nudearia, classed by some in the Aphrothoraca, by others in the Proteomyxa, has an amoeboid body and pseudopodia without axes. As described above (p. 177 and Pig. 80), a Nuclearia-stage occurs in the development of Arcella. Especially remarkable are certain genera which indicate a close relation- ship between Heliozoa and Flagellata. An account of several such forms is given by P&aard (302), in addition to which the following may be noted : Ciliophrys, Cienkowski, has two phases ; in the one it appears as a typical Heliozoon with stiff radiating pseudopodia; in the other it is a typical flagellate. In the process of transformation the Heliozoon-form retracts its pseudopodia, its body becomes amoeboid, and a flagellum grows out ; finally THE SARCODINA 249 the animal becomes a pear-shaped flagellate swimming by means of its flagellum (Schewiakoff, 863 ; Caullery, 300). CUiophrys thus recalls Pseudo- . spora in its two pnases (p. 218), and there can be little doubt that the two forms are closely allied. Dimorpha nutans, Gruber(Fig. 104), has radiating pseudopodia strengthened by axial rods, and hi addition a pair of flagella arising close together at one pole of the body. Both flagella and pseudopodia arise from a centrosome situated near the flagellated pole ; the single nucleus is also excentric and placed close beside the centrosome. The animal uses one of its flagella for attach- ment, while the other remains free (Schouteden). These facts appear to indicate an origin for the Heliozoa from Flagellates such as those of the genus Midticttia (p. 270, Fig. 113), in which the body bears radiating flagella planted evenly over the surface ; transformation of the flagella into stiff pseudopodia would produce the Heliozoon - type of organism. On such a view two peculiarities of the Heliozoan pseudopodia receive explanation : the power of nutation and bending which they fro- Fio. 104. — D\morpha nutans. After Schouteden. quently possess ; and their insertion on a " central grain," which would then represent the blepharoplast, pure and simple, of a flagellate. On this view the pseudopodia of the Heliozoa would appear to be structures quite different in nature from the similarly-named organs of Lobosa. On the other hand the Heliozoa also show affinities towards forms classed among the Reticulosa or " Proteomyxa," as already noted in the case of CUiophrys and Pseudospora. Przesmycki has described a species, Endophrya rotatorium, parasitic in Rotifers, which he considers as a connecting-link between Nudearia and Vampyrella. The exact systematic position of such genera must bo considered at present an open question. VI. RADIOLABIA. General CJiaracters. — The Radiolaria are characterized, speaking generally, by the same type of form and symmetry that is so marked a feature of the Heliozoa, though in many cases the internal 260 THE PROTOZOA structure of the body, and especially the skeleton, may depart more or less widely from the radiate symmetry which is to be regarded, probably, as primitive for the group. Hence three principal types of symmetry can be distinguished in these organisms : (1) Homaxon (Figs. 13, 105, 107), in which all axes passing through the centre are morphologically equivalent, the symmetry of the sphere ; (2) monaxon (Fig. 109), in which the body has a principal or vertical axis round which it is radially symmetrical, the type of symmetry of the cone ; (3) bilaterally symmetrical (Fig. 106), in which the body r / _ FIG. 105. — Acanthometra dastica, Haeckel. sp., Radiating spines of the skeleton (twenty in number, but only twelve are seen in the figure) ; pa., pseudopodic ; c., calymma ; c.c., central capsule ; N , N., nuclei ; x, yellow cells ; my., myo- phrisks. After Biitschli, Leuckart and Nitsche's " Zoologisohe Wandtafeln." can be divided along a principal plane into equivalent right and left halves. With further modification the body may become asymmetrical. Sedentary forms are not known in this group, the species of which are exclusively marine, and occur on the open surfaces of seas and oceans, reaching in many instances a re atively large size and a very high degree of structural differentiation. In the internal structure, the most salient feature is the division of the body by means of a membranous structure, termed the central capsule (Fig. 13, CK), into a central medullary region and a peripheral cortical zone — hence distinguished as the intracapsular THE SARCODINA 261 and extracapsular regions of the body. The intracapsular medulla contains the nucleus or nuclei, and is the seat of reproductive processes. The extracapsular cortex is the seat of assimilation, excretion, food-capture, and of such locomotor processes as these organisms are able to perform, consisting chiefly of rising or sinking in the water by means of changes in a hydrostatic apparatus presently to be described. The Radiolaria are an exceedingly abundant group represented by a great number of species both at the present time and in past ages ; over vast tracts of the ocean-floor their skeletons are the principal, almost the able constituents of the ooze ; and the same must have been true in past times, since in many geological deposits the rocks are composed of the same materials. Every microscopist is familiar with their skeletons, which on account of their beauty and variety of form are favourite objects for microscopic study and demonstration. Corresponding with the variety of forms and species, the internal structure shows a range of variation and differentiation which it is impossible to deal with adequately in a short space ; it must suffice, therefore, to describe here the main structural peculiarities of this group in a general manner, and to indicate briefly the principal variations of structure which are of importance for the classification of the group For further information the reader must be referred to the larger treatises and special monographs. Structure. — Tne central capsule, absent in rare cases, may be a thin, delicate structure, visible only after treatment with reagents, or may be fairly thick. In homaxon forms it is generally spherical, but may assume various shapes correlated with the general body- form, and even may be lobed or branched. It is perforated by openings which place the intracapsular protoplasm in communica- tion with the extracapsular ; the openings may take the form of fine pores scattered evenly over the whole surface (Peripylaria) ; of similar pores aggregated into localized patches, pore-areas or pore- plates (Acantharia) ; of a single pore-plate at one pole of an asym- metrical capsule (Monopylaria, Fig. 106) ; or of one principal and two lateral apertures (Tripylaria). The intracapsular protoplasm contains the nuclear apparatus, either one nucleus of very large size or a number of smaller nuclei (Fig. 105). In addition, various bodies of metaplastic nature, serving as reserve-material for the reproductive processes, are found in this region, in the form of fat-globules, oil-drops, concre- tions, crystals, etc. The extracapsular region consists of three zones, from within outwards : (1) an assimilative layer or matrix immediately sur- rounding the capsule ; (2) a vacuolated layer, known as the "cal- ymma," hydrostatic - in function; (3) a protoplasmic layer from which the pseudopodia arise. 1. The assimilative layer contains pigment, representing ex- cretory substances and ingested food-material in the shape of small 252 THE PROTOZOA organisms captured by the pseudopodia and passed into the body, to be digested in this region. In the Tripylaria an aggregation of food-material and excretory substances produces a characteristic greenish or brownish mass concentrated round the main aperture of the central capsule, and known as the phceodium, whence this suborder is sometimes known as the Phseodaria. 2. The calymma is composed for the most part of a great number of vacuoles containing fluid, the function of which is hydrostatic ; the contents of the vacuoles are stated to be water saturated with carbon dioxide, causing the animal to float at the surface, and enabling it to regulate its position in relation to conditions of environ- ment. In rough weather the vacuoles burst or are expelled from the body, and the animal sinks into deeper and quieter layers of water; there fresli vacuoles are formed, enabling it to return again to the surface if the conditions are favourable. Contractile vacuoles of the ordinary type are not present. In addition to the vacuoles, the calymma contains numerous " yellow cells," generally regarded as sym- biotic organisms of vegetable nature, and named " zooxanthellae " or Hertwig, showing a bilaterally « ZOOchloreUae," according to their symmetrical skeleton consisting of a simple siliceous ring pro- colour. Absent in the Tripylaria, longed into spicular processes, these yellow cells are found, as a ak., Skeleton ; c.c., central cap- , , , sule ; pf., pore-area, surmounted ru*6* m the calymma, but m by a conical structure (c.), the Acantharia they occur in the intra- %*&£?T^3S^ caPsular P^toplasm (Fig. 105, x). After Butsohli, Leuckart and The nature of the yellow cells of Nitsche's "Zoologische Wand- Acantharia has been much disputed, tafeln. . r ' and many observers have regarded them as an integral part of the organism itself ; this view has recently been revived by Moroff and Stiasny, who bring forward evidence to prove that the yellow cells of Acantharia are a developmental phase of the organism. Still more recently this view has been extended by Stiasny to the colony-forming Sphaerozoa in the first place, and then to Badiolaria generally. The difficulty in the way of such an interpretation which arises from the co-existence, in Zhalassicolla and other genera, of yellow cells in the calymma, with an undivided nucleus in the host- organism, is met by supposing -that in such cases developmental FIG. 106. — Lithocircua productus, THE SARCODINA 253 stages of other Radiolarians have penetrated into the calymma, and live there symbiotically — a supposition which is certainly in need of further proof bsfore it can be accepted. 3. The most external layer of the body is a protoplasmic envelope from which the pseudopodia radiate. In Radiolaria, speaking generally, the pseudopodia are straight, slender, and filamentous, composed of motile protoplasm entirely (" myxopodia ") ; but in Acantharia some of the pseudopodia are, like those of Heliozoa, axopodia supported by stiff axial rods of organic substance, which originate deep within the central capsule and pass through the calymma along the axis of the pseudopodium, but without reaching as far as its distal extremity. In some Acantharia (Acanthometrida) are found also peculiar modifications of the bases of certain of the pseudopodia in the form of groups of rod-like bodies, " myonemes " or " myophrisks " (Fig. 105, my.), clustered round each of the spicules of the skeleton. As their name implies, the myonemes are contractile elements which, by their contraction or expansion, alter the hydrostatic balance of the organism, and enable it to rise or sink in the water. According to Moroff and Stiasny, the myonemes are formed in the interior of the central capsule, and are derived from nuclei. In a certain number of Radiolaria a skeleton is absent altogether. The Acantharia have a skeleton composed of a substance which was formerly supposed to be of organic nature, and was termed acanthin by Haeckel, but which consists of strontium sulphate according to Biitschli (310). In other Radiolaria the skeleton, when present, is siliceous. In Acantharia the skeleton invades the intracapsular region, and consists typically of a system of twenty spines or spicules radiating from the centre of the body (Fig. 105). It is a simple and enticing view to regard such a skeleton as origin- ating phylogenetically from a modification of the axis of pseudo- podia. Union of outgrowths from radially-directed spicules gives rise to a lattice-work forming a spherical perforated shell, and as the animal grows in size several such concentric spheres may be formed, one within the other, supported by radial bars which represent the original radiating spicules (Fig. 107). In Radiolaria other than Acantharia the skeleton is usually entirely extracapsular, and exhibits a variety of form and structure which cannot be dis- cussed further here. In some of the Tripylara foreign bodies are utilized for building up the skeleton, either to form the basis of spines secreted by the animal or to construct a coat of armour on the exterior of the body (Borgert). Life-History. — Reproduction of the Radiolaria is effected in some instances by binary fission — namely, in those forms in which a skeleton is lacking or consists of loose spicules. The nucleus 254 THE PROTOZOA divides by a mitosis remarkable for the vast number of chromo- somes, of which there may be over a thousand, and the apparent absence of a centrosome. The more usual method of reproduction, however, is formation of flagellated swarm-spores by a process of rapid multiple fission within the central capsule. Two kinds of swarm-spores are produced, which are known respectively as " isospores " and " anisospores." The isospores (Fig. 108, A), which are probably agametes, are all similar in size and appearance, and frequently contain a crystal in their protoplasm, and are hence sometimes termed " crystal-spores." The anisospores (Fig. 108, SO. Fio. 107. — Actinomma asteracanthion : 3emi-diagrammatic to show the mode of growth of the skeleton. S.1, S.2, S.3, Three concentric lattice- work shells, connected by sp., radial bars which are prolonged beyond the outermost shell as spikes ; N., nucleus ; c.c., central capsule ; pa., pscudopodia. After Butschli, Leuekart and Nitsche's " Zoologische Wandtafeln." B, C), probably gametes, are of two kinds, smaller microspores and larger macrospores ; they differ in structure from the isospores, and lack the characteristic crystal. The swarm-spores vary in struc- ture in different species, but usually have two flagella. Isospores and anisospores are formed in different individuals, but it is still a moot point whether an alternation of generations occurs. Micro- spores and macrospores may be formed in the same individual in some species ; in others they are produced by different individuals. Previous to formation of the swarm-spores the extracapsular region of the body disintegrates, and the central capsule with its contents THE SARCODINA 255 sinks to a considerable depth. The swarm-spores are liberated by the breaking-up of the central capsule. The subsequent develop- ment of the swarm-spores when set free has not been made out. While the main features of the reproductive process are as stated above, the cytological details of the formation of the swarm-spores is still a matter of dispute. The subject is dealt with in the recent memoirs of Moroff on the one part, and Hartmann and Hammer, Harlmann (60), and Huth, on the other. The formation of the anisospores is generally regarded as a breaking- up of the primary nucleus into chromidia, from a part of which the secondary nuclei arise, which become those of the swarm-spores (compare Foraminifera). But according to Hartmann and his adherents, the huge primary nuclei seen in many Radiolaria are polyenergid nuclei or polykarya (p. 121) containing a vast number of nuclear energids ormonokarya, Consisting each of chromatin, in the form of a twisted thread or so-called " chromosome," and a centriole. In the gamete-formation a great number of such monokarya are set free from the primary nucleus to become the gamete-nuclei ; hence the so-called " generative chromidia " set free from the nucleus are interpreted as secondary nuclei or monokarya already formed within the primary nucleus. A similar interpretation is given to the mitosis seen in the process of binary fission ; the huge mitotic figure, composed of mor-e than a thousand chromosomes, is interpreted as being in reality made up of as many mitotic figures as there are chromosomes, since each so-called " chromosome " is regarded'as a single Fio. 108. — Swarm-spores of Cottozoum inerme. A, Crystal-bearing swarm-spores, agametes ; B, C, swarm-spores without crystals, gametes ; B, microspores (microgametes) ; C, macrospores (macrogametes). After Hertwig. nuclear energid or monokaryon with its own centriole, the whole number of energids dividing independently but synchronously to form the supposed mitotic figure. According to Moroff and Stiasny, in Acanihometra pellucida a process of multiplication is proceeding continually within the central capsule, until it is entirely filled up with cells, from which the swarm-spores arise. In this multipli cation, termed by the authors " schizogony," trophic nuclei (" macro- nuclei ") and generative nuclei (" micronuclei ") are formed. The trophic nuclei are the " yellow cells," which ultimately degenerate. Hence the Acan- tharia are considered not to be single individuals, but colonies of animals which have the extracapsular protoplasm, pseudopodia and skeleton in common. Finally, attention must be drawn to the peculiar organisms found in certain Radiolaria, and regarded by some authorities as parasitic Flagellata (Silico- flagellata, Borgert), by others as developmental stages, of the Radiolaria themselves. See Delage and Herouard (6, p. 371). The Radiolaria are classified as follows : SUBORDER I. : PERIPYIARIA SEU SPUMELLARIA. — Central capsule spherical, perforated by evenly-distributed pores. Extracapsular region well developed. Skeleton wanting or consisting of scattered spicules or of lattice-work shells, developed in the extracapsular region, siliceous. Legion 1 : Collodaria.— Skeleton wanting or simple in structure ; monozoic forms. Five families. Examples : Thalassicolla (Fig. 13), Thalassophysa. Legion 2: Sphcerdlaria. — Skeleton complex, usually with lattice-work ; monozoic, generally small. Four families. 256 THE PROTOZOA Legion 3 : Sphcerozoa sen Polycyttaria. — Colonial forms consisting of numerous individuals embedded in a common jelly ; their central capsules are distinct, but their extracapsular regions anastomose. The colonies reach a length of several centimetres. Two families. Example : Collozoum. SUBORDER II. : ACANTHARIA. — Skeleton composed of strontium sulphate, typically in the form of spicules radiating from the centre of the body, within the central capsule ; in addition lattice-work shells may be developed. Central capsule with pores evenly developed, or grouped in areas. A number of families are recognized, grouped in different ways by different authorities. Example : Acanthometra (Fig. 105). \ Fia. 109. — Eucyrtidium cranioides, Haeckel : entire animal as seen in the living condition. The central capsule is hidden by the beehive- shaped siliceous shell within which it is lodged. From Gamble, magnified 150. I. : MONOPYLARIA SETT NASSELLAKIA. — Central capsule monaxon in form, with the pores aggregated at one pole into a pore-plate, and the walls of the pores thickened to form a conical structure directed inwards into the central capsule. Several families. Examples : Lithocircus (Fig. 106), Eucyrtidium (Fig. 109). SUBORDER IV.: TRIPYLARIA SEU I'H^ODARIA.— Central capsule with a principal aperture (astropyle) and two accessory apertures (parapyle). A mass of pigment (phseodium, p 252) surrounds the principal aperture. Divided by Hacker into six legions and numerous families. Example : Aulacantha. Bibliography. — For references see p. 483. CHAPTER XII SYSTEMATIC REVIEW OF THE PROTOZOA : THE MASTIGOPHORA THE distinctive feature of the class Mastigophora is the possession of one or more flagella as organs of locomotion and food-capture, not merely during early stages of development, but in the active phases of the adult organism also. In other classes, as has been pointed out in a previous chapter, flagella may be present in the young stages, but are absent in the adult phases. In the Masti- gophora a flagellum is a permanent feature of the organization, though even in this class it may be temporarily lost, either in active phases, when the animal may become amoeba-like, or in resting phases, especially in parasitic forms of intracellular habitat. The Mastigophora are divided into three subclasses, of which the first, the Flagellata, contains the more typical forms, and con- stitutes the nucleus, so to speak, of the class ; while the two remain- ing subclasses, the Dinoflagellata and Cystoflagellata, may be regarded as specialized offshoots of the primitive flagellate stem. It is convenient, therefore, to deal with the Flagellata in a general manner first, and then to describe the special features of the other two subclasses. SUBCLASS I. : FLAGELLATA (EUFLAGELLATA). General Characters. — The members of this group are for the most part of minute size, and seldom attain to considerable dimensions ; forms of relatively large size, such as the species of Euglena and allied genera, are small as compared with the larger species of the Sarcodina and other classes. As a rule the Flagellata are free- swimming organisms ; a certain number, however, are sedentary in habit, attaching themselves to a firm basis, and using their flagella for food-capture alone. There is a great tendency to colony- formation in this group. In the process of multiplication by fission of the ordinary type, separation between the daughter-individuals may be incomplete, so that they remain connected together, either by means of a common envelope, house, or gelatinous matrix, or by organic, protoplasmic union, or in both ways. Repeated fission 257 17 258 THE PROTOZOA of this kind leads to the formation of a colony which may attain, to dimensions relatively large, though composed of individuals of minute size. The colony may be free-swimming or fixed, and in the latter case is frequently arborescent in form. In many cases the colonies of Flagellata show a differentiation of the constituent individuals into vegetative and generative individuals — the former not capable of reproduction, but purely trophic in function ; the latter destined to be set free, and to produce new colonies, with or without going through a process of syngamy. Bionomics. — In their modes of life the Flagellata exhibit all the four types described in Chapter II. (p. 13), different forms being holozoic, holophytic, saprophytic, or parasitic ; and one and the same form may live in different ways during different periods of its life-history, according to circumstances. The parasitic flagellates have attracted a great deal of attention of recent years, on account of their importance in causing disease in man and animals. Ectozoic parasites may occur in aquatic forms, as for example Costia, para- sitic on the skin of fishes. The entozoic forms are parasitic for the most part in the digestive tract, or in the blood and lymph or their host*. Parasitic flagellates are found in the intestines of practically all classes of the Metazoa, and especially in arthropods and vertebrates ; those parasitic in blood and lymph are found especially in vertebrates, and constitute an important group commonly termed as a whole the Hsemoflagellates, to which a special chapter will be devoted. From forms which were probably parasitic originally in the blood have arisen secondarily forms parasitic in cells which in their intracellular phase lose their flagellum entirely (Leishmania). Many of the intestinal flagellates, especially in vertebrates, are probably not true parasites at all, but for the most part scavengers. In any case their pathogenic role appears to be very limited ; but in some cases a pathological condition of the host may be combined in a suspicious manner with great numbers of the parasites (compare Bohnc and Frowazek, Noc). It is worthy of note that in some cases an intestinal parasite may pass from the intestine into the blood or lymph under pathological conditions of the host. This condition seems to have been noticed •first by Panilewsky, who described cases of frogs and tortoises which had been kept long in captivity and were in bad condition, thin, and with cedematous swellings in the muscles and transudation of lymph into the peritoneal cavity; in such animals there were found in the blood and lymph, especially in the oedemata and tra.ns- udations, abundant flagellates of the genus Hexcuniius ( — Octomitus, Fig. 116), of a species which in normal, healthy animals is found only in the intestine. A number of similar cases have been recorded by Plimmer (383, and Presi- dential Address to the Royal Microscopical Society, 1912), who found both Octomitus and Trichomoncts in the blood of various batrachia and reptiles. The conditions under which these intestinal parasites pass into the blood appears to be strictly comparable to those under which the Leydenia-lonn. of Chlamydophrya passes into the ascitic fluid (p. 237). Whether In such oases the migration of the parasite is the cause of the diseased state of the host, or whether, as seems more Hkely, the abnormal condition of the host gives the parasite an opportunity of spreading into fresh pastures, must remain for the present an open question ; but, according to Plimmer, the Sresence of intestinal flagellates in the blood-circulation is associated with efinite and recognizable lesions of the intestinal wall. In any case, the fact that intestinal flagellates can pass into the blood is a point which is probably of phylogenetic as well as of practical importance (p. 322). THE MASTIGOPHORA 259 Structure. — The body-form is of three principal types : (1) An envelope or tough cortex may be entirely absent, and the body is then amoeboid, as in the Rhizomastigina (Figs. 38, 40) ; (2) a thin cuticle may be present, insufficiently rigid to inhibit changes of body-form due to contractility of the living substance (Fig. 15) ; (3) a thicker cuticle necessitates a constant body-form, which is either rigid and unalterable or sinuous and permitting movements of flexion and torsion. In the second type are comprised forms termed commonly " metabolic," on account of the changes of form they exhibit ; contractions of the superficial layer of the body pass, as it were, in waves from the anterior to the posterior end of the body, in a manner similar to the peristaltic contractions of the intestine, producing rhythmic form-changes in the body. In species in which the cuticle is thin or absent, a constant body- form may nevertheless be maintained by internal form-giving organs, such as the axostyle of Trichomonas (Fig. 5), LopTtomonas (Fig. 45), etc. True internal skeletons, however, do not occur. An external shell or house may be present, enclosing the whole body. The protoplasmic body shows, in the amoeboid forms sdch as the Rhizomastigina (p. 268), distinct ectoplasm and endoplasm. But as a general rule the thin ectoplasm is converted into a firm cuticle, or periplast, enclosing the body and containing contractile elements — myonemes. Hence the ectoplasm appears at first sight to be absent, and the protoplasmic body to consist of endoplasm alone. In larger forms the myonemes can be made visible by suitable treatment (Fig. 28), but as a general rule in such minute organisms the existence of myonemes or other contractile mechan- isms can only be inferred from the movements of contractility or flexibility which the body exhibits. The flagella may perform various functions in different cases ; they may serve as organs of locomotion and of food-capture, as organs of temporary attachment, and as tactile organs. As stated above (p. 52), they may be distinguished by their relation to the progression of the organism, af tractella, anterior, and pulsella, posterior in movement. The flagella vary in number and in arrange- ment in different species, and for the different types of the flageUar apparatus a number of technical terms are in use : monomastigote, with a single flagellum (Fig. 38) ; isomastigote, with two or four flagella of equal length (Fig. 43) ; paramastigote, with one long principal flagellum and a short accessory flagellum (Fig. 15) ; Tieteromastigote, with one or more anterior flagella directed forwards, and a " trailing flagellum " directed backwards (Figs. 5, 25) ; polymastigote, with a tuft of flagella (Fig. 45) ; and holomastigote, with numerous flagella scattered evenly over the body (Fig. 113). Of these various types of arrangement, the heteromastigote con- 260 THE PROTOZOA dition, with a backwardly-directed trailing flagellum (" Schlepp- geissel "), deserves special attention, since by attachment of the trailing flagellum to the body an undulating membrane (p. 56) may arise ; and that it has actually so arisen in some cases is indicated by the existence of pairs of similar forms, in which a Fio. 110. — Codonoaiga botrytis. A, Young specimens attached singly to the stalk of a Vortioella ; B, colony of six individuals on a common stalk ; C, stalked individual which has recently divided into two, pro- ducing a dichotomous division of the stalk, c.v., Contractile vacuole. After Stein. trailing flagellum, free from the body, in the one form is represented by the marginal flagellum of an undulating membrane in the other — as, for exaxtrple, Trichomastix and Trichomonas (Fig. 5) , Prowazekia (Fig. 141), and Trypanoplasma (Fig. 36). tn one group of flagellates — hence known as the Choanoflagellata THE MASTIGOPHORA 261 or Craspedomonads (Fig. 110) — a peculiar structure occurs, known as the " collar," a delicate protoplasmic tube or funnel which arises along a circular base-line of which the insertion of the flagellum is the centre, and so forms a cup, sleeve, or collar-like structure surrounding the flagellum for about a third or a half of its length. It is stated, both for Choanoflagellates and for the very similar collar-cells of sponges, that the collar is a membrane folded in a spiral manner, its insertion running along the body and round the base of the flagellum ; but the spiral structure is not easy to make out. The Choanoflagellates are sedentary forms which, if set free temporarily from their attachment, swim with the flagellum directed backwards, doubtless the mechanical result of the presence of the collar. The function of the collar is probably connected with the capture and absorption of food-particles wafted towards the body by the flagellum. The collar is retractile, but is not capable of active movements such as are seen in an undulating membrane. The organs of nutrition must be considered in connection with the four modes of life already mentioned. (a) In holozoic forms the organism captures and ingests other organisms of various kinds. In some forms the ingestion of food- particles may take place at any point on the body surface ; examples of this are the amoeboid forms, such as Mastigamasba, which capture their food by means of their pseudopodia, like an amoeba ; the holo- mastigote genus Multicilia (Fig. 113) ; the parasitic Lophomonas (Fig. 45), and possibly others. But in most cases food-particles are ingested at the base of the flagellum, the spot towards which they are propelled by the activity of the flagellum itself. There may, however, be no special aperture for food-ingestion, particles which impinge upon the soft protoplasmic body being simply absorbed directly with formation of a food-vacuole. With a more advanced type of organization, a special aperture or cytostome for the ingestion of food-particles is found at the base of the flagellum. The cytostome may be a simple aperture leading through the cuticle directly, or by means of a funnel-shaped depression, into the proto- plasmic body, or it may, in more highly organized forms, lead into a special tube, termed an " oesophagus " or " cytopharynx," which receives the evacuations of the contractile vacuoles, and serves for excretion as well as ingestion (Fig. 84). In any case the oesophagus ends blindly in the fluid endoplasm. There is no special anal aper- ture for expulsion of faecal material, which is expelled at any point of the body-surface in primitive forms, or through the oesophagus and cytostome in those more highly organized. (6) In holophytic forms the organs of nutrition are those of the plant-cell (p. 188) — namely, chromatophores, or corpuscles con tam- ing chlorophyll or allied pigments ; pyrenoids, small glistening bodies 262 THE PROTOZOA embedded in the chromatophores, the centres of the formation of amyloid substances ; and grains of amyloid nature formed by the constructive metabolism of the organism. It is also common to find in the holophytic flagellates a peculiar red spot, or stigma, placed near the anterior end of the body, and probably sensitive to light (p. 205). In general, two types of holophytic flagellates can be recognized : first, forms in which, in addition to the organs already mentioned, those pertaining to the holozoic mode of nutrition are also present ; secondly those possessing only the holophytic apparatus. The first type may be regarded as more primitive forms in which the holophytic habit of life has not become so engrained as to exclude any other mode of nutrition ; but a change is still possible, and the organism can combine or vary the holophytic with the holozoic or saprophytic method. In the second type the organism has be- come plant-like, to the complete exclusion of other methods of nutrition ; the body is generally enclosed completely in a firm cellu- lose envelope, allowing diffusion of liquids and gases, but without apertures through which foreign bodies can pass into the interior. Such forms, if they lose their flagellum in the adult state, are classed as unicellular Algae, and the young flagellated individuals are termed " zoospores." The transition from holophytic flagellates to plants is a gradual one, and the border-line is simply fixed by the characters of the " adult," and is therefore as arbitrary as that between Sar- codina and Mastigophora discussed in a previous chapter. (c) In saprophytic and parasitic forms no special organs of nutri- tion are present, since the food is absorbed in a fluid condition from the surrounding medium. Contractile vacuoles are commonly present in those flagellates which inhabit fresh water. In the more primitive forms the vacuoles empty themselves direct to the exterior. In more highly organized types the vacuoles open into the oesophagus. In Euglena the two contractile vacuoles open into a reservoir- vacuole, which, according to Wager (213), is in open communication with the oesophagus (Fig. 84). The nuclear apparatus consists, as a rule, of a single nucleus of vesicular type, with a distinct karyosome. Chromidia are generally absent, but are found in a few cases (Rhizomastigina). The relations of the nuclear apparatus and the flagella have been discussed above, and are briefly as follows : 1. There is a single nucleus with a single centriole, which functions at the same time as centrosome and blepharoplast. Then either (a) the centriole is within, or connected intimately with, the nucleus, in which case the fla- gellum appears to arise directly from the nucleus, as in Mastigina (Fig. 38) ; or (b) the centriole, and the flagellum it gives off, are quite independent of the nucleus, as in Mastigetta (Fig. 40). THE MASTIGOPHORA 263 2. There is a single nucleus with its centrpsome, and in addition one or more blepharoplasts in relation to the flagellar apparatus. Then (a) at division the old blepharoplasts and flagella are lost, and new blepharoplasts arise during or after nuclear division from the centrosomes ; or (6) the blepharo- plasts and flagella persist, and the former divide independently to form daughter- blepharoplasts from which new flagella arise (Fig. 43). 3. In a certain number of Flagellata, grouped provisionally as Haemo- flagellates or Binucleata (see next chapter), two nuclei, each probably possess- ing its own centrosome, are present : a principal or trophic nucleus and an accessory or kinetic nucleus. In Type 2 the blepharoplast attains to a greater or less degree of indepen- dence of the centrosome, and divides independently of it for many generations of ordinary vegetative reproduction by fission. But there are probably in all cases periods in the life-cycle when the entire nuclear apparatus is reduced to a single nucleus and centriole, from which the condition in the adult, whatever it may bo, arises. For the so-called fourth type of Hartmann and Chagas (62), see below (p. 273). Reproduction and Life-Cycle. — The commonest method of repro- duction is simple or binary fission in the free state. The products of the fission are of equal size, and the division of the body is in- variably longitudinal (Senn, 358) — that is to say, along an axis continuing the direction of the principal flagellum or flagella. In addition to this, the typical method of reproduction, other types of division occur. Multiple fission in the free active condition is known in some parasitic forms, such as Trypanosoma leivisi and Lophomonas blattarum (Janicki, 70). On the other hand, fission may sometimes take place in a resting, non-flagellated condition, or within a cyst ; in the first case it is frequently, in the second perhaps always^ of a multiple type. The occurrence of syngamy in the life-cycle is a point which has been disputed, probably owing to the fact that in forms of simple structure it takes place only at long intervals in the life- cycle, or under special conditions. Moreover, the longitudinal division prevalent in this group makes it practically very difficult to decide, except by continuous observation, whether two conjoined flagellates are individuals about to fuse in syngamy or to separate after fission. In the colonial Phytomonadina, where highly-differ- entiated gametes are found, the occurrence of syngamy lias long been known, but the existence of sexual processes in other flagel- lates has been doubted by high authorities. In recent years, how- ever, it has been observed in a number of forms, and there can be no doubt of the existence of sexual processes in flagellates generally. A summary of recent observations, with full references, is given by Dobell (335, pp. 109-111). The available data are as yet insufficient to make it possible to give a connected account of syngamic pro- cesses in Flagellata generally, and only a few typical cases can be dealt with here. A simple type of syngamy has been described in Copromonas 264 THE PROTOZOA subtilis (Fig. Ill) by Dobell (335). In this species the two gametes appear perfectly similar to each other, and are not, in fact, distin- guishable in any way from ordinary individuals of the species. Two such individuals come together and unite by their anterior or flagellar extremities. In one gamete the flagnllum is lost, and the couple swims about by means of the re- maining one ; this is the only difference between the two gametes which could be interpreted as one of sex. While fusion of the bodies is still incomplete, the nucleus of each gamete divides by a simple type of promitosis (p. 109). One of each pair of sister- nuclei thus produced is a reduction - nucleus, which degenerates ; the other persists. The per- sistent nucleus of each gamete then divides a second time, but into two very unequal halves ; the smaller nucleus in each case degenerates as a reduction -nucleus, while the larger persists as the pronucleus. The bodies of the gametes are now completely fused, and the fusion of the pronuclei follows. The zygote may become en- cysted at once, or may continue to live a free life. In the first case the fusion of the pro- nuclei takes place within the cyst, from which it is ultimately set free as an ordinary individual which feeds and multiplies vegeta- tively. In the second case the zygote becomes an ordinary free individual at once, the interlude of encystment being omitted. Fio. 111. — Life-cycle of Copromonas subtilis. A, Ordinary adult form ; B, C, D, " vegetative " reproduction by binary fission ; E — J, stages of reduction and syngamy : F , 0, H, reduction ; /, J, fusion of the two pronuclei ; the /.ygote (/) may develop into an ordinary free-swimming individual, or (J) may retract its flagellum and become encysted ; K, cyst ; L, liberation of an adult form from the cyst. After Dobell (335). THE MASTIGOPHORA 265 The syngamy of Copromonas is thus seen to be a case of perfect isogamy, and is probably to be regarded as representing a very primitive type, whence the more complex sexual processes of other Flagellata have been evolved — (1) by greater specialization and differentiation of the gametes in their relation to other phases of the life-cycle (gamete-formation) and to one another (sexual differ- entiation) ; (2) by correlation of the sexual phases with definite crises, to which they become restricted, in the general life-cycle. In the Rhizomastigina sexual processes occur of a type resembling those found in the Sarcodina to such an extent as to indicate that the affinities of this group is rather closer to some of the primitive Rhizopods than to typical Flagellata. The life-cycle (Fig. 112) has been worked out in full detail in Mastigetta vitrea by Goldschmidt (41). Vegetative reproduction in the free state is by binary fission of the ordinary type, and occurs when food is abun- dant ; a falling-off in the supply of nutriment leads to gamete-formation and syngamy. In the earliest stages of the sexual generation a differentiation of the individuals into macrogametocytes and microgametocytes is to be observed, though externally they are similar to ordinary individuals and continue their vegetative life during the early stages of gamete-formation. In the macrogametocyte, first a quantity of nucleolar substance, and then of chromatin, is set free from the nucleus ; these two substances unite to form a chromidial mass from which a number of secondary nuclei are formed. The secondary nuclei become scattered through the cytoplasm, and each becomes surrounded by a protoplasmic body. The small cell thus formed is a macrogamete, which goes through reducing divisions. The still active macrogametocyte, which has its cytoplasm crammed with the small gametes, now becomes encysted. Within the cyst the gametes acquire flagella and become motile. At this stage the original nucleus of the gametocyte breaks up and disappears rather suddenly. Finally the cyst- wall is ruptured and the flagellated gametes escape. The formation of the microgametes takes place in a manner essentially similar to that already described for the macrogamctes, but with a few differences in detail. The microgametocytes become encysted at the very beginning of the process ; then formation of chromidia begins, and as soon as it is completed the primary nucleus degenerates ; the microgametes have no flagella, and are shot out of the cyst when it bursts. The free macrogametes measure on the average about 3'6 p diameter, and have a flagellum 15 to 18 /* in length ; the microgametes are 2*8 p in diameter, and have no flagellum. A macrogamete seeks out a microgamete and fuses with it, cytoplasm and nucleus. The zygote retains the flagellum of the macrogamete, and becomes a small, monad-like individual which multiplies by fission as such. After several generations the monads cease to multiply, and each grows up into an adult Mastigetta. A development similar in the main is described by Goldschmidt for Mastigina, but some of the phases escaped his observation. Comparing the sexual cycle of Mastigetta (Fig. 112) with that of Copromonas (Fig. Ill), the chief difference is seen to be that in the former an ordinary individual does not become a gamete directly but a gametocyte, which by a process of multiple fission gives rise to a generation of minute swarm-spores, the gametes. In the two sexes a slight differentiation of the gametes is seen. Further, in the life-cycle of Mastigella considered as a whole, there are two forms of individuals, each capable of multiplying vegetatively for many 266 THE PROTOZOA generations — namely, the monad form, product of syngamy, and the adult, mastigamoeba-form, which ultimately produces the monad-like gametes. Hence the life-cycle in such a type is an alternation of generations (metagenesis), which, as in so many other FIG. 112. — Life-cycle of Mastigella vitrea, diagrammatic. 1, 2, and 3, Different forms assumed by the adult " vegetative ' type" of individual ; 3a, 36, repro- duction by binary fission; 4 — 10, gamete - formation ; a (in each case), microgamete-forraation, 6, macrogamete-formation ; in the former the gamont becomes encysted, and the principal nucleus degenerates early in the process ; in the latter the gamont remains motile and the principal nucleus persists to the last : 4 — 6, extrusion ot chromidia from the nucleus and formation of secondary nuclei ; 7, 8, formation of the gametes round the secondary nuclei ; 9, extrusion of the gametes ; 10a, the small, non-flagellated micro- gametes ; 10&, the larger, flagellated macrogametes ; 11, copulation of tho gametes ; 12, 12o, 126, multiplication by binary fission of the monad-like zygote ; 13, 14, growth of the monad-form, after a period of multiplication, into the adult mastigamceba-form. After Goldschmidt (41). oases in the animal kingdom, appears to have come about by mul- tiplicative processes taking place in a larval type, phylogenetically older — namely, the monad form, the only form of individual that THE MASTIGOPHORA 267 occurs in the life-cycle of Copromonas. In Mastigina, on the other hand, the monad form developed from the zygote apparently does not multiply by fission, but develops directly into the adult form — perhaps a more primitive state of affairs. A very instructive series is furnished by the colony-forming Phytomonads of the family Volvocidce. At one end of the seriec are primitive types, such as Stephanosphcera, where the colony is composed of eight monad individuals, all alike, which may be agamonts in one colony or gamonts in another. Each agamont multiplies by fission to form eight small cells, which remain con- nected together and grow into full-sized monads, thus giving rise directly to new colonies. In the gamont-colonies each gamont (gametocyte) gives rise by multiple fission to a large number of minute biflagellate swarm-spores, the gametes, which are set free and copulate. The syngamy is perfectly isogamous. The zygote grows in size, and finally multiplies to form the eight monads of a new colony. At the other end of the series are the species of the genus Volvox, in which the colony is composed of a great number of individuals, which may be of three kinds, not necessarily all present in the same colony : (1) The ordinary " somatic " monads, locomotor and trophic in function, which do not reproduce themselves in any way ; (2) agamonts, so-called " parthenogonidia," which multiply by fission to form daughter-colonies ; (3) gamonts or gametocytes, which are sexually differentiated as " microgonidia " and " macro- gonidia." The microgonidia produce by multiple fission a swarm of small biflagellate microgametes, comparable to the gametes of StephanosphoBra. In the macrogonidia, on the other hand, multi- plicative processes are in abeyance, and each becomes a single, ovum- like macrogamete, which is fertilized by the relatively minute microgamete. Thus, the syngamy in Volvox is anisogamous to the highest degree ; and, as in other cases among Protozoa, this condition appears to have arisen from a primitive isogamy in which, in both sexes, the gametocytes sporulated to produce a swarm of minute gametes, by the process of sporulation becoming altogether suppressed in one sex — namely, the female — while retained in its primitive form in the other. The colonies of Volvox, with their differentiation of individuals, exhibit a condition transitional to that of the Metazoa. The trophic, non-reproductive individuals, taken as a whole, may be compared to the Metazoan soma, the repro- ductive individuals to the germen. In Pleodorina californica dis- tinct male, female, OP parthenogenetic colonies occur (Chatton), as is the case in some species of Volvox. Classification. — The Flagellata are classified in different ways by different authors, and in the present state of our knowledge of the group no system can be regarded as in any way final. As in other groups of Protozoa, there 268 THE PROTOZOA are a certain number of well-defined orders and families characterized by the possession in common of certain features of organization which leave no doubt as to their taxonomic homogeneity. On the other hand, there are a large number of primitive forms whose characteristics are mainly of a negative order, and of which the affinities are in consequence vague and uncertain, the systematic position debatable. There is, moreover, frequently an element of uncertainty, in the case of many forms, as to whether they represent truly specific adult forms, or merely developmental stages of some other soecies of the Flagellata or Sarcodina. Finally there are a certain number of species and genera concerning which it is still debated whether they should be assigned to the Mastigophora or some other class of Protozoa. Hartmann and Chagas (62) have proposed to utilize the relations of the flagellar to the nuclear apparatus for systematic classification of the FlageUata, as suggested also by Prowazek (354). But, apart from the fact that these relations have as yet been investigated in very few flagellates, and that in such minute objects the details are very difficult to make out and liable to be a subject of dispute, it may be doubted whether these points of structure are sufficiently constant to be of classificatory value in this subclass, since they appear to vary considerably in allied forms. Thus in Copromonas subtilis, according to Dobell (335), the blepharoplast persists through division- phases, and divides independently of the nucleus ; but in C. major, according to Berliner, the old blepharoplast and flagellum are lost at each division, and a new blepharoplast, from which the new flagellum grows out, is formed by division of the nuclear centriole in each daughter-individual. Again, the third type of flagellar insertion (p. 263) is found in the TrypanosomidcR, allied to the CercomonadidcB, and in the trypanoplasms, which belong to the family Bodonidos, as shown in the next chapter. Classification by these characters is, therefore, at least premature, if not fallacious. Compare also Senn (358). The classification adopted here is in the main that of Doflein (7), with certain modifications. For convenience a number of forms are put together in the Pantastomina, without, however, claiming that this order is anything more than a cataloguer's makeshift for disposing of a number of forms of dubious position and uncertain affinities. ORDER I. : PAKTASTOJHNA. — Holozoic, with no definite mouth-opening ; food-particles ingested at any point on the surface of the body. Suborder 1 : Rhizomastigina. — Body amoeboid ; food captured and ingested by means of pseudopodia. Several genera, only known as yet from fresh water, are referred to this very interesting group ; such are Mastigamceba, F. E. Schulze, Mastigina, Frenzel (Fig. 38), and Mastigetta, Frenzel (Fig. 40), distinguished from one another by the nature of their amoeboid movement and the characters of their pseudopodia. In appearance the species resemble amoebae which possess a long and well- developed flagellum, or in Dimastigamceba two, in Trimastig- amceba (Whitmore, 280) three flagella. Locomotion and food-capture are carried on for the most part as in an amoeba, and the flagellum appears to function chiefly as a tactile organ in the adult mast igamceba- phase ; in the young monad-phase, on the other hand, the flagellum is the sole organ of locomotion and food-capture, as in an ordinary flagellate. The relation of the flagellum to the nucleus is of Type 1 described above (p. 263), a single centriole which functions both as centrosome and blepharoplast ; in Mastigina and Mastigamceba the flagellum arises from the nucleus (Type la) ; in Masti- gella the origin of the flagellum is distinct from the nucleus (Type 16). The life-cycle of Mastigdla is described above (p. 265). In many points, especially in the formation of secondary gamete-nuclei from chromidia, the develop- ment resembles more that of the Sarcodina than that of the Flagellata, and by many authorities the affinities of the Rhizomastigina are considered to be rather with the first of these two classes. The mastigamcebae certainly link the true flagellates with the Proteomyxa and Mycetozoa ; and if the flagellum were lost in the adult phase, they would be classed in the Sarcodina without hesitation. Fio. 113. — A, Multicilia lacuslris, after Lauterborn. /I., Flagclla, one of which is curled up into a loop ; ps., pseudopodium-like process ; N., one of the nuclei (the others are hidden by the ingested food-masses) ; C., ingested Chlamydomonads ; c., chlorophyll-bodies, the remains of other Chlamydo monads in process of digestion. B, Multicilia palustna, after Penard. N., The single central nucleus. 270 THE PROTOZOA Suborder 2: Uolomastigina. — With numerous flagella radiating from a spherical or approximately spherical body. This suborder contains the single genus Multicilia, Cienkowski, to which several species, some fresh-water, some marine, have been referred. The number of flagella varies in different species, and their precise relation to the nuclear apparatus remains to be made out. M. lacustris, Lauterborn (Fig. 113, A), is multinucleate ; M. palustris, P6nard (Fig. 113, B), has a single nucleus. The body is not covered by a cuticle, and may throw out pseudo- podia, or even become amoeboid (Lauterborn). Nothing is known of the life-cycle, but in M. lacustris Lauterborn observed reproduction by simple fission (plasmotomy ?). In the present state of our knowledge adequate data are lacking for discussion or the affinities of this genus. Done in (7) regards it as a form lying at the root of the Infusorian stem, and derives the most primitive Ciliata from a form similar to Midticilia, in which the numerous flagella become specialized in structure and movement to give rise to an even coat of cilia ; Penard (302), on the other hand, considers Multicilia allied to the Heliozoa (p. 249). It is clear that the genus is one which would repay further study. ORDER II. : PBOTOMONADENA. — Flagellates for the most part of small or minute size ; with a single flagellum ; or with a principal and one or two acces- sory flagella ; or with two flagella, one directed anteriorly, the other pos- teriorly as a trailing flagellum. Nutrition holozoic, saprophytic, or parasitic ; in the first case the food-particles are ingested at the base of the flagellum, where a definite mouth-opening may be present or absent, but without a distinct oesophagus in any case. The contractile vacuole Is generally single, if present, and empties itself direct to the exterior. This order .com p. ises a vast assemblage of genera and species, subdivided by Doflein into eight families, one of which, the Trypanosomidce, including the important parasitic genus Trypanosoma, is discussed in detail in the next chapter. The cuticle is generally thin, and the body is often capable of amoeboid or metabolic movements ; if amoeboid, however, the flagellum is the organ of locomotion, so long as it is present, and not the pseudopodia. The relations of the flagellum to the nuclear apparatus are, in general, of the second type (p. 263), according to Hartmann and Chagas (62) — that is to say, with distinct centrosome and blepharoplast ; but it is extremely probable that in the simpler forms Type 1 occurs also (compare Alexeieff, 327), and in the Trypanosomidce the distinctive feature is the possession of Type 3, with trophonucleus and kinetonucleus, as also in some of the Bodonidce (Prowa- zelnd). The life-cycle of the free-living forms is probably in general of a simple type, similar to that described above in Copromonas (Fig. Ill) ; but observations on the sexual processes are at present very scanty. For a detailed description of the forms included in this order the reader must be referred to the larger treatises, especially Biitschli (2) and Senn (320) ; it must suffice here to mention some of the more typical forms. Cercomonas, type of the family Cercomonadidce (Fig. 1 14), has a single flagellum ; the hinder end is frequently drawn out into a long tail-like process, and is capable of change of form. (Ecomonaa (Oikomonos) differs in having the body rounded. Monos, type of the family Monodidce, has a principal flagellum and one or two accessory flagella. Clodomonos and Spongomonos (Figs. 41, 42) form arborescent colonies ; the constituent monads have two flagella of equal size, both directed forwards. Alexeieff (327) considers that the Monodidce should be placed in the suborder Chrysomonadina (see below). Bodo (Fig. 115), type of the family Bodonidce, has two flagella, one directed forwards, the other backwards as a trailing flagellum ; the species of this genus are free-swimming and do not form colonies ; they occur both free- living and parasitic, for the most part in the digestive tracts of various animals. Bodo locertoB, from the cloaca of Locerfa spp., has been studied by Prowazek (354), who has described a process of autogamy, but doubt has been cast upon his observations by Dobell (336). Note also the oocurrenca of Bodo-liko forms in the development of Cryptodifflujiv (p. 230, aupra). The flagellate THE MASTIGOPHORA 271 recently described by Wenyon (361) from a culture of human faeces, and referred by him to the genus Cercomonas, would appear rather to belong to the genus Bodo. To the family Bodonidce must be referred also the genera Prowazekia and Trypanoplasma, dealt with in greater detail in the next chapter. Hdcomastix, Senn (358), is to be referred to the Bodonidce or made the type of a distinct family ; its two flagella of unequal length are both directed backwards in move- ment. Finally, mention must be made of the group of flagel- lates characterized by the pos- session of a collar (see p. 261, supra), and hence commonly known as " choanoflagel- lates" or " craspedomonads." They are sedentary forms, attached by the end of the body opposite to the flagellum, and may remain single, but more usually form colonies often of considerable extent (Fig. 110). The flagellum is used mainly for food-capture, in which the collar also pro- bably plays an important Flu. 114. — Cercomonaa crossicavda, Dujardin, part ; but an individual may showing amoeboid changes of form. After become detached from its Stein, support, and swim freely, the flagellum being then directed backwards. The systematic position of the choanoflagellates has been differently estimated by different authors ; by some they have been ranked as a primary subdivision of the Flagellata, which are then divided as a whole into Choanoflagellata and Lissoflagellata. the second of these divisions being used to include all other flagellates. Since, however, the choanoflagellates scarcely differ from ordinary monads except in the possession of the characteristic collar, a specialization of the food- capturing function related to a sedentary life, they are now generally ranked as a family of the Protomonadina, the Choanoflagellidce. ORDER III. : POLYMASTIGINA. — Flagella from three to eight in number, usually all more or less equal in size ; in other points of structure similar to the last-mentioned order. Two families, which are sharply marked off from one another, are referred to this order. 1. Tetramitidce, with three or more flagella, which all arise at the anterior end close together. The flagella may all be directed forwards, or one of them may be turned backwards as a trailing flagellum ; in the latter case the trailing flagellum may or may not be united to the body by an undulating membrane. The species referred to this family are for the most part parasitic. Endoparasitic forms of common occur- rence, especially in the digestive tracts of vertebrates, are Trichomastix, with three anterior flagella and a free trailing flagellum, and Trichomonas (Fig. 5), with the same number and arrange- ment of the flagella, but having the trailing flagellum united to the body by an undulating membrane. These two forms occur frequently in the same host, and are perhaps to be interpreted as two developmental phases of the same FIG. 115.— A Bodo saltans, Ehren- berg. B. Bodo gracilis, Stein. After Stein. 272 THE PROTOZOA organism rather than as distinct generic types. Trichomonas hominis is ontozoic in the human intestine, T. vaginalis in the human vagina ; they appear to be harmless scavengers rather than parasites. The encyst ment of Trichomonas has been the subject of some controversy. According to Alexeieff (326), the supposed cysts of Trichomonas described by various authors are in reality independent vegetable organisms, of the nature of yeasts. In some species of Trichomonas the anterior flagella are four in number (Alexeieff, 323); for such forms Parisi (A.P.K., xix., p. 232) has founded a subgenus Tetratricho- monas. The genus Macrostoma, according to Wenyon (362), differs from Trichomonas in having the undulating membrane wedged in a deep groove ; M. mesnili occurs in the human intestine. According to Alexeieff (324), Macrostoma is a synonym of Tetramitus. Mono- cercomonas, including a number of common intestinal parasites, has four anterior flagella °f equal length, or two longer, two shorter (Alexeieff, 325). Costia necatrix, also referred to this family, is ectoparasitic on the skin of fishes. According to Moroff, it has four flagella in two pairs, two larger and two smaller, all of which serve for locomotion ; but the larger pair are used also for fixation, and the smaller pair for wafting into the mouth the food-particles, which consist chiefly of dead epithelial cells torn away from the epidermis (see also Neresheimer). 2. Octomitidoe.* — With six or eight flagella, arranged in pairs ; the body is bilaterally symmetrical in structure. Entozoic forms, for the most part of intestinal habitat. The remarkable bilateral symmetry of the species of this family is not merely an external characteristic of the body, but affects the internal structure as well, and the entire nuclear structure is doubled, with right and left halves. Octomitus (synonym, Hexamitus ; see Dobell, 236), with four pairs of flagella (Fig. 116), includes a number of entozoic species — e.g., 0. intestinalis, from the cloaca of the frog and other animals. Lamblia intestinalis (synonym, Megastoma entericum, Pig. 117) is a common inhabitant of the human intestine. It becomes encysted, and is probably disseminated in this form. Within the cyst it divides into two (Rodenwaldt). L. sanguinis, described by Gonder (A.P.R., xxi., p. 209) from the blood of a falcon, is probably an intestinal parasite gone astray (vide p. 258). The order Polymastigina differs little from the Protomonadina except in the complication of the flagellar apparatus, correlated probably with the entozoic habit. Hartmann and Chagas propose to merge the Polymastigina * Doflein terms this family the Polymaatigidce, but the name is clearly in- admissible, since the genus Polymostix belongs to the preceding family, and is closely allied to Trichomonas, but has six anterior flagella and no trailing flagellum (compare Alexeieff, 325). FIG. 116. — Octomitus dujar- dini. W.1, Anterior blep- haroplast, from which the first and second flagella of that side of the body arise ; W.2, second blepharoplast, giving off the flagellum of the third pair ; #., left-hand nucleus ; ax., left axostyle ; W.3, third blepharoplast, at the extremity of the axo- style, giving off one of the flagella of the fourth pair. All the structures indicated are paired, and the letters indicate the member of each pair on the left side of the body. After Dobell (236). THE MASTIGOPHORA 273 in the Protomonadina, and then to divide the order into two suborders ; the first, entitled the Monozoa, would include the Protomonadina as constituted above, with the exception of the Trypanosomidce (" Binucleata "), and with the addition of the Tetramitidce. The second suborder, Diplozoa, would in- clude only the Octomitidce. This arrangement certainly seems more natural than that which is usually adopted, so far as the Tetramitidce and Octomitidce are concerned. ORDER IV. : EUGLBNOIDINA. — Larger forms, with mouth.-aperture and oesophagus ; with a complex vacuole-system opening into the oesophagus ; often with holophytic apparatus, chromatophores, stigma, etc. This order represents, so far as structural complication of the individual is concerned, the highest type of organization among Flagellata. The body, may be metabolic, or of definite contours, with thick cuticle. The free-living FIG. 117. — LanMia intestinalis. A, Ventral view ; B, side view. N., One of the two nuclei ; ax., axostyles ; /I.1, /Z.2, ft.3, ft.*, the four pairs of flagella ; «., sucker- like depressed area on the ventral surface ; x, bodies of unknown function. After Wenyon (277). forms are either holozoio or saprophytic, if colourless, or holophytio if pro- vided with chromatophores, in which case they may be capable of nourishing themselves by more than one method. The flagellum may be single, or there may be a second flagellum, usually smaller than the principal flagellum, and sometimes directed backwards as a trailing flagellum. The attachment of the flagellum is of the second type (p. 263), with blepharoplast distinct from the centrosome. According to Hartmann and Chagas (62), in Peranema trichophorum the centrosome first divides to furnish a blepharoplast, and the latter, having become completely independent of the nucleus, divides into two, a distal blepharoplast or basal granule of the flagellum, connected by a rhizoplast (centrodesmose) with the proximal blepharoplast or anchoring granule. The authors consider that this should be regarded as a fourth type 18 274 THE PROTOZOA of flageliar insertion, characteristic of this order ; but it is simplest to regard it merely as a secondary complication of the second type, and one which is not universal in this order, since in Copromonas subtilis the blepharoplast remains undivided, so that this species shows a flageliar attachment strictly of the second type. In Euglena, according to Wager (213), the flagellum passes through the oesophagus and becomes attached to the wall of the reservoir- vacuole by a bifurcate base. On one of the branches is a distinct thickening in close contact with the stigma (p. 205). The thickening is prob- ably the blepharoplast, and the two branches represent the rhizoplast. The sexual processes of the Euglenoidina are but little known, and Copro- monas is the only genus in which the complete life -cycle has been worked out ; in this species it is of a simple type (p. 264, Fig. 111). The order comprises three families. The first, Euglenidce, contains forms provided with chromatophores, ho'ophytic, saprophytic, and parasitic (Haswell) in habit. Examples: Euglena (Fig. 4), Phacus (Fig. 118). The second family, Astasiidce, contains the genus Astasia (Fig. 15), colourless and saprophytic or parasitic. The third family, Per- anemidce, contains numerous genera without chro- matophores, holozoic or saprophytic. Examples : Peranema, Copromonas (Fig. 111). The subfamilies Heteronemince and Anisonemince are heteromastigote. Example : Anisonema (Fig. 25). OBDEBV. : CHBOMOMONADINA. — Small forms, with- out O3sophagus or vacuole-system, with delicate cuticle and one or two flagella ; their characteristic feature is the possession, usually, of one or two conspicuous chromatophores, green, yellow, or brownish, in colour. The nutrition, for the most part holophytic, may be also holozoic or saprophytic. Divided into two suborders. Suborder 1 : Chrysomonadina. — With one or two flagella and one or two yellowish-brown chroma- tophores ; body often amoeboid or metabolic ; colony- formation frequent ; nutrition holozoic and holophytic. Three families. Examples : Chrysamoeba, Chromulina, Dinobryon, etc. According to Scherffel, Chrysamoeba is the amoeboid, non-flagellated phase of Chromulina ; compare also Lauterborn (345'5). To this suborder must be referred also the Coccolithophoridce, marine flagellates which secrete the calcareous shells known as coccoliths (vide Lohmann). Suborder 2 : Cryptomonadina. — Small forms with one or two flagella, colour- less, or with chromatophores ranging in colour from yellowish- brown to olive- green or blue-green. Holophytic or saprophytic, not holozoic. Examples : Chilomonas, colourless ; Cryptomonas, some species of which are symbiotic in Sarcodina (p. 15). Doflein refers the Silicoflagellata to this order (p. 255). ORDER VI. : PHYTOMONADINA SEU PHYTOFLAQELLATA. — Completely and exclusively holophytic, with cellulose envelope and without mouth-aperture. This orcjer comprises the most plant-like flagellates, to all intents and purposes unicellular algae which retain throughout life their flageliar apparatus and their motility. The individual is generally small, and the body is, except in one family, of definite form and enveloped in a rigid cellulose envelope which may stand off from the body, and is perforated by pores through which the flagella pass out to the exterior. The flagella are usually two in number, sometimes tour, of equal size. The cytoplasm generally contains a large green chromatophore and a red stigma. The flagellar insertion, according to Hartmann and Chagas, is of the second type, as in Protomonadina. The reproduction may take the form of multiple fission within the body-envelope to form numerous swarm-spores, which when set free may be gametes or agametes. Colony-formation is frequent in this order (p. 257). FIG. 118. — Phacua triqueter. tes., (Eso- phagus ; c.v., con- tractile vacuole ; at., stigma ; N., nucleus. After Stein. THE MASTIGOPHORA 275 Three families are recognized. The first, represented by the genus Pyra- mimonas, contains primitive forms in which the body is metabolic and the cellulose envelope is absent. The second family, CMamydomonididce, com- prises non-colonial forms such as Chlamydomonas, Hcematococcus, etc. Nephro- B l'io. 119. — Gonium pectorale: colony of sixteen individuals, each with two nagella. A, In surface view ; B, in side view. N., Nuclei ; c.v., contractile vacuoles ; st., stigmata. After Stein. selmis. referred by Senn (358) to this family, has two flagella, on which it creeps like a Bodo. The third family, Volvocidce, comprises colony-forming species in which the individual is similar in structure to the Chlamydomonads, and the colony is composed of individuals ranging in number from four, eight, 276 THE PROTOZOA sixteen, or thirty-two, up to many thousands. Examples are Qonium (Fig. 119), Stephanosphcera, Volvox, etc. In addition to the six orders of flagellates enumerated above, there remain Home peculiar parasitic forms, the systematic position of which is extremely doubtful. Such are the family Lophomonadidos, represented by Lophomonas blattarum, a common parasite of the end-gut of the cockroach and other Orthoptera, and the Trichonymphidce. including the genus Trichonympha and allied forms, parasitic in the end-gut of termites of various species. Lophomonas blattarum, which has recently been studied by Janicki (70), bears a tuft of flagolla arising at the anterior pole of the body from a double ring, or rather horseshoe, of blepharoplasts^ situated at the edge of a funnel- shaped or cup-like structure, the calyx, which is prolonged into an axostyle (Fig. 45). The nucleus hes within the calyx, which is surrounded in its turn by a peculiar thickening or support, termed the " collar," consisting of free, radially-disposed rods crowded together to form an aureole -like figure, approxi- mately spherical. The nutrition is holozoic, and food-particles are ingested at any point on the body-surface, as in the Pantastomina. Multiplication takes place by binary or multiple fission in the free state ; and division of the nucleus up to eight within a cyst has been observed, but the entire life-cycle has not been worked out. Associated with L. blattarum, another form, L. striata, occurs, but it is doubtful if this is a distinct species, or a phase or condition of L. blattarum. The group or family Trichonymphid.ee comprises a number of peculiar parasites found in the digestive tract of various species of Termitidce ; such are the genera Joenia, Lophophora, Calonympha, Devescovina, etc., and finally the genus Trichonympha, from which the family takes its name. The chief peculiarity of these forms is the possession of numerous flagella, which may be disposed in tufts at the anterior end of the body, in a manner similar to Lophomonas (which by some authorities is included in this family), or may be distributed over the whole body, like a coat of cilia, as in the genera Trichonympha, Dinenympha, etc. According to Hartmann, Trichonympha hertwigi occurs under two forms, which he believes to represent male and female gamonts. They multiply by binary fission, and also by a process of sporulation to produce swarm- spores which are believed to be gametes. Dinenympha also exhibits sexual dimorphism, according to Comes (333). From Janicki's investigations, there can be no doubt that Lophomonas is a true flagellate, possibly allied to Trichomonas, possibly, however, to the Pan- tastomina. The genus Joenia, parasitic in Calotermes flavicollis, was thought by its discoverer, Grassi, to connect Lophomonas and Trichonympha ; the recently-described genus Lophophora (Comes, 332) also has points of resem- blance to Lophomonas, but is remarkable for the presence of undulating mem- branes running the length of the body. By some authorities, however, the Trichonymphidae have been placed with the Ciliata, while Hartmann considers that they should rank as an independent class of the Protozoa. SUBCLASS II. : DINOFLAGELLATA SEU PERIDINIALES. The characteristic feature of this subclass is the possession of two flagella, which arise close together about the middle of the body. One flagellum (Fig. 120, e) runs longitudinally backwards as a trailing flagellum ; the other (Fig. 120, d) runs transversely round the body. It is further characteristic of this group for the cuticle to be greatly thickened, forming a tough cuirass, or lorica, investing the body. The two flagella are usually lodged in grooves in the cuirass, the longitudinal flagellum in a longitudinal groove or #ulcits, the transverse flagellum in a circular groove, or annulus. THE MASTIGOPHORA 277 The transverse flagellum executes undulating movements which were formerly mistaken for those of a ring of cilia ; hence the name Cilioflagellata formerly applied to this group. The cuirass, composed of cellulose or an allied substance, is in its typical form a perfectly rigid structure, and is often prolonged into spikes and processes which cause the body as a whole to assume strange or even monstrous forms (Fig. 121). Detailed studies on the skeleton have been published by Kofoid in a series of memoirs (374-383). The nutrition is for the most part holophytic, but in some species ingestion of solid food has been observed. A great many parasitic forms have been made known of recent years (Chatton, 366-369 ; Caullery, 364) ; these are for the most part forms which, in the vegetative, parasitic phase are inert bodies with no sign of locomotor organs, often fixed and pedunculate when ecto- parasitic ; but in their reproductive phases they betray their affinities by the formation of numerous flagellated swarm-spores exhibiting the typical Dinoflagellate structure. The pelagic species generally possess chroma- tophores, and frequently a red stigma, which in some genera — Pouchetia (Fig. 31), Eryihropsis — is modified into an eye-like organ. The deep-sea forms, on the other hand, are colourless. In many Dinoflagellates a peculiar system of vacuolea is found (Fig. 122), consisting of two sacs containing watery fluid, each of which empties itself to the exterior by its own duct. They differ from ordinary contractile vacuoles in possessing a dis- tinct envelope and in not performing rhythmical contractions, and have hence been given the special name of " pusules " (Schiitt). One of these organs, termed the " collecting-pusule," consists of a reservoir- vacuole surrounded by a ring of smaller vacuoles which empty themselves into it ; the other, termed the " sack-pusule," is a large cavity which takes up a great part of the interior of the cuirass. The function of these organs is probably hydrostatic. The commonest method of reproduction is binary fksion in the transverse plane of the body, in which each daughter-individual receives a half of the cuirass of the parent and regenerates the hah* that is wanting. Fission rapidly repeated may lead to the formation of chains of individuals. In other cases multiple fission within the cuirass has been observed, leading to the formation of swarm-spores which are possibly gametes ; but little is known of the sexual processes of these organisms. The Dinoflagellates are an exceedingly abundant and widespread group, Fio. 120. — Olenodi- nium cine turn, Ehrenberg. a, Amyloid granules ; b, stigma ; c, chro- matophores ; d, flagellum of the transverse groove ; e, flagellum of the vertical groove ; v., vacuole. From Lankester. Fio. 121. — Ceratocorys horrida : cuirass. After Stein, from Lankester. 278 THE PROTOZOA highly differentiated as regards forms and species. The vast majority are pelagic in habit, and constitute an important element of the plane ton fauna, both marine and fresh-water. A certain number of species are adapted to parasitic life. They are divided into two orders. ORDER I. : ADINIDA (Prorocentraceae). — Primitive forms in which the typical peculi- arities of Dinoflagellate organization are not fully developed. The body-envelope consists of a bivalve shell without furrows. The two Hagella emerge through an aperture between the two valves, and one flagellum projects freely into the water, while the other twists round it at the base. Example : Prqrocentrum. ORDER II. : DINIFEBA. — With the typical characters of the subclass, as described above. Families: (1) Oymnodinidce, without a well- developed cuirass — example : Gymnodinium ; the marine genus Oxyrrhis (Fig. 123) is referred to this family by Senn (358) ; it is holozoic in habit. (2) Peridinidce, with a well- developed cuirass made up of definite plates — examples : Glenodinium (Fig. 120), Ceratium, Ceratocorys (Fig. 121), Peridinium, etc. ; Pyrodinium (Plate, 385) is remarkable for its intense phos- phorescence ; at the hinder pole, between the chromatophores, the cytoplasm contains a body, the "Nebenkorper" of Plate, surrounded by numerous oil-drops, which are perhaps the seat of the luminosity. (3) Dinophysidce, oceanic species with the cuirass divided by a sagittal suture, often of extraordinary form — example : Dinophysis, etc. (4) Blastodinidce, a family created by Chatton (366, 367) for certain parasitic forms ; such are Blastodinium, an internal parasite of various cope pods, and Apodinium mycetoidfs, an ectoparasite of appendicularians (Fritittaria). The parasitic vegetative form, without organs of locomotion, gives rise by periodic segmentation of mother- cells to successive generations of swarm-spores, which in their structure resemble Gymnodinium. iu^ /E5Ev?«*I^*' SUBCLASS III. : CYSTOFLAGELLATA SEU RHYNCHOFL AGELL ATA . This group comprises a small number of forms all marine and pelagic in habitat. Their chief peculiarity is that, like so many other pelagic organisms of all classes, the body is inflated, as it were, with watery gelatinous substance, so that it attains to a size which far exceeds the actual bulk of the living substance con- tained in it. In consequence of the secondary increase in size, the powers of locomotion are feeble, and these organisms float more or less helplessly on the surface of the sea. FIG. 122. — Peridinium diver- gens : ventral view showing the vacu&le-system. c.p., The collecting-puaule sur- rounded by a rosette of still smaller pusules which open into it ; s.p., the large sac- pusule, or reservoir ; both opening into the fund us (/.), from which both the trans- verse flagellum (t.), lying in the annulus (a.), and the longitudinal flagellum (I.), arise. After Schutt, from Lankester. ea. Fio. 123. — Oxyrrhia marina, Duj. P., Peristome ; N., nucleus; /. v. f ood- vacuoles ; ex., excretory mass about to bo ejected. After Blochmann, from Senn (slightly modified) ; magnification 1,000. THE MASTIGOPHORA 279 The best known form is the common Noctiluca mttiaris of our coasts. The adult Noctiluca is about the size of an ordinary pin's head (1 to 1'5 millimetres in diameter). The spherical body consists chiefly of jelly, with at one pole a superficial concentration of the protoplasm containing the nuclei and giving off the locomotor organs. Prom this central mass of protoplasm strands extend in an irregular network through the whole body, which is limited by a thin pellicle. The central protoplasm bears the so-called " peristome," a deep groove containing the mouth-aperture near one end. The mouth is bordered by pro- jections known as the " tooth " and the " lip," and near it arise two motile organs — a small flagellum, and a large tentacle-like process which shows a transversely striated structure and performs twisting and lashing movements. The tentacle is sometimes named the " flagellum," and the true flagellum the " cilium " ; the former probably serves as the organ of locomotion, the latter for food-capture. The nutrition is holozoic. Noctiluca reproduces itself by binary fission, and also by multiple fission producing a brood of small flagellate swarm-spores. The formation of the latter has been stated to be preceded by isogamous conjugation of the adults, but the matter is open to doubt, and it is possible that the swarm-spores them- selves represent the gametes. Other genera of Cystoflagellata are Leptodiscus and Craspedotdla (Kofoid, 373), both remarkable for their superficial resem- blance to medusae. No tentacle like that of Noctiluca is present in either of these forms, and locomotion is effected by rhythmic contractions of the disc-like body. Bibliography. — For references see p. 486. CHAPTER XIII THE H32MOFLAGELLATES AND ALLIED FORMS General Characters and Principal Types. — Under the term "Hsemo- flagellates " are grouped together a number of forms of which the characteristic, though by no means invariable, habit is alternating parasitism in the blood of a vertebrate and in the digestive tract of a blood sucking invertebrate host. The group must be regarded, however, as one founded on practical convenience rather than on natural affinity — as a method of classification comparable to that of the gardener rather than of the botanist. The existence of a parasitic habit common to a number of different forms is in itself no proof of genetic affinity or community of descent, and it is highly probable that more than one line of ancestry has contributed, through divergent adaptation, to the composition of the group Hsemoflagellates. The name itself has, moreover, lost much of its significance, since closely allied to the forms parasitic in blood, and inseparable from them in a natural scheme of classification, are other forms parasitic only in invertebrates, or even free-living. The chief morphological characteristic of the Huemoflagellates is the possession of two nuclei, a trophonucleus and a kinetonucleus, and the relation of the locomotor to the nuclear apparatus is of the third type distinguished in the preceding chapter (p. 263) ; on this account they are ranked by Hartmann and Jollos (390) as a distinct order of the Flagellata termed the Binucleata. The Haemo flagellates as a group comprise a number of forms which represent in some cases distinct generic types, in others merely developmental phases alternating with other forms in the life-cycles of particular species. The following six generic names represent the more important of these types : 1. Trypanosoma (Fig. 126, etc.), with a single flagellum which arises near the kinetonucleus, at the extremity of the body which is posterior in progression, and runs forward as the marginal flagellum of an undulating membrane. At the anterior end of the body the flagellum is usually continued as a free flagellum, but in some cases it ends with the undulating membrane. A vast number of species parasitic in the blood of vertebrates and in the digestive tract of 280 THE H^EMOFLAGELLATES AND ALLIED FORMS 281 invertebrates alternately are comprised in this genus. Trypano- some-forms also occur as developmental phases in the life-cycle of species parasitic solely in the digestive tracts of insects. 2. Trypanoplasma (Figs. 36, 134), with two flagella arranged in a heteromastigote manner, and with the posterior trailing flagellum united to the body by an undulating membrane for the greater part of its length. A number of species are known, which by their dis- tribution fall into three sections : (1) Species parasitic in the blood of fresh-water fishes, with alternating parasitism in the digestive tractvof leeches ; (2) species parasitic in the digestive tract of marine fishes ; (3) species parasitic in various invertebrates. 3. Crithidia (Fig. 135), with a single flagellum which arises near the kinetonucleus, at about the middle of the body, in front of or close beside the trophonucleus, and runs along the pointed anterior end of the body to form the marginal flagellum of a relatively short, often rudimentary, undulating membrane, beyond which it is continued as a free flagellum. As an independent genus this type comprises species parasitic in the digestive tracts of various insects ; but the majority of the so-called species of Crithidia aro merely phases in the developmental cycle of trypanosomes. 4. Leptomonas (Herpetomonas — Figs. 124, 136), with a single flagellum arising at the anterior end of the body, and with no trace of an undulating membrane. As an independent generic type this form occurs as a parasite of invertebrates, chiefly insects ; secondarily also in the latex of plants (Euphorbiacese). It occurs also as a developmental form of the next genus in the invertebrate host or in cultures. 5. Leishmania (Fig. 138), with an oval body containing a tropho- nucleus and kinetonucleus, but with no flagellum. As a generic type this form is an intracellular parasite of a vertebrate host, multiplying .there by fission and developing into a typical Lepto- monas-torm. On the other hand, as a developmental phase this form represents simply a non-flagellated, resting stage which may occur in the life-cycle of either Trypanosoma, Crithidia, or Leptomonas. 6. Prowazekia (Fig. 141), with two flagella arranged in the hetero- mastigote manner, as in Trypanoplasma, but with the trailing flagellum quite free from the body, without an undulating mem- brane. Prowazekia is therefore quite similar in its morphology to Bodo, with which it was formerly confused, if, indeed, it is really distinct, and it differs from Bodo only in the possession of a kineto- nucleus. Several species are described, free-living or intestinal in habitat. Considering the above six types as a whole from a morphological standpoint, it is seen that there are two types of structure amongst them — the cercomonad or monomastigote type, represented by 282 THE PROTOZOA Trypanosoma, Crithidia, and Leplomonas, of which Leishmania may be regarded as the resting, non- flagellated phase ; and the bodonid or heteromastigote type with two flagella, seen in Trypanoplasma and Prowazekia. We shall return to this point in considering the affinities of the group as a whole and of its constituent genera. The six types enumerated above are given with the nomenclature and definitions most commonly accepted, but it is necessary to state that the application and significance of the names Crithidia, Leptomonas, and Herpeto- monas, are much disputed and are far from being settled. The type of the genus Herpeto- monas of Saville Kent is a species found in the digestive tract of house-flies, H. muscce- domesticce (Fig. 124). According to Prowazek (557), this form possesses normally two flagella, which are connected together by a membrane ; according to Patton (551) and many others, the biflagellate condition is due to precocious division of the normally single flagellum as a preparation for division of the body (compare Strickland, 558 ; Wenyon, 84). Those who follow Prowazek in regarding the biflagellate condition oi Herpetomonas as its normal adult form employ the older genus Leptomonas of Saville Kent* for forms with, a single flagellum (Chatton,Boubaud, Prowazek). The main source of the confusion in the nomen- clature arises from the uncertainty which still exists in many cases as to whether a given form or structural type is to be regarded as an in- dependent specific or generic type, or as a developmental phase of another species. This applies especially to the genus Crithidia, founded by Leger (543) for a species, C. fasciculata, from the intestine of Anopheles maculipennis, and defined as a small uniflagel- late form shaped like a grain of barley (Greek, icpidr)). Such forms, however, occur as developmental forms of trypanosomes or of leptomonads, and it is extremely probable that the species on which Leger founded his genus was simply a phase of this kind, which Wood- cock (527) has proposed to call the " trypano- monad" phase, in the development of a trypanosome. On this ground Dunkerly (535), who has recently discussed the whole question, considers that the name Crithidia cannot be used as a generic name at all, but must be merged in Leptomonas, the name that should be used for all the uniflagellate parasites of insect-guts ; while Herpetomonas should either become a synonym of Leptomonas, or should be used solely for Prowazek's biflagellate type, if that prove to be a distinct generic type. On the other hand, Leger and Duboscq (646, p. 232, footnote) consider that Crithidia should be retained, and Leptomonas ranked as a * The genus Leptomonas was founded by Saville Kent, '' Manual of Infusoria," vol. i., p. 243, for L. biitscMii, parasite of the nematode worm Trilobus gracilis ; the genus Herpetomonas was founded on p. 245 of the same work for H. muscce- domesticoe and H. lewisi ( = Trypanosoma lewisi). Leptomonas is therefore techni- cally the older genus. B Fio. 124. — Herpetomonas muscce-domesticce (Burnett). A, Motile individual with two flagella ; B, cyst : »., nucleus ; bl, kinetonucleus. After Prowazek. THE H^MOFLAGELLATES AND ALLIED FORMS 283 synonym of it. The question has given rise to a controversy which has been carried on by some of the participants in an acrimonious and even unseemly manner, and which it would be unprofitable to discuss further here, since the question is one which must be decided ultimately by facts, and not by personal opinions or tastes. The various forms comprised in the Haemoflagellates may now be considered in detail, beginning with the most important type. I. THE GENUS TRYPANOSOMA. Occurrence. — Trypanosomes were first discovered as blood - parasites of cold-blooded vertebrates — fishes and batrachia ; the type-species of the genus Trypanosama is T. rotatorium (synonyms, T. sanguinis, Undidina ranarum) of the frog (Rana esculenta). Trypanosomes are now known, however, to occur commonly as blood-parasites in all classes of vertebrates. In a wild state many species of mammals, birds, and other vertebrate animals, are often found to harbour trypanosomes in their blood, though frequently in such scanty numbers as to render the detection of the parasites extremely difficult. It may be almost impossible in some cases to find trypanosomes in the blood of an animal by direct microscopic examination, owing to their great scarcity ; but in such cases an artificial culture made from the blood may reveal the presence of the parasites, since in a few days the trypanosomes originally present in small numbers in the blood multiply, under favourable conditions, to produce a swarm of flagellates. The cultural forms are quite different, as a rule, from the blood-forms which gave rise to them, and appear generally as crithidial or trypanomonad types ; thus, cultures furnish evidence of the existence of a trypanosome in a given host, but give no indication whatever of the type of parasite actually present in the blood. In some cases the trypanosomes appear to be present in the peripheral circulation of the vertebrate host only at certain periods, and at other times they are only to be found in the internal organs or tissues of the host, such as the spleen, bone-marrow, liver, lungs, etc. The trypanosome of Athene noctua — T. noctuce, for example — is to be found during the winter only in the bone-marrow of its host, and appears in the peripheral circulation during the summer months, and then most abundantly in the night-time (Minchin and Wood- cock, 42). Hence, for various reasons, it may often be extremely difficult to decide whether a given animal is infected with trypano- somes or not ; and in recent years trypanosomes have been dis- covered in animals in which their presence was previously quite unsuspected — for instance, in calves (Crawley, Carini, 423, Stockman ; see also Bulletin of the Sleeping Sickness Bureau, No. 29, p. 320) and in sheep (Woodcock 527, p. 713, footnote). -fl' B FIG. 125. — Trypanosoma mega, from the blood of African frogs, fl.1, Marginal flagellum of the undulating membrane ; fl.2, free flagellum ; m., myoneme-striations (it is doubtful whether the granular streaks or the clear interspaces correspond exactly to the actual myonemes) ; n, kincto nucleus ; N, space in which the trophonuoleus lies, but, not being stained, it is not clearly defined in the preparation. After Minchin, magni- fied 2,000; compare Figa. 11 and 12 at the same magnification. THE ILEMOFLAGELLATES AND ALLIED FORMS 285 Effects on the Host. — The trypanosomes found infesting wild animals in Nature are, as a rule, quite specific to a particular host, and, so far as can be observed, perfectly harmless to it. If the relations between host and parasite had always been of this type in all cases, our knowledge of trypanosomes would be in a much more backward state even than it is. Of recent years a vast amount of attention has been attracted to these parasites owing to the diseases of man and animals caused by certain species of trypano- somes, and hence termed comprehensively " trypanoso aliases." The greater number of these pathogenic species belong, from the structural point of view, to a type which may be called the brucii- type (Fig. 12) ; such are T. brucii, cause of tsetse-fly disease ; T. gam- bienae, of sleeping sickness ; T. evansi, of surra ; T. equiperdum, of dourine ; and many others. The structural similarity of these species renders their identification a matter of extreme difficulty. Of a slightly different type is T. equinum, of " mal de caderas " in South America, with a very minute kinetonucleus ; but the recently- described T. hippicum of " murrina " (Darling, 428) appears to be a typical member of the 6n«cn-group. T. theileri, on the other hand, from cattle, is very distinct in size and appearance from the members of the 6rMC»»-group. Finally, T. eruzi, the cause of human trypanosomiasis in Brazil, stands apart from all the others in peculiarities of reproduction and development, which have led to its being ranked in a distinct subgenus, Schizotrypanum. The problem of the pathogenic trypanosomes has been touched upon in Chapter II. From a survey of trypanosomes in general, it is clear that the normal type of these parasites is one which is specific to one or to a limited number of species of hosts, to which it is quite harmless. The pathogenic species are to be regarded as aberrant forms not yet adapted to their hosts, as an instance of a disharmony in Nature. They are species which have probably established themselves but/ recently hi the hosts to which they are pathogenic. As contrasted with the natural, non-pathogenic forms, their most striking peculiarities are that they are nonspecific to one host, but can flourish in a great number of different species of hosts, and that in susceptible animals their power of multiplication has no limit. T. brucii, so deadly to many domestic animals, is known to occur also as a natural parasite of wild animals, to which it is harmless. Structure. — The constitution of the trypanosome-body is of a very uniform type in its general traits, though subject to great variation in different cases as regards size, form, and minor details of structure. The body is typically long and sinuous, with the anterior end tapering gradually to a fine point, while the posterior extremity is usually broader, and tapers more abruptly, or ends bluntly ; but hi different forms, even of the same species, there may 286 THE PROTOZOA be great variation, from long, slender to short, stumpy types, and in some cases the posterior end is also greatly drawn out and attenu- ated. The principal nucleus or trophonucleus is usually situated near the middle of the body. The kinetonucleus is almost invariably behind the trophonucleus,* sometimes close behind it, but more usually near the posterior extremity, separated from the tropho- nucleus by about half the length of the body. The flagellum arises from a centriole (blepharoplast) which is in connection with the kinetonucleus. In the more primitive type of arrangement the blepharoplast is lodged within the kinetonucleus itself, and then the flagellum appears to arise from the kineto- nucleus directly (Wenyon, 84). In most cases, however, the blepharoplast is situated close beside, and usually in front of, the kinetonucleus, connected with it by a delicate rhizoplast. When the blepharoplast is distinct from the kinetonucleus, it is at present an open question whether the kinetonucleus contains a centriole of its own, in addition to the blepharoplast, or whether the blepharo- plast represents a centriole which belongs to the kinetonucleus, but has migrated to the exterior of this body. Passing from the blepharoplast to the surface of the body, the Hagellum forms the free border of the undulating membrane, which runs forward from the vicinity of the kinetonucleus to the extreme anterior end of the body as a fin-like ridge or fold of the periplast, of variable width (cf. Fig. 126). The flagellum may in some cases end with the undulating membrane at the anterior end of the body, but more usually it is prolonged forward beyond this point, so that a free portion of variable length is to be distinguished from the mar- ginal portion contained in the undulating membrane. The sinuous body, the undulating membrane, and the flagellum, are alike in a state of incessant movement during life, and in larger forms con- tractile myonemes are clearly visible in the periplast of the body (Fig. 28, p. 58) ; in the more minute individuals the presence of such elements must be inferred from their movements, but cannot always be demonstrated optically. The movements of a trypanosome, speaking generally, are of two types : travelling movements, when it progresses with the free flagellum forwards, sometimes very fast, shooting across the field of the microscope in a straight line (mouvement en fleche), sometimes, on the other hand, pushing its way s lowly through the blood-corpuscles, with the flagellum directed either forwards or backwards in movement ; and wriggling movements, when the animal writhes incessantly in serpentine contortions with little or no displacement * The only known exceptions are furnished by certain forms of the recently- described T. rhodesienae (vide Stephens and Fantham), and by some of the small forms seen during the multiplication of T. lewiai (Fig. 127, L). It is needless to point out that the statement made above applies to the typical trypanosome-form as found in the vertebrate blood, and not to the developmental forms through which they pass iu the invertebrate host (crithidial and other types). THE H^MOFLAGELLATES AND ALLIED FORMS 287 from a given spot. Many trypanosomes, especi?lly the large stout forms, are very sluggish in their movements, and show but little power of progression. At the opposite extreme, in this respect, is the African parasite of cattle, well named by Ziemann T. vivax, which, according to Bruce and his collaborators (411, iii.), " dashes across the field of the microscope with such rapidity that it is impossible to follow its movements, cyclone -like leaving a clear path, the corpuscles in its track having been flung on either side. If it remains at the same spot for a time, as it sometimes does, it has an appearance of great energy and power, throwing the surrounding red blood-corpuscles about in wild confusion." In the foregoing paragraphs the terms " anterior " and " posterior," as applied to the trypanosome-body, have been used strictly with reference to its mode of progression. It is pointed out below, in the comparison with other types such as Trypano plasma and Crithidia, that the extremity Fio. 126. — A, Trypanosoma tineas of the tench ; note the very broad undulating membrane in this species; B, C, T. pence of the perch, slender and stout forms. After Minohin, X 2,000. of the body which is anterior, in the strictly morphological sense, in one species, may conceivably be posterior in another case. Hence some writers avoid the use of the words " anterior " and " posterior," and substitute for them ".flagellar " and " aflagellar " respectively, to denote the two poles of the body. There is as yet, however, no concrete evidence for regarding the flagellar extremity as morphologically posterior in any known species of trypanosome. The undulating membrane is to be regarded as a fold of the periplast or ectoplasm, into which the granular endoplasm may extend a short way in some cases ; it arises from the body along a line which is sometimes spoken of as " dorsal," an unnecessary refinement of terms. The free edge of the membrane, with its marginal flagellum, can be shown by direct measurements to exceed considerably in length that portion of the body to which it is at- tached ; consequently its free edge is thrown into folds or pleats more or less marked. In preparations, trypanosomes are seen to lie, speaking generally, 288 THE PROTOZOA in one of the three ways ; a. certain number show the body extended nearly in a straight line, with the free edge of the membrane much pleated, but as a rule the body is curved, and then either with one principal bend, like a C, or with several S-like serpentine bends. In either case the undulating membrane is seen almost invariably to run on the convex side of each curve. In C-liko forms (Fig. 126, A) the membrane runs evenly along the outside of the principal curve, and the myonemes parallel to it. In S-like forms (Fig. 125, B) the membrane is often seen distinctly to be spirally twisted round the body, the myonemes also exhibiting the same twist. In life the undulating membrane performs, as its name implies, movements like those of a sail flapping in the wind. Wave-like undulations run along it from one end to the other, but not always in the same direction ; it has been observed that reversals of the move- ments may take place, the waves first running in one direction for a time, and then suddenly undergoing a change and running in the opposite direction (Minchin and Woodcock, 42). Much confusion exists hi the nomenclature of the parts of the trypanosome- body, more especially with regard to the small body for which Woodcock's term " kinetonucleus " (" Geisselkern ") is here used — a confusion due to differences of cytological interpretation. While it has never been doubted that the larger body (AT.) is a true nucleus, various views have been held with regard to the smaller body (n.), which, summarized briefly, are as follows : The older writers regarded it merely as an organ of the periplast from which the flagellum arose. Stassano and Bradford and Plimmer re- garded n. as a body of nuclear nature, and termed it the " micronucleus," comparing it with the similarly-named body of Infusoria. Lave ran and Mesnil (464, 391), on the other hand, regarded n. as the " centrosome," the name by which it is generally known in France. Schaudinu (132) emphasized strongly its nuclear nature, and stated that n. was not a centre-some, but nevertheless used for it the term " blepharoplast," by which it is still generally known in Germany, although a true blepharoplast is a body of centrosomic nature. Moore and Breinl (484) reverted to the centrosomic view, and termed n. the " extranuclear centrosome," believing that it arose by division of the intranuclear centrosome contained in the principal nucleus (N.). Hartmann and Frowazek (63), on the basis of their nuclear theory of the centrosome (see Chapter VL, p. 95), regarded n. as a body both of nuclear and centrosomic nature, using for it the term " blepharoplast " ; so also Rosen busch. Finally, Doflein (7), who is not convinced of its truly nuclear nature, continues to employ for n. the term " blepharoplast." With these many conflicting views with regard to the nature ot n., the basal granule has been either ignored or overlooked, or considered as a mere " end- bead " of no particular importance, or ranked as a centriole, as it doubtless is. The nomenclature used here is based on the general theory that a centrosome, or its equivalent, a blepharo- plast, is an achromatinic body of nuclear origin, but not equivalent to an entire nucleus, and on the conviction that n. is a true nucleus, and therefore is not to be regarded either as a centrosome or a blepharoplast. For a fuller dis- cussion of these points, see Robertson and Minchin (80). The trophonucleus of a trypanosome is typically a vesicular nucleus con- taining a karyosome in which is lodged a centriole. The karyosome varies ia size in different species, and is sometimes double or multiple ; in T. granu- losum the smallest forms have a single karyosome which buds off others as the animal increases in size (Minchin, 478). By the method which is most in vogue, however, for making permanent preparations of trypanosomes — namely, the various modifications of the Romanowsky-stain — this structure is seldom to be made out, and the trophonucleus appears generally as an evenly-stained mass or as a dense clump of stained granules. It contains a centriole, difficult to make out in the resting condition, owing to its being embedded in the substance of the nucleus. The kinetonucleus consists mainly of a mass of plastin impregnated with chromatin, staining very deeply, rounded, oval, or even rod-like in shape. According to Rosenbusch, the chromatinic mass of the kinetonucleus is to be regarded as representing THE H^MOFLAGELLATES AND ALLIED FORMS 289 a karyosome, and it is surrounded by a space, sometimes purely virtual, which represents the nuclear vacuole, bordered by a delicate nuclear mem- brane, on or close to which the basal granule of the flagellum is lodged. In some species of the brucii-gtoup, an axial filament, apparently) a sup- porting structure of the nature of an axostyle, has been described (cf. Swel- lengrebel, 514). The system of fibrils, however, with which Prowazek decorates the trypanosome-body are probably artefacts (cf. Minchin, 479). Many trypanosomes contain granules in their cytoplasm which stain similarly to chromatin, so-called " chromatoid grains." According to Swel- lengrebel (514), they are of the nature of volutin (p. 68, supra). The division of a trypanosome is initiated, as a rule, by the division of the blepharoplast or basal granule of the flagellum, and following close on this a reduplication of the flagellum takes place, the exact method of which is disputed. In some cases the old flagellum appears to split ; in others the parent -flagellum remains unaltered, and a daughter-flagellum grows out from the daughter-blepharoplast. It is asserted by some that in all cases the new flagellum really arises as an independent outgrowth of a blepharoplast, and that the splitting of the old flagellum is only apparent, and due to the daughter-flagellum growingout at first in its sheath, from which it separates later (cf. Wenyon, 84). The division of the kinetonucleus follows hard on that of the blepharoplast, and next, as a rule, the trophonucleus divides. When the division of flagellum and nuclei is complete the body divides, begin- ning to do so at the flagellar end ; the two sister-trypanosomes are often connected for a time by the posterior extremities. The division of the kinetonucleus is a simple constriction into two ; that of the trophonucleus is of a simple type, in which first the centriole and then the karyosome divides. The two daughter-karyosomes travel apart, and the nucleus follows suit. The two daughter-nuclei sometimes remain connected for a time by a long centrodesmose, which is finally severed. Such, at least, is the mode of division of the two nuclei as it has presented itself to the majority of investigators, and the nuclear division of trypanosomes is to be regarded as amitotic, or at least not further advanced towards mitosis than that of Coccidium described above (p. 106, Pig. 51). According to Rosenbusch, however, the division of the nuclei, both trophic and kinetic, takes place by true mitosis. This author is in advance of his contemporaries upon this point, and his statements require independent confirmation before they can be accepted unreservedly, since in objects of such minuteness, requiring delicate and elaborate technique, imagination may all too readily outrun perception. Life-History. — The transmission of trypanosomes from the blood of one vertebrate host to another is effected, probably for every species of these parasites, by the agency of a blood-sucking inverte- brate of some kind. When the host is a terrestrial vertebrate, the transmitting agent is generally an insect, such as a mosquito or some biting fly or bug, or an ectoparasite of the host, such as a flea, louse, or possibly a tick in some cases ; the trypanosomes of aquatic vertebrates, on the other hand, are transmitted by leeches in all cases that have been investigated. In addition to inoculative transmission (p. 24) of this kind, trypanosomes may pass directly from one vertebrate host to another during coitus ; this is known to occur in the case of the parasite of " dourine " in horses (T. equi- perdum), and has been suspected, but not proved, to take place in other cases also. It is also possible for the vertebrate to become infected by devouring animals containing living trypanosomes, 19 290 THE PROTOZOA whether it be the blood-sucking invertebrate, or possibly the flesh or organs of another vertebrate infected with trypanosomes. Two methods of inoculative transmission of trypanosomes have been distinguished ; in the one, known as the " direct " or " mechan- ical " method, the parasites merely become contained in or adhere to the proboscis of the blood-sucking intermediary when it sucks blood from an infected animal ; and when it feeds a second time the try- panosomes pass directly, and without having undergone any change or development, into the second host ; in the other, known as the " indirect " or " cyclical " method, the trypanosomes, when taken up by the blood-sucking invertebrate, go through a developmental cycle in it, at the end of which, but not before, they are " ripe " for inocu- lation into a suitable vertebrate host. Comparing natural with artificial processes of infection, in the direct method the blood- sucking invertebrate may be said to play the role merely of an injection-syringe, but in the indirect method it acts also as a culture- medium, in which the parasite passes through various phases and assumes forms quite different from those occurring in vertebrate blood. Patton (393) has put forward the view that transmission is always by the. direct method, and that the crithidial and other forms found in the blood-sucking invertebrate are parasites of the invertebrate alone, and have no connection with the trypanosomes found in vertebrates ; but the number of cases in which it has now been shown clearly that trypanosomes go through a definite cycle in the invertebrate host disproves Patton's contention, and renders it unnecessary to discuss it further. It is rather the direct method that stands in need of further demonstration ; though undeniably possible as a laboratory-experiment, it may be doubted if it ever really occurs in Nature, and in any case it is probably to be regarded as a purely accidental rather than a normal occurrence. It has been frequently asserted or assumed that trypanosomes can pass from parent to offspring, by so-called " hereditary trans- mission," in the invertebrate host, but convincing proof of this state- ment is as yet lacking entirely. Attempts to prove hereditary trans- mission by direct experiment have given, for the most part, negative results, and the observation so frequently made, that leeches, tsetse- flies, fleas, mosquitoes, etc., bred from the egg and not exposed to infection, are entirely free from parasitic flagellates, affords cumu- lative evidence against the existence of any such method of trans- mission (cf. Kleine and Taute, 459). Brumpt (419), however, asserts that T. inopinatum is transmitted hereditarily from parent to off- spring of the leech Helobdella algira. According to Porter (554), " Crithidia " mdophagia of the sheep-ked is also transmitted from parent to offspring in this insect ; and if, as is extremely probable, the flagellate in question is the developmental phase of the trypano- THE H^MOFLAGELLATES AND ALLIED FORMS 291 some of the sheep, it would furnish another instance of hereditary transmission. Hence' this mode of transmission must, apparently, be reckoned with in some instances, though it is evidently an ex- tremely rare phenomenon in trypanosomes generally. Just as a given species of trypanosome is, in Nature, capable of maintaining itself only in a particular species, or limited group of species, of vertebrate hosts, so it may be said, as a general vrule, that in transmission by the cyclical method the parasites are specific in the same way to certain invertebrate hosts, in which alone they are able to go through their full natural cycle. Amongst the many blood-sucking invertebrates which may prey upon the vertebrate, we may distinguish " right " and " wrong " hosts ; in the right host or hosts the parasite establishes itself more or less easily, and passes through a full and complete developmental cycle ; in the wrong host it either dies out immediately or goes through only a part of its cycle. The distinction between right and wrong hosts must not, however, be taken in an absolute sense, but as implying only that, amongst many possible hosts, there is one at least to which the parasites have become better adapted than to any other ; but the trypanosomes may sometimes succeed in maintaining themselves in other than the right host sufficiently long to pass back again into the vertebrate. Thus, in the case of the rat-trypanosome (T. lewisi) the right host is a rat-flea (Cerato- phyllus fasciatus, or possibly other species) ; but it may persist in the rat-louse (Hcematopinus spinulosus), and even pass from it, though rarely, back into the rat again. The following are a few well-established examples, in addition to that of T. lewisi already cited, of trypanosomes and their right hosts. Many pathogenic species of trypanosomes in Africa are transmitted by tsetse-flies — e.g., T. gambiense and T. vivax by Glossina 'palpalis, T. brucii by G. morsitans* etc. The recently- described T. cruzi of Brazil was discovered in its invertebrate host, a blood-sucking hemipterous insect, Conorhinus megistus, before it was found in the blood of human beings. The trypanosomes of certain fresh-water fishes — namely, goldfish, perch, etc. — pass through their developmental cycle in the leech Hemiclepsis mar- ginata (Robertson, 503). T. raice of skates and rays develops in the leech Pontobdella muricata (Robertson, 500, 502). The trypano- some of African crocodiles, T. grayi, develops in the tsetse-fly Olossina palpalis (Kleine, 458 ; Kleine and Taute, 459), and stages in its life-cycle have consequently been confused with those of T. gambiense in the same fly. The trypanosomes of birds are prob- ably transmitted for the most part by mosquitoes, but the details of * According to Taute, O. morsitans can act as a true boat for T. gambiense, and, conversely, according to Fischer, O. palpalis can do the same for T. brucii. 292 THE PROTOZOA their transmission have not yet been worked out in a satisfactory or conclusive manner. It must be considered for the present an open question whether true try- panosomes occur as parasites of an invertebrate host exclusively ; the answer to the question will depend on the significance given to the expression " true trypanosome." It is now practically certain that many leptomonads have a trypaniform phase in their development (see p. 314, infra), so-called " Icpto- trypanosomes." In Drosophila confusa, a non- biting, muscid fly, Chatton and Alilaire (compare also Chatton and Leger) found in the Malpighian tubules a trypaniform type of flagellate which they consider as a " eutrypanosome," as a species of Trypanosoma distinct from the Leptomonas occurring in the gut of the same fly (Fig. 137). Wenyon (84) also found similar forms in the Malpighian tubules of house-flies in Bagdad, and considered that they might belong to the cycle of the Leptomonas (Herpetomonas) in the same host. In both cases the phase in the Malpighian tubules is a little stumpy trypanosome- like form, very similar in its characters to T. nanum. The fact that these " eutrypanosomes " are so far known only to occur in flies which are infected also by a species of Leptomonas indicates that, like the " leptotrypanosomes," they are merely a phase in the cycle of the Leptomonas. From the foregoing it is seen that the complete life-cycle of a trypanosome is an alternation of generations corresponding to an alternation of hosts. One part of the cycle is passed in the blood of a vertebrate, in which the predominant form is the trypanosome- type of flagellate ; the second part is passed in the digestive tract of an invertebrate, and here the predominant form is the crithidial or trypanomonad type. We may consider the life-history, therefore, under these two principal phases : 1. As a type of the life-cycle in the vertebrate host, that of the common rat-trypanosome may be taken. After infection, natural or artificial, of the rat, the trypanosomes make their appearance in the blood about the fifth, sixth, or seventh day. What the para- sites have been doing during this time, the so-called " incubation- period " in the rat, cannot as yet be stated definitely ; it may be that the relatively few trypanosomes inoculated by the flea or syringe have merely been multiplying steadily, in the manner presently to be described, until they become sufficiently numerous in the blood to be detected by microscopic examination ; there may, on the other hand, be phases of the parasite as yet unknown during this period, and, according to recent statements (Carini, 422), a process of schizogony takes place in the lung similar to that dis- covered by Chagas in Schizotrypanum cruzi (see below). When the trypanosomes first appear in the blood, their most striking peculiarity is the extraordinary diversity in type which they exihibit. Besides " ordinary " individuals of the normal dimensions of the " adult " form, there are others smaller or larger, the extremes of size being relatively huge in one direction, very minute in the other. These differences of size are due to the fact that the try- panosomes are multiplying actively, the large forms being those H J K FIG. 12.7. — Various forms of multiplication in Trypanosoma lewisi from the blood of the rat. A, Trypanosome of tho ordinary type ; B, small form resulting from division ; C, stage in equal binary fission ; the nuclei have divided and two flagella arc present, but division of the body is beginning, and is indicated by a lighter streak down the middle of the body ; D, final stage of binary fission, which is complete except for a bridge of protoplasm, much drawu out, connecting the hinder ends of the two sister-trypanosomes ; E, form with hinder end drawn out (longocaudenae type), the result of binary fission as seen in the last figure ; F, unequal binary fission of a large trypanosomc ; O, H, continued fission of the same type ; in 0 a parent and three daughter- individuals, in H a parent and seven daughter-individuals, can be distin- guished ; the parent-individual in each case is marked by the possession of a fiagellum of the full normal length, while the daughter-individuals, formed by successive divisions, have flagella varying in length ; /, a small form, similar to B, but with the kinetonucleus in front of tho trophonucleus ; J, binary fission of a form similar to / ; K, further division of a similar form producing a rosette of seven individuals still connected together. From preparations made by Dr. J. D. Thomson ; magnified 2,000 diameters. 294 THE PROTOZOA which are about to reproduce themselves by some form of fission, while the small forms are those which ha^Te resulted from a recent act of reproduction. The multiplication of T. lewisi in the rat's blood takes various forms (Fig. 127). In some cases a trypanosome divides by equal binary fission (C, D), but this is comparatively rare. More usually the fission is markedly unequal, and of a multiple type. Small daughter-forms are 'split off from large parent -individuals, and usually many at a time ; the nucleus of the parent- form divides several times, and subsequently the body divides into as many portions as there are nuclei, thus producing rosette -like forms (Fig. 127, F, O, H) in which the original parent can usually be distinguished by its long flagellum from the small daughter-individuals with their flagolla growing out. The small forms are sometimes set free with a crithidial type of struc- ture, the kinetonucleus in front of the trophonucleus (Fig. 127, L), and these immature forms may proceed to reproduce themselves rapidly again by either binary or multiple fission, in the latter case forming rosettes in which no large parent-form can be distinguished (Fig. 127. K). A curious type of trypanosome found during the multiplication -period of T. leunsi is a form with the posterior end prolonged to a great length, so that it almost resembles a second flagellum (Fig. 127, E), and has sometimes been mistaken for such. This form has been described by Lingard as a dis- tinct species under the name T. longocaudense. These forms appear to arise by binary fission (Fig. 127, Z>) ; they are of constant occurrence and very numerous at a certain stage of the multiplication-period. The multiplication of T. lewisi in the rat's blood is most active from the eighth to the tenth day after infection, after which it is on the decline and gradually ceases. The relative number of forms of ordinary size increases steadily, while those of unusual dimen- sions, whether great or small, become continually scarcer, until about the twelfth or thirteenth day the tiypanosomes, now usually present in vast numbers in the blood, are of uniform size and appearance, exhibiting, apart irom occasional abnormalities, indi- vidual variations only of a comparatively slight character ; and all multiplication has ceased 'entirely, never to recommence in the same host. The trypanosomes swarm in the blood of the rat for a certain time, which varies in different cases, but is usually one or two. months. The infection of the rat is sometimes spoken of as " acute " when the trypanosomes are multiplying, and as " chronic " when multiplication has ceased, not, however,t very well-chosen terms, since the trypanosomes soon begin to diminish in number, and finally disappear altogether ; sometimes the diminution is very gradual and slow, sometimes it takes place with great rapidity. In either case the rat gets rid of its infection entirely sooner or later, without having suffered, apparently, any marked inconvenience from it,* and is then immune against a fresh infection with this species of tcypanosome. * Instanced arc on record of lethal epizootics of rats ascribed to T. lewisi ; but the proof that this parasite was really the cause of the disease is lacking. Under normal circumstances rats show no perceptible pathological symptoms whatever THE ELEMOFLAGELLATES AND ALLIED FORMS 295 A type of development in the vertebrate host contrasting in many points with that described in the foregoing paragraphs Is seen in T. cruzi (Fig. 128), the cause of human trypanosomiasis in Brazil. In this case the ordinary or adult forms of the trypanosome found in the* general circulation do not multiply there ; but the investigations of Chagas and of Hartmann have made known two types of multiplication which take place in the internal organs of the body. The first type of multiplication proceeds in the capillaries of the lung (Fig. 128, 6 — e). An adult trypanosome loses its flagellum, and in some cases its kinetonucleus also ; its body then becomes rounded off into an oval mass ; the trophonucieus, and also the kinetonucleus, if present, multiply by sue cessive divisions to form eight nuclei of each kind ; and finally the body divides within its own periplast into eight' minute daughter-individuals, so- "called " merozoites." The merozoites are stated to exhibit a dimorphism // i FIG. 128. — Phases of T. (Schizotrypanum) cruzi in vertebrate blood, a, The two forms of the adult trypanosome, " male " (upper) and " female " (lower), from human blood ; b, preparations for schizogony ; c, schizont ; d, division of the nucleus of the schizont ; e, division of the schizont into eight merozoites ; /, merozoite in a blood-corpuscle ; g, intracorpuscular phase in late stage of growth ; h, similar phase escaping from a corpuscle, the flagellum not yet formed ; t, similar phase, the flagellum in process of formation. Stages 6 — e are found in the lung, the others in the peripheral blood. After Chagas (425). which Chagas regards as sexual ; those produced by trypanosomes which retained thejr kinetonucleus have both trophic and kinetic nuclei and a rudiment of a flagellum (male forms) ; those derived from trypanosomes which lost both flagellum and kinetonucleus have only a trophonucleus (female forms) ; in the latter case the single nucleus divides into two unequal parts, of which the smaller becomes the new kinetonucleus, and a flagellum is formed subsequently. • In either case the merozoites penetrate into blood- f rnm even the most swarming infection with T. lewisi (for the action of the " ren- furc£s " strains see p. 28). Those who study habitually the lethal species of trypanosomes often display a natural bias, not in the least justified, to assume that a similar virulence is an inseparable attribute of all other species of these parasites. If that were so, it would be necessary to consider practically every specimen of pike, bream, perch, or tench, in the Norfolk Broads, for instsnce, to be in a diseased condition. 296 THE PROTOZOA corpuscles, and so pass into the general circulation. Within the corpuscle they grow into the adult form, which is finally set free from the corpuscle as a trypanosome of normal structure. The adult trypanosome (Fig. 128, a), swimming freely in the blood- plasma, may either be taken up by the inverte- brate host in which it develops, or may repeat the process of multiplication by schizogony. The second type of multiplication was first described by Hartmann from hypertrophied endothelial cells of the lung ; Chagas (426) has since found it in the tissues of the body, more especially in the cardiac muscle, central nervous system, and striped muscle. In this type the parasite is intracellular, and has the appearance and structure of a Leishmania (cf. Fig. 138), a rounded body containing a trophonucleus and a kinetonucleus, but no flagellum or undulating membrane. On account of its power of multiplication by schizogony, Chagas has made T. cruzi the type of a special genus, Schizotrypanum ; the type of multiplication observed in the lung-capillaries is not essentially different, however, from that of T. leurisi in the blood, except for its alleged sexual dimorphism ; and, accord- ing to Carini (424), similar processes of schizogony occur in other trypanosomes The intracellular multiplication in the tissues, however, recalls strongly that of the parasite of kala-azar (see p. 316, infra). Schizotrypanum thus forms an important link between a typical blood-trypanosome, such as T. lewisi, and a tissue-parasite, such as the species of Leishmania, in which the free trypanosome-phase no longer exists, apparently. Chagas considers the multiplication of Schizotrypanum cruzi in the tissues as non-sexual, and serving to increase the number of parasites in the host, but that which takes place in the lung- capillaries as a process of gametogony whereby the sexually differentiated adult forms are produced. His grounds for this interpretation are, first, that in human blood the adult trypanosomes exhibit a dimorphism rarely found in guinea-pigs infected artificially, in which also schizogony in the lung is seldom observed ; secondly, that the invertebrate host, Conorhinus, is always rendered infective if fed directly on infected human blood, but very rarely becomes infective if fed on guinea-pigs, even when these animals show an intense infection. He suggests that the greater resistance of the human organism to the parasite stimulates the production of sexual forms which the trypanosome may cease to produce in a less resistant host. In the more familiar pathogenic species, such as T. brucii, T. gambiense. etc., the development in the vertebrate host takes the form mainly of continued multiplication by binary fission simply. Reproduction of this kind may pro- ceed until the trypanosomes swarm in the blood ; or, on the contrary, the trypanosomes may be at all times relatively few in number, even when fatal to their host. T. brucii, for example, may produce in different hosts an acute or a chronic form of disease ; in the latter case the infected animal may live a long time, and the parasite exhibits very limited powers of multi- plication. The behaviour of the parasite in the natural hosts to which it is harmless has not been studied. In many pathogenic species, periods of multiplicative activity, during which the trypanosomes are abundant, alternate with periods during which the parasites pass into a resting condition in the internal organs, and become scarce or disappear in the general circulation. In this phase they are alleged to lose their flagellum, dimmish in size, and become small, rounded " latent borl Sea," which, according to Moore and Breinl (484), have only a single nucleus ; but according to Fantham they are Leishmania-like, with distinct tropho- nucleus and kinetonucleus. From resting stages of this kind the actrvs trypanosomes are developed again. Laveran (462), however, denies that there is a non-flagellated stage of development in the vertebrate host, and considers that the elements described as "latent bodies" represent involution-stages of the parasites — that is to say, forms which have become deformed in structure owing to unfavourable conditions, but not to such an extent as to be incapable of recovery if the conditions improve. THE ILEMOFLAGELLATES AND ALLIED FORMS 297 In the vast majority of trypanosomes in their natural hosts, such as birds, fishes, etc., the mode of multiplication and the developmental cycle remains a mystery, although the sizes of the individual trypanosomes and their numbers are observed to vary at different times in the same host. Considerable light has been thrown upon this question by the recent investigations of Machado upon the multiplication of Trypanosoma rotatorium of frogs, a species re- markable for the polymorphism it exhibits. The results obtained by Machado may be summarized briefly as follows : Trypanosomes of any size may divide by binary fission when free in the blood (supposed " non-sexual " reproduction). On the other hand, trypanosomes of large size may become rounded, flattened, leaf -like forms, losing 'their flagellum ; such forms undergo a process of schizogony in the internal organs, chiefly in the liver or kidneys, sometimes in the spleen, sometimes even in the circulating blood. The kinetonucleus approaches the trophonucleus, and may (1) remain distinct from it, so-called " male " type ; or (2) may pass into the trophonucleus, in which the karyo- some breaks up to form a small secondary karyosome ; the kinetonuclear karyosome then fuses with, or becomes closely adherent to, the secondary trophonuclear karyosome — so-called " female " type. A multiplication of the nuclei then takes place : in the " male " type by independent divisions of the kinetonucleus and trophonucleus ; in the " female " type by divisions of the single mass formed by fusion of the kinetonuclear and trophonuclear karyoeomes, followed by budding off of small nuclei from the originally single nucleus. Thus the body of the rounded-off trypanosome becomes filled, within its periplast, with nuclei varying in number from five to seven- teen ; then round each nucleus ("female ") or each pair of dissimilar nuclei (" male ") the protoplasm becomes condensed to form as many merozoites, which are finally set free by rupture of the periplast. The merozoites of " male " type develop a flagellum ; in those of " female " type the single nucleus divides into two nuclei of unequal size, a larger trophonucleus and a smaller kinetonucleus, and from the latter a basal granule is budded off from which the flagellum grows out (Fig. 30, G). In either case the mero- zoites (which may divide further after being liberated from the parent body) become transformed finally into the smallest forms of trypanesomes, which then grow up into the larger forms found in the blood. Machado' s observa- tions of fact, apart from his theoretical interpretations, explain the many different forms found in the frog's blood, which have recently been studied in detail by Lebedew ; compare also Mathis and Leger. In other cases there may be three well-marked types of form — long and slender, short and stumpy, and intermediate or indifferent forms, as in T. gambiense (Fig. 12 ; cf. Minchin, 477, Hindle, 450, Bruce, 405) ; or there may be every gradation in size from small to large forms, as in T. gramdosum of the eel (Fig. 129) ; or, finally, the trypanosomes may be practically uniform in size and structure, as in T. lewisi after the multiplication-period, T. vivax, etc. A satisfactory explanation of the polymorphism has not been found in all cases ; the various forms may be in some instances stages of growth related to multiplication, as in T. lewisi during the multiplication-period ; in other cases the polymorphism — for example, of T. gambiense — may be sexual differentiation which is related to the subsequent development in the in- vertebrate host ; a third possibility is that in some cases the propagative forms, destined for multiplication in the invertebrate host, are differentiated from the other forms found in the vertebrate host, as in T. noctuce (Minchin and Woodcock, 42). Different explanations must probably be sought in different cases. 2. The cycle in the invertebrate host always takes place entirely or mainly in the digestive tract, though the extent to which this region is invaded varies greatly. In the development of T. leurisi in the flea the parasites pass down as far as the rectum, and there 298 THE PROTOZOA undergo the principal phase of their cycle. In the development of the trypanosomes of fresh-water fish in the leech Hemiclepsis, the parasites do not pass farther back than the crop (Robertson, 503). Finally, in the many species of pathogenic trypanoso'mes which are transmitted by tsetse-flies of various species, two types of develop- mental cycle can be distinguished : in the one, the parasite invades Fid. 129. — Trypanosoma granvlosum of the common eel : four different sizes, probably stages of growth. After Minchin (478), x 2,000. the whole alimentary canal of the* fly ; in the other it undergoes the greater part of its development iir the proboscis and pharynx alone. The details of the developmental cycle in the invertebrate host are very inadequately known, and have only been studied in a very few instances. As a rule the characteristic form of this part of the life-history is a crithidial or trypanomonad type, repre- THE H^MOFLAGELLATES AND ALLIED FORMS 299 senting the principal multiplicative phase in the invertebrate host ; it is a form in which the kinetonucleus is placed in front of, or close beside, the trophonucleus, and in which, consequently, the undu- lating membrane is confined to the anterior region of the body, and may be quite rudimentary. As a rule the body of the trypano- monad is shorter, stiffer, more pear-shaped, than in the typical try- panosome-form ; no longer sinuous and flexible, it is held straight and rigid in progression, which is effected almost entirely by the flagellum. In many cases, however, the free flagellum is very short, and used to attach the organism to the lining of the digestive tract. Besides the trypanomonad form, the developmental cycle may also include many other types of form, and often exhibits a degree of polymorphism which is most bewildering, and compared to which the diversity of form seen in the vertebrate host is but slight. Taking the development of T. lewisi in the rat-flea as a typical example, the parasites when taken up by the flea pass with the ingested blood into the stomach (mid-gut) of the insect. In this part they multiply actively in a peculiar manner, not as yet de- scribed in the case of any other trypanosome in its invertebrate host (Fig. 130) ; they penetrate into the cells of the epithelium, and in that situation they grow to a very large size., retaining their flagellum and undulating membrane, and exhibiting active metabolic changes in the form of the body, which in early stages of the growth is doubled on itself in the hinder region, thus becoming pear-shaped or like a tadpole in form, but later is more block-like or rounded. During growth the nuclei multiply, and the body when full-grown approaches a spherical form, and becomes divided up within its own periplast into a number of daughter-individuals, which writhe and twist over each other like a bunch of eels within the thin envelope enclosing them. When this stage is reached, the flagellum, which hitherto had been performing active movements and causing the organism to rotate irregularly within the cell, disappears altogether, and the metabolic movements cease ; the body becomes almost perfectly spherical, and consists of the periplast-envelopa within which a number of daughter-trypanosomes are wriggling very actively ; the envelope becomes more and more tense, and finally bursts with explosive suddenness, setting free the flagellates, usually about eight in number, within the host-cell. The products of this method of multiplication are full-sized trypanosomes, complete in their structure, and differing but slightly in their characters from those found in the blood of the rat. They escape from the host-cell into the lumen of the stomach. To the intracellular multiplicative phase in the stomach a crithidial phase in the rectum succeeds (Fig. 131 . In the fully- established condition the rectal phase consists of small pear-shaped 300 THE PROTOZOA forms with the flagellum very short, in some cases projecting scarcely at all from the body at its pointed end. These forms are found attached by means of their flagella, often in vast numbers, to the wall of the rectum, sometimes also in the intestinal or pyloric region ; they multiply by binary fission, and form a stock, as it were, of the parasites, which persists for a long time in the flea — probably, under favourable conditions, for the whole life of the insect. Experi- ments have shown that a flea once rendered infective to rats can FIG. 130. — Trypanosoma lewisi: developmental phases from the stomach of the rat-flea. 0, Ordinary form from the blood of the rat ; A — F, intracollular stages : A, a trypanosome curled on itself ; B, similar form in which the body has become rounded ; G, multiplication beginning, division of kinetonucleus and trophonucleus, daughter-flagellum growing out ; D, further stage — three nuclei of each kind, two short daughter-flagella, and a long pa rent- flagellum wrapped round the body ; E, six nuclei of each kind, five daughter-flagella, parent- flagellum wrapped round the body ; F, eight nuclei of each kind, the daughter-flagella running parallel with the parent- flagellum ; 0, the type of trypanosome resulting from the process of multiplication seen in the fore- going figures ; this is the form which passes down the intestine into the rectum. Magnified 2,000. N.B. — The drawings in this figure and in Fig. 131 are made from prepara- tions fixed wet with Schaudinn's fluid and stained with iron-hsematorylin ; in such preparations the trypanosomes always appear appreciably smaller than in films stained with the Romano wsky-stain (see Minchin, 479) ; con- sequently these figures, though drawn to the same magnification as Figs. 1 1 , 127, etc., arc on a slightly smaller scale ; compare the trypanosome drawn in 0 with those in Figs. 11, A, and 127, A. remain so for at least three months, without being reinfected. From the rectal stock trypahiform individuals arise by a process of modification of the crithidial forms, in which the flagellum grows in length, the anterior portion of the body becomes more drawn out, the kinetonucleus migrates backwards behind the trophonucleus, taking with it the origin of the flagellum, and an undulating mem- THE H^MOFLAGELLATES AND ALLIED FORMS 301 brane running the length of the body is established. The trypani- fonn individuals thus formed are of small size and broad, stumpy form ; they represent the propagative phase which passes from the flea back into the rat. From the rectum they pass forwards into the stomach, and from the stomach they appear to be regurgitated into the rat's blood when the flea feeds. Experiments show that the flea becomes infective to the rat in about six days after it first took up the trypanosomes from an infected rat. The intracellular phase is at the height of its develop- ment about twenty-four hours after the flea takes up the trypano- somes ; the rectal phase begins to be established towards the end of FIG. 131. — Trypanosoma lewisi : developmental phases from the rectum of the rat- flea. A, Early rectal form ; B, crithidial form attached to wall of rectum ; C, D, division of crithidial form ; E, clump of orithidial forms detached from wall of rectum, hanging together by their Hagella, one of them beginning to divide ; F, O, H, crithidial forms without free flagella ; /, rounded form Without flagellum ; J, K, L, M, series of forms transitional from the crithidial to the final trypaniform type ; N, the last stage in the flea. Magnified 2,000. the first or beginning of the second day ; and the stumpy, trypani- form, propagative phase is developed in the rectum towards the end of the fifth day. The account of the development of T. lewisi in the flea given in the fore- going paragraphs is based upon investigations, some of them as yet unpub- lished, carried on in conjunction with Dr. J. D. Thomson by the author (480-482). Some of the phases of the parasite have also been described by Swellengrebel and Strickland (517). A number of investigators — namely, Prowazek (497), Breinl and Hindle, Baldrey (396), Rodenwaldt, and others — have studied the development of this trypanosome in the rat-louse (Hcemato- pinus spinulosus). Experiments have shown that this insect is also capable of transmitting the trypanosomo from rat to rat, but only, to judge from the 302 THE PROTOZOA published results, in rare instances, in striking contrast with the ease with which the transmission is effected by the rat-flea. The rat-louse may be regarded, therefore, as a host in which the trypanosome establishes itself only exceptionally, and by no means as the host to which it is best adapted. Crithidial and other forms have been seen in the louse, but the intracellular phase has not been observed, and it is probable that most of the forms de- scribed from this host are degenerating forms maintaining a feeble and pre- carious existence under adverse conditions, and destined to die off and dis- appear sooner or later. The developmental cycle of Schizotrypanum cruzi in the bug Conorhinua megistus has been described by Chagas,* and is briefly as follows (Fig. 132) : FIQ. 132. — Phases of Schizotrypanum cruzi in the bug Conorhinus megistus. a, b, and c, Forma transitional from the ordinary trypanosomes to the rounded forms ; d, clump of rounded forms ; e and /, change of rounded into crithidial forms ; g and h, crithidial forms ; t, trypaniform type from the salivary glands ; j, encapsuled form from the intestine. After Chagas (425). The trypanosomes taken up by the bug into its stomach change in about six hours ; they lose their flagellum and contract into rounded, Leishmania- like forms, which multiply actively by fission. After a time multiplication ceases, and the rounded forms become pear-shaped, develop a flagellum at the pointed end, and change into typical crithidial forms which pass on into the intestine, and there multiply by fission. In this way the characteristic condition of the infected bug is produced, with the intestine containing a swarm of trypanomonad individuals multiplying actively. The final stage in the insect is a small trypaniform type which is found in the body- cavity and salivary glands, whence it doubtless passes into a vertebrate host again. * A critical summary and review of the memoir of Chagas is given by Minchin in Nature, voL Ixxxiv., pp. 142-144 (August 10, 1910), with three text-figures. THE H^MOFLAGELLATES AND ALLIED FORMS 303 The three principal phases in the development of T. cruzi in the bug may be compared, without difficulty, with those of T. lewisi in the flea, though differing in minor details ; in both cases an early multiplicative phase in the stomach is followed by a crithidial phase, also multiplicative and constituting the principal stock of the parasite, in the hinder part of the digestive tract ; to this succeeds a propagative trypaniform phase, which in the case of T. lewisi passes forwards to the stomach, but which in the case of T. cruzi appears to pass through the wall of the alimentary canal into the body- cavity, and so into the salivary glands. Other developmental forms have been described by Chagas, but their relation to the cycle of the parasite, if indeed they really belong to it, is not clear. The developmental cycle of the trypanosomes of fresh- water fishes in the leech Hemidepsis marginata (Robertson, 603) begins also by active multi- plication in the crop about six to nine hours after the flagellates have been ingested. The trypanosomes divide by repeated binary fission of unequal type, budding off small individuals which are crithidial in type and multiply in their turn. In a few days the crop is populated by a swarm of trypano- monad forms of various sizes, multiplying actively. Towards the end of the digestion, the propagative phase begins to appear in the form of long, slender trypaniform individuals which arise directly from the crithidial forms, and pass forwards in great numbers from the crop into the proboscis-sheath, whence they are inoculated by the leech into a fresh host. A certain number of the crithidial forms remain behind in the crop, however, where during hunger- periods they may pass into a resting Leishmania-form ; when the crop is again filled with fresh blood, these forms begin to multiply again, repopulating the crop with crithidial forms, from which a fresh batch of trypaniform propagative individuals arise towards the end of digestion again. In the development of T. raice in the leech Pontdbddla muricata (Robertson, 500, 502), the ingested trypanosomes multiply in the crop in a similar manner by unequal binary fission, budding off small individuals which, however, are rounded and leishmanial in type, and which pass down from the crop into the intestine, where they develop a flagellum, become crithidial in type, and multiply actively. During hunger-periods they become leishmanial, resting forms which persist when all other forms have succumbed and died out, becom- ing crithidial again when the supply of food is renewed. From the crithidial forms arise the long, slender trypaniform individuals of the propagative phase, which pass forward into the proboscis to be inoculated into the fish. The development of T. vittatae, from the blood of the Ceylon tortoise, Emyda vittata, in the leech Glosfiphonia sp., is of a similar type, but takes place almost entirely in the crop (Robertson, 501). The development of T. gambiense in the tsetse-fly, GRossina palpalis, so far as it has been described by Kleine (457), Kleine and Taute (459), and Bruce and his collaborators (415), presents some peculiar features not quite intelli- gible at present. The whole development takes a long time, about eighteen to twenty-five days or more, a fact which, together with the low percentage of. flies which become infected, accounts for the existence of a developmental cycle having been missed by so many investigators, until it was first dis- covered by Kleine. From five to seven days after the infection of the fly the trypanosomes disappear or become scarce in its digestive tract, indicating, possibly, an intracellular stage yet to be discovered. Later, in a small percentage of the flies, the trypanosomes reappear in the digestive tract in enormous numbers. The flagellates at this stage vary greatly in size, form, and appearance, but crithidial forms are stated to be very rare, a feature in which the development contrasts with the usual type seen in other trypano- somes. Finally an invasion of the salivary glands takes place, though in what way it is brought about is not clear ; short, stumpy trypaniform individuals are found in the glands, which represent the ripe, propagativ* phase destined to be inoculated into the vertebrate host. These ripe forms first make their appearance, according to Kleine, in the intestine. 304 THE PROTOZOA In many species of trypanosomes transmitted by tsetse-flies, a peculiar mode of development occurs, as already stated, in the proboscis, termed by Roubaud, who discovered it, a culture d'attente. The trypanosomes taken up from the vertebrate change very rapidly into trypanomonad ("leptomonad," Roubaud) forms, with the kinetonucleus far forward, and attach themselves by the tip of the flagellum to the wall of the proboscis tube. In this situation they multiply in the salivary fluid by binary fission, until great numbers are present. In some cases this culture in the proboscis appears to be the sole form of developmental cycle in the fly, as, for example in T. cazalboui (Roubaud, 506, Bouffard), T, vivax (Bruce, 411, iii.) ; this type is termed by Roubaud evolution par fixation directe. In other species (T. dimorphon, T. pecaudi) the parasite multiplies first in the digestive tract of the fly, and then spreads forward into the proboscis— -evolution par fixation indirecte of Boubaud ; in this case, however, the possibility does not seem to be excluded that the forms seen in the digestive tract may have belonged to the developmental cycle of a distinct trypanosome. Development of this kind has only been observed in tsetse-flies. According to Bouffard, T. cazalboui can be transmitted mechanically by Stomoxys, but goes through its developmental cycle only in the proboscis of Olossina palpalis ; Stomovys may therefore cause epidemics of the disease (" souma "), but endemic areas are always in regions where O. palpalis occurs. The tsetse-fly is not infective until six days after first feeding on an infected animal, and it then remains infective permanently, or at least for the greater part of its existence. Hence the proboscis- cycle is a rapid develop- ment, comparable, as regards the time it requires, to that of T. lewisi in the flea rather than to that of other trypanosomes in the digestive tract of the tsetse. Finally,* mention must be made of the cysts of T. grayi, described by Minchin (476), occurring in the hind-gut of Olossina palpalis. The cysts result from the encystment of a crithidial form, and are very similar to the cysts of Herpetomonas, described by Prowazek (Pig. 124), from the hind-gut of the house-fly ; their mode of formation indicates that they are destined to pass out of the rectum to the exterior with the faeces, and Minchin has suggested that a contaminative method of spreading the infection may occur in addition to the usual inoculative method. The possibility must be reckoned with, however, that the cysts in question may be part of the cycle of a distinct flagellate parasite, perhaps peculiar to the fly alone, and may not belong at all to the life- cycle of T. grayi, which has now been shown to be the developmental form of the trypanosome of the crocodile (cf. Cystotrypanosama, Roubaud, 557 -5). According to Kleine and Taute, trypanosomes, not encysted, may be found in the faeces of infected tsetses. Apart from the somewhat aberrant development of the members of the 6rwctt-group, which require further elucidation, the cycle of a trypanosome in the invertebrate host appears to consist typically of three principal phases : (1) An initial multiplicative phase, which may be trypaniform, as in T. lewisi, or Leishmania - like, as in T. cruzi, or may take the form of unequal division of large trypani- form individuals to produce either small crithidial forms directly, as in fish-trypanosomes in the leech Hemiclepsis, or rounded Leishmania-torma which later become crithidial, as in T. raice and T. vittacet ; to this initial phase succeeds (2) a crithidial phase, which may pass farther down the alimentary canal, and which in any case multiplies by fission and constitutes the principal stock of the * The development described by Schaudinn (132) for T. noctuee is dealt with in a subsequent chapter (p. 390). THE H^EMOFLAGELLATES AND ALLIED FORMS 305 parasite, keeping up the infection of the invertebrate host. In hunger-periods the flagellates may persist as simple, rounded, Leishmania-like forms. Sooner or later many, it may be the greater number, but not all, of the crithidial forms become modified into the trypaniform individuals, which represent (3) the propagative phase of the parasite, and pass forwards to be inoculated into the verte- brate host. Those crithidial forms which do not become trans- formed into the propagative individuals remain to multiply and replenish the stock. A very much debated question in this development is that relating to the occurrence of sexual phases and syngamy, which, purely on the analogy of the malarial parasites, are assumed almost universally to occur in the invertebrate host. Not in a single instance as yet, however, has the sexual act been proved satisfactorily to take place in the development of trypano- somes. The fertilization described by Schaudinn (132) in " T. noctuce " is the well-known conjugation of Hctiteridium, which can be observed without difficulty ; and though Schaudinn described so-called "male " and " female " types of trypanosomes in the mosquito, he expressly stated that they did not and could not conjugate. The process of syngamy described by Prowazek (467) for T. leurisi in the rat-louse, though " confirmed " by Baldrey (396), Gonder(445'5), and Rodenwaldt, is almost certainly the agglomeration of degenerating forms (Swellengrebel, 516 ; compare Reichenow, 78, p. 268). Less biassed investigators, who have studied the developmental cycle of trypanosomes with great care, such as Chagas, Robertson, and others, have been quite unable to observe sexual processes of any kind. .The liability to error in the interpretation of observations is greatly increased, first by the fact that trypanosomes divide longitudinally and often unequally, secondly by the phenomena of agglomeration (p. 128), which occur readily under un- favourable conditions. Consequently the adhesion together of two trypano- somes may be due to quite other causes than sexual affinity. In some cases the alleged occurrence of syngamy has been based merely on the fact that non- flagellated forms have been seen, which, on the analogy of the malarial parasites (p. 362), are termed " ookinetes " and interpreted as zygotes. It is certainly remarkable, in view of the paucity of data, that so many investigators, following Schaudinn's lead, should persist in ascribing all form- differentiation in trypanosomes to sex, and should be unable, apparently, to conceive of any other cause of polymorphism in parasites which have to adapt themselves, in the course of their life-cycle, to a great diversity of conditions (compare also Doflein, 430). It must be emphasized that the only true criterion of sexual polymorphism is sexual behaviour, and until that has been established it is premature to speak of sexual differentiation. Some investigators have upheld the unfashionable view that the syngamy of trypanosomes occurs in the vertebrate host ; so Bradford and Plimmer, and more recently Ottolenghi, who has described in T. brucii, T. equinum, T. gambiense, and T. equiperdum, the following process of sexual conjugation : Two trypanosomes of very different size and appearance attach themselves to one another by their hinder ends. One, regarded as the microgamete, is more slender, and contains one trophonucleus or a larger nucleus of this kind and two smaller (reduction-nuclei) ; the other, the macrogamete, is much larger, and contains also a larger nucleus near the kinetonucleus and two or more other nuclei in process of degeneration. The macrogamete also has usually three, sometimes two or four, nagclla and undulating membranes. After the two gametes have united by their hinder ends, a small nucleus is budded off from the principal nucleus of the microgamete, passes over into the body of the macrogamete, and fuses with its principal nucleus. Subsequently the microgamete appears to degenerate, and the fertilized macrogamete to 306 THE PROTOZOA divide up into trypanosomes of the ordinary type. Those who consider that syngamy can only occur in the invertebrate host will doubtless regard the process described by Ottolenghi as phenomena of agglomeration and de- generation. In the present state of our knowledge, however, it is best to keep an open mind on this question, and to await further investigations. In T. gambiense, Moore and Brein) (484) have described a process of fusion between the kinetonucleus and trophonucleus in the formation of the " latent bodies," and have interpreted this as a sexual process, a suggestion hardly to be taken seriously. A similar process alleged to occur in the multiplication- forms of T. lewisi has been interpreted by Schilling as the inevitable autogamy. All that can be said at present, with regard to sexual processes in trypano- somes, is that, on the analogy of other Protozoa generally, syngamy may be expected to occur in some part of the life-cycle. It remains, however, for further research to establish definitely the conditions under which syngamy takes place, and the nature of the process in these organisms ; nor can it be considered as sound reasoning, in the absence of concrete observations, to at- tempt to limit the possible occurrence of syngamy, or to infer the exact form it takes, either by analogies more or less far-fetched with one or another group of Protozoa, or by the mere existence of form-differentiation, and still less by the arbitrary interpretation of certain forms as zygotes or ookinetes. A very variable feature in the development of trypanosomes is the sus- ceptibility of the invertebrate host. In the case of T. leurisi, only about 20 per cent., approximately, of the fleas fed experimentally on infected rats become infective in their turn, and in the case of tsetse-flies and pathogenic trypanosomes the percentage is much smaller. There are also grounds for suspecting that a certain condition or phase of the trypanosome in the blood of the vertebrate is sometimes necessary for establishing the developmental cycle in the invertebrate ; compare the observations and conclusions of Chagas with regard to Schizotrypanum cruzi, mentioned above (p. 296). In Trypano- soma noctuce the summer form which appears in the blood is of a type distinct from the winter forms found in the bone-marrow (Minchin and Woodcock, 42). On the other hand, in the case of the trypanosomes of fresh- water fishes, Robert- son (503) found that every leech became infected that was fed on an infected fish ; so that the simplest method of determining whether a fish was infected was to feed a newly-hatched Hemidepsis on it. A question often discussed is whether trypanosomes in any part of their development may pass through " ultramicroscopic " stages. Schaudinn (132) expressed the opinion that some stages of trypanosomes investigated by him were small enough to pass through bacterial filters ; though he did not put this suggestion to an experimental test, it is often quoted as a proved fact.* Moore and Breinl (484) also asserted, without experimental data, that infected blood remained infective after filtration. On the other hand, attempts by Bruce and Bateman to obtain experimental verification of these statements gave negative results (compare also Report XI., p. 122, of the Sleeping Sickness Commission). Recently it has been asserted by Fry that T. brucii can throw off granules which, when liberated, possess a certain motility of their own in the blood ; this process is regarded as " essentially of a vital and not a degenerative nature." That a trypanosome or any other living cell might excrete grains which when set free could exhibit movements due to molecular or other causes is highly probable ; but that such grains represent a stage in the life- history of a trypanosome is far from being so ; nor can analogy with spiro- chsetes be considered as a legitimate argument in favour of any such con- clusion. There remains for consideration the development which trypanosomes undergo in artificial cultures, in which they exhibit a series of forms quite different from those seen in the blood of the vertebrate, and so far resembling the cycle in the invertebrate host in that the predominant phase is a crithidial * It is doubtful whether the forms of which Schaudinn made this statement were really trypanosomes or spirochaetes. THE H^MOFLAGELLATES AND ALLIED FORMS 307 or trypanomonad type of flagellate. Until the cultural development of a trypanosome has been compared in detail with its natural development in the invertebrate host, it is impossible to estimate precisely the bearing of the cultural series of stages from the point of view of the physiology and mor- phology of the parasite. The only investigator who has attempted this is Chagas (425), who found in cultures of Schizotrypanum cruzi the same three principal phases — namely, rounded, crithidial, and trypaniform — that occvu in the natural cycle, and in the same order of sequence. At present, therefore, it would be unprofitable to discuss in detail the series of forms occurring in artificial cultures, and it must suffice to refer the reader for further infor- mation to the principal works on the subject, namely, those of Novy and McNeaJ (489), Bouet, Franca (438, 443), Rosenbusch, Thomson (525), Wood- cock (527), Lebedew, and Doflein (431). As already pointed out above, the cultural method is often of the greatest practical value in determining whether, in a given case, an animal is infected with trypanosomes or not. Lebedew has described what he believes to be syngamy in the cultural phases of T. rotatorium ; compare also the account of Leishmania below (p. 319). The genus Trypanosoma comprises a vast number of species, parasitic in the blood of animals throughout the vertebrate series ; and several attempts have been made to subdivide and classify Fid. 133. — Endotrypanum schaudinni from the blood of Cholwpua didactylus. A — E, Various forms of the intracorpuscular parasite ; F, trypanosome from the blood of the same host. After Mesnil and Brimont, magnified about 1,500 diameters. this comprehensive genus into smaller groups. Such attempts have either taken the course of splitting off particular forms, char- acterized by some special peculiarity, from the main group, or of subdividing the group as a whole on some principle of morphology or development. An example of the first method is tAe foundation by Chagas (425) of the genus Schizotrypanum, as already mentioned, for T. cruzi, on the ground that it multiplies by schizogony and possesses intracorpuscular phases. The genus Endotrypanum was proposed by Mesnil and Brimont for a peculiar form which was discovered by them within the red blood-corpuscles of a sloth (Cholcepus didactylus), and which is very probably an intracorpus- cular phase of a trypanosome found free in the blood-plasma of the same host. The life-cycle of Endotrypanum is not yet known. Chagas considers it not improbable that it should be placed in the 308 THE PROTOZOA same genus as T. cruzi, in which case the name Endotrypanum has the priority over Schizotrypanum. In the present state of know- ledge, data are lacking for deciding how far it is possible to employ either multiplication by schizogony or an intracorpuscular habitat as characters for defining genera of trypanosomes* An intra- corpuscular habitat is probably commoner in trypanosomes than has usually been supposed. It has been described quite recently by Buchanan in T. bntcii. Attempts to subdivide the genus Trypanosoma as a whole have been based on the possibility that the trypanosome-type of structure may have had two distinct phylogenetic origins, one through Leptomonas and Crithidia from a cercomonad ancestor, the other through Trypanoplasma from a heteromastigote or Bodonid type. The trypanosome-form might be imagined to have arisen from either of these two types. It could be derived from a form like Trypanoplasma by loss of the free anterior flagellum, in which case the flagellum of a taypanosome is to be regarded as posterior ; on the other hand, if, in a form like Leptomonas, the kinetonucleus and with it the origin of the flagellum, be shifted backwards to the neighbourhood of the trophonucleus, and if at the same time the flagellum runs forwards along the body connected to it by an un- dulating membrane, a Crithidia-like form results, from which, by still further displacement backwards of the kinetonucleus and flagellum to near the posterior end of the body, a trypanosome- form is produced in which the single flagellum is to be regarded as anterior. It is therefore conceivable that the trypanosome-form may comprise two morphological types, structurally indistinguish- able, but entirely different in origin, and opposite in morphological orientation of the body. From this point of view, Woodcock (395) subdivided trypano- somes into two genera : Trypanomorpha, with cercomonad ancestry and flagellum morphologically anterior ; and Trypanosoma, in a restricted sense, with heteromastigote ancestry and flagellum morphologically posterior. The genus Trypanomorpha included only one species, T. noctuce of Athene noctua ; all other species of trypanosomes were left in the genus Trypanosoma sens, strict. Liihe put forward a classification based on similar conceptions with different interpretations, and proposed three genera of trypano- somes : Hcematomonas (Mitrophanow) for the trypanosomes of fresh- water fishes believed to have a heteromastigote ancestry ; Trypano- zoon for the trypanosomes of mammals, such as T. lewisi, T. brucii, etc., regarded as having a cercomonad ancestry and an anterior flagellum ; and Trypanosoma sens, strict, for the trypanosomes of frogs and reptiles. T. noctwe, on the other hand, he regarded, in agreement with Sch&udinn (see p. 390, infra), merely as a develop- "THE H^MOFLAGELLATES AND ALLIED FORMS 309 mental stage of Hcemoproteus. Although, however, it is quite possible that some trypanosomes may have a heteromastigote ancestry, all the developmental facts hitherto discovered indicate a cercomonad ancestry with a single anterior flagellum, and there is no concrete evidence of a heteromastigote origin for any species that has been studied up to the present. Trypanoplasms, so far as they have been studied, preserve their biflagellate, heteromastigote type of structure throughout their development in all active phases, and never pass through a trypaniform or crithidial phase. Try- panosomes, on the other hand, show constantly a crithidial phase in the invertebrate host, but have not been observed in any case to be heteromastigote or even biflagellate, except temporarily during division, in any phase of the life-history. Consequently, attempts to subdivide trypanosomes on a -morphological or phylogenetic basis mast be regarded at present as premature (compare also Laveran, 461). II. THE GENUS TRYPANOPLASMA. The peculiar distribution and occurrence of the species of this genus has been pointed out above. Originally founded for forms parasitic in the blood of fishes, it now comprises a somewhat heterogeneous collection of species, some of which were formerly referred to other genera of Flagellates. Of recent years, the number of species known to be parasitic in invertebrate hosts has increased, and is increasing rapidly. Such are T. (" Trypano- phis ") grobbeni, found in the gastro vascular system of Siphdnophora (Keysselitz, 453) ; T. (" Bodo ") helicis, from the receptaculumseminis of Helix pomatia and other snails (Friedrich) ; T. dendroccdi, from the digestive tract of Dendroccdum lacteum (Fantham and Porter, P.Z.S., 1910; p. 670) ; T. vaginalis, from the female genital organs of leeches (Hesse, C.R.A.8., cli., p. 504) ; and T. gryllotalpce, from the end-gut of Gryllotalpa vulgaris (Hamburger). These examples show that the genus, as at present defined, is of widespread occur- rence. It may be doubted, however, if the various species described should all be placed together. The species of Trypanoplasma parasitic in blood are only known as yet from fresh-water fishes ;* they have an alternation of hosts, being transmitted by leeches. The life-history of the intestinal trypanoplasms has not been investigated, but in all probability they have but a single host, which acquires the infection by swallowing accidentally their cysts or other resting stages passed out from a * The " Trypanoplasma " stated by Bruce and his colleagues (412, pp. 495, 496) to occur in the blood of birds and in the digestive tract of tsetse-Hies was in reality a Leucocytozoan. 310 THE PROTOZOA former host. T. helicis, according to Friedrich, passes from one snail to another mechanically in the spermatophores during coitus. The following account refers mainly to the blood-inhabiting species : The body of a trypanoplasm is relatively broader and shorter, less sinuous and serpentine, than that of a trypanosome, and is at the same time softer and more plastic, being limited by an extremely thin periplast. The contractile, often slightly metabolic, body yields readily to pressure, and exhibits in consequence passive form-changes when moving among blood-corpuscles or B FIG. 134. — A, Trypanoplaama abramidis from the blood of the bream ; B and C, T. keysselitti from the blood of the tench : B, small ordinary form ; C, large form. After Mincnin, magnified 2,000. other solid particles. The principal structural feature is the possession of two flagella, which arise close together at the anterior extremity from a pair of blepharoplasts or diplosome, or from a single basal granule (Martin). One flagellum projects freely for- wards ; the other turns more or less abruptly backwards, and passes down the side of the body at the edge of an undulating membrane to the hinder end, beyond which it projects freely backwards to a variable extent in different species. In T. gryttotalpoe the un- THE H^EMOFLAGELLATES AND ALLIED FORMS 311 dulating membrane only extends along two-thirds of the length of the body, after which the posterior flagellum becomes free. The kinetonucleus, situated at the extreme anterior end of the body, is relatively very large, usually exceeding the trophonucleus in size, and is sometimes constricted into two or three portions, but is generally a compact mass which stains deeply in preparations. In T. helicis, according to Jollos, it is prolonged backwards into fibrils, usually two in number, which extend some way down the body, and are probably comparable to an axostyle. The tropho- nucleus has a vesicular structure with a conspicuous karyosome. Its position in the body varies, being in some species close behind the kinetonucleus, in others near the middle of the body. It often appears to be lodged completely in the undulating membrane, which in this genus is often very broad and less sharply defined than in a trypanosome, appearing as the border of a flattened body. The cytoplasm frequently contains numerous " chromatoid grains." Trypanoplasms in the blood of fishes often exhibit marked polymorphism, with two extremes of size, small and large (Fig. 134, B, C). According to Keysselitz (454), the large forms are the gametes which conjugate in the leech, and are distinguishable as male and female forms, but the statement requires confirmation. From the investigations of Robertson (503) on the development, it appears more probable that the large forms are simply full-grown individuals, ripe for multiplication by fission. Unfortunately, next to nothing is known of the reproduction of the parasites in the vertebrate host, though it has been observed that their numbers are subject to considerable fluctuations, and that a fish showing at one time a very scanty infection of the blood may have a " relapse," and appear later well infected. Keysselitz accuses these parasites of pathogenic properties, but this charge is founded on observations on fish in captivity, in which weakened powers of resistance may lead to abnormal activities on the part of the parasite (compare also Neresheimer). The development of blood - trypanoplasms in the invertebrate host, which is in all known cases some species of leech, appears to be of a comparatively simple type as compared with that of trypano- somes, and consists of little more than rapid multiplication by binary fission to produce a swarm of relatively small trypano- plasms, some of which, more slender and elongate in form, pass forwards into the proboscis, and are inoculated by the leech into a fish. Conspicuous in this development, as compared with that of trypanosomes, is the entire absence of any uniflagellate forms, crithidial or other. So long as a trypanoplasm is in an active state, it is invariably biflagellate. Resting forms without a locomotor apparatus may occur. In T. helicis, Friedrich describes winter 312 THE PROTOZOA forms with a single nucleus, which is in some cases the tropho- nucleus, in others the kinetonucleus. The accounts given of the process of division are somewhat conflicting. According to Martin, division of T. congeri is initiated by the division of the single basal granule of the flagella, followed by splitting of each flagellura longitudinally. Next the trophonucleus divides amitotically, the karyosome becoming first drawn out into a band, after which the nucleus as a whole is constricted into two. Lastly the kinetonucleus becomes elongated, and divides simply by a transverse constriction into two pieces. Jollos, however, following Bosenbusch's statements for trypanosomes, affirms that the division of both nuclei is mitotic in T. helicis. Alexeieff, on the other hand, denies that the kinetonucleus of Trypanoplasma is a nucleus at all. This author also describes a series of chromatinic blocks at the base of the undulating membrane of T. intestintUis, similar to those seen in Trichomonas (compare Pig. 6). Keysselitz (464) has described syngamy in the development of T. " borrdi " in the leech Piscirola, but the description and figures are unconvincing, and the matter requires reinvestigation. No other investigators have found sexual processes of any kind in trypanoplasms. III. THE GENUS CRITHIDIA. The distinctive structural feature of Crithidia (Fig. 135, A) is the relatively short undulating membrane which, with the single flagellum, arises in the middle of the body from the vicinity of a kinetonucleus situated beside, or in front of, the trophonucleus. The form of the body varies from a relatively long, slender type to the short, " barley-grain " form from which the name of the genus is derived. As already pointed out, the application of the name Crithidia as the denomination of a genus is involved in considerable confusion and perplexity — partly because the distinctive morphological characters shade off by imper- ceptible gradations into those of trypanosomes on the one hand, and leptomonads on the other, but still more because a certain number of the " species of Crithidia " are unquestionably de- velopmental stages either of trypanosomes or leptomonads, and others are justly suspected of being so. In the present state of know- ledge, it is safest to presume that any " Crithidia " from the digestive tract of a blood-sucking insect is a stage of a trypano- some from the blood of a vertebrate, until the contrary has been clearly established. At the same time the possibility must always be taken into account that a blood-sucking invertebrate may harbour flagellate parasites peculiar to itself in addition to those — VL Fio. 135. — Crithidia minuta, I^ger, from the gut of Tabanua tergestinue. A, Or- dinary motile indi- vidual; B, O, young forms, with flagel- lum short or rudi- mentary. After Leger. THE H^EMOFLAGELLATES AND ALLIED FORMS 313 which it takes up in vertebrate blood, and that in this way stages of the life-cycle of two or more distinct parasites may be confused together. Up to the present, however* no blood-sucking insect has been proved satisfactorily to harbour flagellate parasites not derived from vertebrate blood. After deducting doubtful species of Crithidia, there remains a residue which appears to comprise genuine, independent species, parasites of the digestive tract of insects. As examples of such species may be cited C. campanulata, recorded from the digestive tract of Chironomus plumosus (Leger, A.P.K., ii. 1903, p. 180), from that of the larva of Ptychoptera (Leger and Duboscq) and of caddis- worms (Mackinnon, 547) ; 3. gerridis, from Oerris spp. (Patton, 550 ; Porter, 555) ; and possibly others. The life-cycle of C, gerridis has been investigated by Patton and Porter. The parasite appears under two principal phases : an active, flagellate phase, which grows to a large size, and multiplies by fission, sometimes very actively, forming rosettes ; and a resting, non- flagellate Leishmania - form. The flagellate forms may be free in the digestive tract, or may attach themselves to the lining epithelium of the gut by their flagella. The non-flagellate forms are found in the crop, where they grow into the adult phase, and in the rectum, where they become encysted. The flagellate phase is found throughout the digestive tract and in the ovaries, but has not been observed to pass into the ova The encysted forms pass out of the rectum, and infect new hosts by the contaminative method. IV. THE GENUS LEPTOMONAS (HERPETOMONAS). The genus Leptomonas comprises typical intestinal parasites of insects, especially Diptera and, above all, Muscidce. Several species are also known in Hemiptera. They are in most cases parasites of the insect alone, having no alternate host, and infection is brought about by the contaminative method, so far as is known, cysts dropped by one host being accidentally devoured by another. But some species are found as parasites of the latex of Euphor- biaceae, and in this case an alternation of hosts occurs. The para- sites are taken up from the plants by bugs (Hemiptera) which suck their juices, and by the agency of the bugs the flagellates are inoculated into other plants again (Lafont ; Bouet and Roubaud, 530 ; Fran§a, 537, 538). There can be little doubt that in this case the bug is the primary, the plant the secondary host. The plants, or the parts of them that are infected by the Leptomonas, suffer considerablv. The term " flagellosis " has been proposed for the disease. 314 THE PROTOZOA The distinctive structural features of this genus are the possession of a single flagellum, arising from close beside a kinetonucleus which is placed far forwards in the body, and the entire absence of an undulating membrane (Fig. 136, B ; Pig. 137, d). As already stated above, however, the application of the names Leptomonas and Herpetomonas is much disputed, and the morphological defini- tion of the genera in question is attended with considerable diffi- culties, chiefly owing to the fact that in one and the same host a great variety of forms may occur, with regard to which it is not possible, in the present condition of knowledge, to state with cer- tainty whether they represent distinct species of flagellates, refer- able even to distinct genera, occurring fortuitously in the same host, or whether they are all merely developmental phases of the same species. The following are the principal forms which may K In A ^D frV* V" E.'1' F © FlQ. 136. — Leptomonas jacvlum, Leger, from the intestine of Nepa cinerea. A, B, Monad forms ; G, division of a monad form ; D, monad form with short flagellum ; E, F, O, gregarine-like forms : E, in division, F, attached to an epithelial cell by the rudimentary flagella, which resemble the rostra of gre- garine sporozoites. After Leger. occur together in the same host : (1) Large, biflagellate individuals (Fig. 124, A), often with a distinct pair of rhizoplasts connected with the two flagella, the type to which, according to one set of opinions, the name Herpetomonos should be restricted, but which on another view represents merely an early stage in binary fission, with a daughter-flagellum precociously formed ; (2) smaller flagel- lates with a single flagellum (Fig. 136, B ; Fig. 137, d), the type for which the name Leptomonas is employed by those who regard the true Herpetomonas as typically biflagellate, while by those who hold the contrary view the two genera are ranked as synonyms ; (3) cri- thidialforms (Fig. 137, pa, mange, and ffirtpfia, a seed, on account of the sores and ulcers of the akin of fishes produced by Myxosporidia, arid the resemblance of their spores to little seeds. 323 324 THE PROTOZOA having nothing in common except the parasitic habit and the adaptations arising from it, more especially the propagation by spores. The modern tendency is rather to split up this vast assemblage into smaller groups, and to abolish the Sporozoa as a primary subdivision of the Protozoa. It is practically certain, at least, that the two main subclasses into which it is always divided are per- fectly distinct in their origin. The class Sporozoa is retained here solely in deference to custom and convenience, and without preju- dice to the affinities and systematic position of its constituents, a question which will be discussed when the group as a whole has been surveyed. The life-cycle of a Sporozoon may be started conveniently from the minute germ or sporozoite which escapes from the spore, or from the corresponding stage when spores are not formed. The sporozoite may have one of two forms : it may be an aincebula, a minute amoeboid organism ; or it may be of definite form, a little rod-like or sickle-shaped animalcule (" falciform body," " Sichel- keiru ") which is capable of twisting or bending movements, but retains its body-form, and progresses by gliding forwards ; for this second type of sporozoite the term " gregarinula " has been proposed in a previous chapter (p. 169). The sporozoite, whatever its form, is liberated in the body of the new host, and begins at once its parasitic career ; it nourishes itself and grows, often to a relatively huge size, at the expense of the host. This phase of the life-history is termed the " trophic phase," and the parasite itself during this phase a trophozoite, by which term is understood a parasite that is actually absorbing nourishment from the host. The trophozoite may be lodged within cells (cytozoic), or in tissues of the body amongst the cells (histo- zoic), or in some cavity of the body in which it either lies free or is attached to the wall (coalozoic). Whatever then- habitat, the trophozoites of Sporozoa never exhibit any organs or mechanisms for the ingestion or digestion of food, but absorb their nutriment in all cases in the fluid state, by osmosis through the surface of the body, from the substance of the host ; if pseudopodia or flagella are possessed by these parasites, they are never used for food- capture, except in so far as pseudopodia, by increasing the surface of the body, may augment its absorptive powers. The parasite may exhibit multiplicative phases in which it reproduces itself actively, so that there may be many generations of trophozoites within one and the same host, which may thus be quite overrun by swarms of the parasites. Multiplication of this kind, which is non-sexual, is known as schizogony ; the trophozoites which multiply in this manner are termed schizonts ; and the minute THE GREGARINES AND COCCTDIA 325 daughter-individuals, products of schizogony, are termed mero- zoites, to distinguish them from sporozoites which they may resemble closely. Sooner or later, however, the propagative phase, destined to infect new hosts, makes its appearance ; so-called sporonts (see p. 330, infra] multiply by sporogony, which is combined with sexual phases, to produce the sporo/oites. The life-cycle of the parasite may be passed entirely in one host, or there may be an alternation of hosts of different species, with a distinct series of phases of the parasite in each. When there is but a single species of host, the method of infection of new hosts is usually contamina- tive (p. 24), by means of resistant spores and cysts ; when there is an alternation of hosts, the infection may be inoculative (p. 23), without resistant phases, as in malarial parasites, or contamina- tive, with resistant phases, as in Aggregata (p. 353). Whether the life-cycle be of simple or complex type, it ends with the production of sporozoites, bringing it back to the starting- point again ; and in the vast majority of cases the sporozoites are enclosed, one or more together, in tough sporocysts to form the characteristic resistant spores. As a rule each spore arises from a single spore-mother-cell or sporoblast. The Sporozoa fall naturally into two subclasses, which' have received various names, according as one or another of their char- acteristic features has been considered diagnostic. It is best to define each subclass by a number of characteristics, since none by itself is sufficiently distinctive. In the first subclass the trophic and reproductive phases are typically distinct — that is to say, the animal becomes full-grown, and ceases to grow further, before reproduction begins, hence Telosporidia (Schaudinn) ; reproduction takes place usually by a process of multiple fission in which the daughter-individuals are budded or split off on the outer surface of the parent-body, hence Ectosporea (Metchnikoff) ; and the germs or sporozoites produced are gregarinulae, hence Rhabdogeniae (Delage and Herouard). In the second subclass the trophic and reproductive phases usually overlap — that is to say, the still-growing or even quite young trophozoite may begin to form spores, hence Neosporidia (Schaudinn) ; the spore-mother-cells are formed by a process of internal gemmation, being cut off within the cytoplasm of the parent, hence Endosporea (Metchnikoff) ; and the- sporozoites produced are amoebulae, hence Amcebogeniae (Delage and Herouard). Of the three contrasted characters by which the two subclasses are distinguished, the most absolutely diagnostic is probably the form of the sporozoite. The names Telosporidia and Neosporidia 326 THE PROTOZOA are, however, in more common use than the other names of the subclasses given above.* The subclass Telosporidia, as mentioned above, includes the three orders Gregarinoidea, Coccidiidea, and Haemosporidia. ORDER I. — GREGARINOIDEA. The chief characteristics of this order are — First, that the tropho- zoites are parasites of epithelial cells in the earlier stages of their growth, but in later stages they become entirely free from the cells, and lie in cavities of the body ; their most frequent habitat is the digestive tract, but sometimes they are found in the body-cavity or the haemocoele. The full-grown trophozoite is of relatively large size and definite form, with a thick cuticle as a rule. In addition to these characters, the reproduction and spore-formation, presently to be described, are quite distinctive in type, the most diagnostic feature being that each spore is the product of a single zygote. The Gregarines are an extremely abundant order of the Sporozoa, highly differentiated in structure, and comprising a great number of species classified into genera and families. They occur most commonly as parasites of the digestive tract or body-cavity of insects, but also as parasites of other classes, such as Echinoderms and Annelids ; in Molluscs they are comparatively rare, and, though, they occur commonly in Prochordata (Ascidians), they are not known from any class of Vertebrata in the strict sense of the word. In the early phases of development, during which the tropho- zoite is a cell-parasite, it may be entirely enclosed in the cell, or only attached to it by one extremity of the more or less elongated body. In the latter case the sporozoite may have the anterior end of the body modified into a definite rostrum, by which it attaches itself to the host-cell, and from which is developed a definite organ of attachment, termed an epimerite (Fig. 142, ep.), often of com- plicated structure, and provided with hooks and other appendages. When the cytozoic phase is past and the host-cell is exhausted, the parasite drops off, shedding its epimerite as a rule. In the earlier phase, in which an epimerite is present, the parasite was termed by Aime Schneider a cephalont (" cephalin "), and in the later phase a sporont (" sporadin "), the original use of this term, now applied in a wider sense to denote in this and other orders of Sporozoa those individuals about to proceed to spore-formation. The body of the Gregarine-sporont always contains a single nucleus, but may be divided into partitions or septa formed as ingrowths of the ectoplasm, and is then said to be " septate " or " polycystid." * The subclass Rhabdogenise, as instituted by Delage and Herouard, included the Sarcosporidia, which, however, arc almost certainly true Amoebogcniae. THE GREGARINES AND COCCIDIA 327 As a rule, in such cases there is but a single septum, which divides the body into two parts termed respectively protomerite and deuio- merite (Figs. 7, 142) ; but in the curious genus Tceniocystis (Leger, 616) there are a number of septa, giving the parasite a superficial resemblance to a segmented worm. The body of a gregarine consists typically of distinct ectoplasm and endo- plasm. The ectoplasm may be further differentiated into three layers : an ex- ternal cuticle or epicyte, a middle layer or sarcocyte, and a deeper contractile layer or myocyte containing myonemes (Fig. 29, p. 58). The epimerite, with its hooks and processes, is derived from the epicyte ; the septa, if present, from the sarcocyte. The endoplasm is usually extremely granular, and contains great quantities of stored-up food material in reserve for the reproductive processes ; chief amongst these substances are para- glycogen - spherules, extremely charac- teristic of these parasites. A remarkable feature of gregarines is the power possessed, by many species, of gliding forward, often at a great pace, without any visible organs of locomotion. Two explanations have been given of these movements: (1) by Schewiakoff, that they are due to extrusion of gelatinous fibres from the hinder end of the body, secreted between the epicyte and sarcocyte ; (2) by Crawley. that the movements are produced by contrac- tions of the myonemes which are only present in motile forms. In motionless forms the ectoplasm is very thin, and consists of epicyte alone. The nucleus of a gregarine is usually very large, spherical, and vesicular in type, with one or more distinct karyo- somes. It is typically single, except in the cases of precocious association men- tioned below — exceptions, however, which are only apparent, since in such cases the gregarine represents in reality two individuals fused into one. In the septate forms the nucleus lies in the deutomerite normally. In Pterocephalus (Nina), however, a second nucleus, which appears to be of transitory nature and to take no share in the repro- ductive processes, has been discovered in the protomerite (Leger and Duboscq, 621). The nucleus-like body observed by Siedlecki in Lankesteria ascidiae, and by Wenyon (84) in L. cidicis, occurring at the point of contact of the two associated sporonts in the cyst, is perhaps a body of similar nature. The nucleus of Callyntrochlamys phronimce is remarkable for being surrounded by a halo composed of radiating processes, each a thin tubular evagination FIG. 142. — Examples of gregarinea in the " cephalont " condition. A, Actinocephalua cHigocanthus ; B, Stylarhynchua longicollis. ep., Epimorite ; pr., protomerite ; d., deutomerite. After Schneider. 328 THE PROTOZOA of the nuclear membrane (Dogiel, 605) ; as a rule the surface of the nucleus is perfectly smooth. Chromidia are stated to occur in the cytoplasm of some gregarines (compare Kuschakewitsch). According to Comes, they are scarce in normal individuals, but become abundant with over-nutrition ; since he states, however, that they arise in the cytoplasm, it is possible that they represent grains of the nature of volutin rather than true chromidia. According to Drzewccki, however, the nucleus of Monocystids may, during the early growth of the trophozoite, break up into chromidia and be re-formed again, or may throw out vegetative chromidia which are absorbed in the cytoplasm ; Kuschake- witsch, however, regards this as a degenerative process. Drzewecki affirms that Stomatophora (Monocystis) coronata, from the vesiculae seminales of Pheretima sp., possesses a mouth-opening in a peristome, and an anal aperture, and takes up solid food in the form of the spermatozoa of its host. If so it is quite unique, not only among gregarines, but among Sporozoa generally. The ingested spermatozoa are stated to be taken up and digested by the nucleolus (karyosome). According to Hesse, the supposed mouth and peristome are parts of a sucker-like organ of attachment. The alleged nucleolar digestion is perhaps a misinterpretation of the extrusion of chroma - tinic particles from the karyosome. The Gregarines are subdivided at the present time into two suborders characterized by differences in the life-cycle. In the first suborder, known as the Eugregarinae, the parasite has no multiplicative phase, but the trophozoites proceed always as sporonts to the propagative phase by a method of reproduction (sporogony) which is combined with sexual processes, and leads to the formation of resistant spores. In the second suborder, the Schizogregarinse, the trophozoites which arise from the sporozoites become schizonts which multiply for several generations non-sexually, by schizogony, before a generation of sporonts (gamonts or gameto- cytes) is produced which proceed to reproduce themselves by sexual sporogony. Stated briefly, the Eugregarinae have only a propaga- tive phase, sporogony, in their life-cycle ; the Schizogregarinse have both a multiplicative phase, schizogony, and sporogony. The sporogony is of essentially the same type in both orders. It is simplest, therefore, first to describe the life-cycle of a eugregarine, and then to deal with the multiplicative phases of the schizogre- garine. The complete life-cycle of a eugregarine may be divided into eight phases. 1. The sporozoites are liberated from the spores in the digestive tract of the host in all cases known, and usually proceed at once to attach themselves to, or penetrate into, the cells of the lining epithelium of the gut ; but in a few cases the sporozoites pass through the wall of the gut into other organs, as does, for example, the common Monocystis of the earthworm, which penetrates into the vesicula seminalis, and finally into sperm-cells. 2. In the early cytozoic phase the trophozoite may be con- tained completely within a cell (Fig. 143, A, B,) or merely attached to it; the former condition, speaking generally, is characteristic THE GREGARINES AND COCCIDIA 329 of the Acephalina, the latter of the Cephalina. In either case, the first effect of the parasite is to produce a hypertrophy, often very great, of the cell attacked (Fig. 143, B) ; later, however, the cell atrophies, dies, and shrivels up (Fig. 143, (7). (a) In the Acephalina the intracellular parasite is set free from the cell by its dissolution, and, if lodged in" the epithelium of the gut, may pass out of the epithelium either on its inner side, into the lumen of the gut again, or on its outer side, into the bloodvessels or body-cavity. (6) In the Cephalina the relation of the parasite to the host- FIG. 143. — Lankesteria ascidice, parasite of Ciona intestinalis. A, Young intracellular stages in the intestinal epithelium; B, older intracellular stage ; C, extracellular trophozoite attached by a process of the anterior end of the body to a withered epithelial cell, ep., Normal epithelial cell ; ep. , hypertrophied epithelial cell containing (G.)the young grega- rine ; »., nucleus of normal cell ; n.', nucleus' of infected cell. After Siedlecki, magnified 750. cell varies greatly, and has been studied in detail by Leger and Duboscq (618 and 620). The sporozoite may merely prick the surface of an epithelial cell with its rostrum (e.g., Pterocephalus) , or may dip a short stretch of its anterior end into the cell (e.g., Pyxinia), or may penetrate so far that the nuclear region of the parasite is within the cell (e.g.,Stylorhynchus), or, finally, may become completely intracellular (e.g., Stenophora). Ultimately, in all cases, the chief mass of the body of thegregarine projects from, or grows out of, the host-cell into the cavity of the digestive tract, and becomes the protomerite and deutomerite in septate forms ; the attached 330 THE PROTOZOA portion of the body develops into an epimerite which may acquire a large size and a complicated structure. Originally attached to one cell, which it destroys, the epimerite may acquire a secondary attachment to other cells of the epithelium, which in this case are not injured by it, as in Pteroceyhalus. Ultimately the epimerite breaks off, and the body of the sporont drops into the cavity of the digestive tract. In some cases (Pyxinid) the early attached stages may free themselves from the epithelium several times, and attach themselves again. 3. When liberated from the host-cell, the trophozoite grows into the adult sporont, which, as its future history shows, is a gamont or gametocyte. A remarkable feature of gregarines at this stage is the tendency to associate together (Fig. 7), a habit from which the name Gregarina is itself derived. In some cases quite a number of individuals may adhere to one another in strings ; such associa- tions, known as " syzygies," are, however, of a temporary nature, passing flirtations, as it were, which have no significance for the life-cycle or development. On the other hand, a true association of individuals destined to form gametes always, apparently, occurs at one time or another in the life of the sporont. In the majority of cases, however, the sexual association does not take place till the end of the trophic phase, when the sporont is full-grown and ripe for reproduction. But in a number of instances the associa- tion takes place early in the trophic phase, between quite young free trophozoites ; and " neogamous " association of this kind may lead to almost complete fusion of the bodies of the two individuals, only their nuclei remaining separate, thus producing the appear- ance of a binucleate trophozoite (Fig. 70, p. 128). In general, the two trophozoites which associate are perfectly similar in appearance, and exhibit no differentiation ; this is so in all cases where they pair side by side. In some cases where there is an early association end to end — that is to say, where one sporont attaches itself by its protomerite to the deutomerite of another (Fig. 7, p. 9), as is common in polycystid forms — the two sporonts may be differentiated one from the other. In Didy- mophyes, for instance, the protomerite of the posterior individual disappears ; in Ganymedes the two sporonts are held together by a ball-and-socket joint (Huxley). It is not known whether these differences stand in any constant relation to the sex of the sporonts. In Stylorhynchiis the two partners attach themselves to one another by their anterior extremities (Leger, 614). 4. As soon as growth is completed, the reproductive phases are in iated by the formation of a common cyst round the two asso- ciated sporonts, which together form a spherical mass (Fig. 144, a). The parasite is now quite independent of its host ; it is, in fact, a THE GREGARINES AND COCCIDIA 331 parasite no longer, and may now be ejected with the faeces. The nucleus of each sporont then divides by repeated binary fission (Fig. 144, 6) into a large number of nuclei, which place themselves at the surface of the body (Fig. 144, c). A question much debated with regard to the life-history of gregarines is whether a single sporont can encyst by itself, without association with another, and then proceed to the formation of spores. It has been asserted frequently that this can occur, and the suggestion has been put forward that the differences in the size of the spore observed in some species may be correlated with double or solitary encystment. Schellack (630) has discussed the question in detail, and is of opinion that in septate eugregarines solitary encystment either does f FIQ. 144. — Schematic figures of syngamy and spore-formation in gregarines. a, Union of two sporonts in a common cyst ; b, various stages of nuclear division in each sporont ; c, formation of gametids beginning (" pearl-stage ") ; d, stages in the copulation of the gametes : in the left upper quadrant of the figure, separate gametes are seen ; in the left lower quadrant the gametes are uniting in pairs ; the right lower quadrant shows fusion of the pronuclei ; and in the right upper quadrant complete zygotes (sporo blasts) are seen ; e, stages in the division of the nuclei of the sporoblasts, which assume an oval form ; a different stage is seen in each quadrant, eight nuclei being present in the final stage ; /, cyst with ripe spores, each containing eight sporozoites ; two spores are seen in cross-section. Modified after Calkins and Siedlecki. not occur, or leads to nothing if it does, but that amongst the Acephalina and schizogregarines it can take place j a clear case has been described by Leger in Lithocystis schneideri, parasite of- Echinocardium ; and in Monocystis pareudrili solitary encystment leading to spore-formation is described by Cognetti de Martiis. In some species cysts containing three sporonts have been seen ; Woodcock also found a specimen of Cystobia irregvlaris with three nuclei. With regard to the differences in the size of the spores, the possi- bility has to be taken into account that in some cases they may be developed parthenogenetically — that is to say, the gametids may each become a sporo- blast directly, without copulation with another. 332 THE PROTOZOA The first division of the nucleus of the sporont has given rise to considerable discussion and has been the object of much study. In the resting state the sporont-nucleus is a body of relatively huge size, but the first spindle formed in the sporont is, like all the subsequent mitoses, a minute structure. Some authors have believed that the sporont contains two nuclei, comparable to those of Infusoria — namely, a very large macronucleus of purely vegetative nature, which takes no part in the subsequent development ; and a minute micronucleus of generative nature, from which the first and subsequent FIG. 145. — Stages in the formation of a generative nucleus (" micronucleus ") from the primary nucleus of Pterocephalua (Nina) gracilis. A, Primary nucleus showing the first appearance of the micronucleus in a clear space ; B, disruption of the primary nucleus ; appearance of the micronucleus in the form of a few chromosomes in the centre of a little island of nuclear substance ; C, further stage in the formation of the micronudeus ; D, micronucleus com- plete with the first centrosome ; the remainder of the primary nucleus in process of absorption. After Leger and Duboscq (621) ; A magnified 800, B, C, D, 1,000, diameters. mitoses arise. Recent researches, however — more especially those of Schnitzler on Qregarina ovata, Schellack (629) on Echinomera hispida, L^ger and Duboscq (621) on Pterocephalus, Robinson on Kalpidorhynchus, Duke on Metamera, and especially Mulsow( 123) on Monocystis roetrata — leave no doubt but that the sporont contains a single large nucleus, which consists chiefly of vegetative chromatin and other substances, but contains also the generative chromatin, relatively minute in quantity in proportion to the whole bulk of the nucleus. THE GREGARINES AND COCCEDIA 333 The generative chromatin may organize itself into a definite secondary nucleus (" micronucleus ") during the break-up of the sporont-nucleus, as in Ptero- cephalus (Fig. 145) ; or the first spindle arises within the sporont-nucleus before it breaks up, as in 0. ovata (Fig. 146) ; or a number of distinct chromo- somes are formed in the sporont-nucleus during the process of its disintegration, which pass to the exterior of the nucleus and form the equatorial plate of a spindle of which the achromatinic elements appear to arise chiefly outside the nucleus, as in Monocystis rostrata. In either case the first spindle consists only of the generative chromatin ; the remainder of .the original sporont-nucleus is disintegrated and absorbed, or is left over in the residual protoplasm of the cyst. The statement of Kuschakewitsch, to the effect that the primary nucleus of the sporont may break up into a mass of chromidia, from which a number of secondary (generative) nuclei are re-formed, has not received confirmation in any quarter. The mitoses in the sporont are remarkable, in most cases, for the very distinct centrosomes (Fig. 147), which appear at the side of the nucleus before FIG. 146. — Two stages in the formation of the first division-spindle of Gregarina ovata, showing its origin from a very small part of the primary nucleus. In A the spindle is seen within the primary nucleus ; in B the spindle is becoming free from it at one point, after which the remainder of the primary nucleus degenerates. After Schnitzler ; magnification 850 diameters. division begins as a grain or a pair of grains placed at the apex of a " cone of attraction " ; in Monocystis rostrata, however, centrosomes appear to be absent. The number of chromosomes in the equatorial plate is usually four ; but in Monocystis rostrata the number appears to be eight, and in Pterocephalus and the allied genus Echinomera there are five chromosomes, frur of ordinary size and one large unpaired chromosome. Unlike the unpaired chromosome of Metazoa, that of the gregarines is present in both sexes ; it gives rise, during the reconstitution of the daughter nucleus, to the karyosome ; and the karyo- some is eliminated from the nuclear spindle at the subsequent mitosis. The significance of the unpaired chromosome is far from clear, and requires further elucidation. 5. Each of the nuclei of the preceding stage grows out from the surface of the body surrounded by a small quantity of protoplasm, and thus a great number of small cells are budded off over the 33* THE PROTOZOA whole body of each sporont. The small clear cells produced stud the opaque body of the sporont like pearls ; hence this stage is often spoken of as the " pearl-stage" (perlage, etc.). The remainder of the body of the sporont is left over as residual protoplasm, which may contain nuclei, but which takes no further direct share in the development. The cells that are produced are known as the "primary FIG. 147. — Stages of nuclear division in the cyst of Pterocephalus (Nina) gracilia. A, Resting nucleus with a centrosome at one pole ; B, division of the centre- some ; O, D, formation of the nuclear spindle and equatorial plate ; ejection of the karyosome ; E, nuclear spindle, with the unpaired chromosome on the left, also the remains of the karyosome ; F, diaster-stage, with the unpaired chromosome stretching across, the karyosome on the left ; the centrosomes have each divided again ; 0, H, later stages of division ; /, J, K, reconstruction of the daughter-nucleus ; the unpaired chromosome forms the karyosome. After Leger and Duboscq (621) ; magnification of the figures, 1,200 diameters. sporoblasts," but a better name for them is the gametids, since each one is destined to become a gamete The amount of transforma- tion which a gametid undergoes in becoming a gamete may be very considerable, or it may be practically nil. In some cases the male gamete develops a special structure, while the female remains THE GREGARINES AND COCCIDIA 335 unmodified ; in other cases both male and female remain in the undifferentiated condition of the gametid. For an account of the gametes of gregarines, see above (Fig. 79, p. 174). Reduction has been described in several cases in the formation of the gametids. In the genus Oregarina the nucleus of the gametid divides twice to form two reduction-nuclei (Leger and Duboscq, 621); Paehler and Schnitzler have also described a reduction-division in the gametids of Ore- garina ovata. In Monocystis rosfrata, on the other hand, the reduction takes place, according to Mulsow (123), in the last nuclear division in the sporont- body, prior to the budding off of the gametids. In this case the ordinary number of chromosomes is eight, as seen in all the divisions of the nuclei ; in the final division the eight chromosomes associate to form four pairs, those of each pair being in close contact, but not fused ; in the mitosis that follows one chromosome of each pair goes to each pole of the spindle, thus reducing the number of chromosomes in each gametid-nucleus from eight to four. 6. When the gametes are ripe, they copulate in pairs, and probably in every case the gametes of each pair are of distinct parentage. This is certainly the case when the gametes show any trace of sexual differentiation, since those of one sex can be seen to arise from one sporont, and of the other sex from the other. In many cases the two sporonts are separated from one another by a partition dividing the cyst into two chambers, in one of which the male gametes are formed, in the other the female ; when the gametes are ripe, the partition breaks down and pairing of the sexes takes place. 7. The zygote becomes oval or spindle-shaped, and a membrane is secreted at its surface to form the sporocyst, which becomes an exceedingly tough and impervious envelope, and is generally composed of two layers — epispore and endospore. Within the sporq- cyst the nucleus (synkaryon) divides usually three times to form eight nuclei, and then the protoplasm of the sporoblast divides up into as many slender, sickle-shaped sporozoites, leaving over a small quantity of residual protoplasm. The sporozoites are usually arranged longitudinally in the spore, with the residual protoplasm at the centre. The number of sporozoites in the spore is almost invariably eight ; exceptions to this rule are only known amongst the schizogregarines. The spores of gregarines differ enormously in different species in form and appearance, and often have the sporocyst prolonged into tails, spines, or processes of various kinds. Various mechanisms may be developed for liberating the spores from the cyst ; for instance, in the genus Gregarina (Clepsydrina) the cyst is provided with sporoducts, and the residual protoplasm derived from the sporonts swells up when the spores are ripe, and forces them out through the sporoducts in long strings. 8. The ripe spore with its contained sporozoites passes out of 336 THE PROTOZOA the body to the exterior. Usually it passes out per anum with the faeces, but when the spores are formed in some internal organ of the body, as in the Monocystis of the earthworm, it may be necessary for the host to be eaten by some other animal, which then scatters the spores broadcast in its faeces. In all cases, so far as is known, the new host is infected by the casual or contaminative method, and in its digestive tract the spores germinate and liberate the sporozoites. In the case of Cystobia minchinii, parasite of Cucu- maria, it is extremely probable that the host acquires the infection by taking up the spores per anum into its respiratory trees, where the spores germinate (Woodcock). The schizogony characteristic of the schizogregarines takes place during either the second or third of the phases described in the foregoing paragraphs, in trophozoites derived from the^ sporo- zoites by growth, and it takes various forms which cannot be described in general terms ; a few examples must suffice. 1. Sdenidium cauUeryi (Fig. 148) : The sporozoite penetrates into a cell of the intestinal epithelium, and grows to a large size, remaining uninucleate. When full-grown, the intracellular parasite gives rise by a process of multiple fission to a great number of motile merozoites which penetrate into epithelial cells, grow, and finally become free sporonts. The schizogony of Merogre- garina amaroucii (Porter) is of a similar type, but fewer merozoites are produced by the schizont. 2. In Schizocystis gregarinoides (Fig. 149) the sporozoite attaches itself by its rostrum to an epithelial ceil, and as it grows in size its nuclei multiply ; it finally becomes a multinucleate schizont of very large size, which may be either vermiform, and is then attached by an anterior sucker-like organ to the epithelium, or massive in form, and quite free. When full-grown, its body divides up into as many small merozoites as there are nuclei. The merozoites may probably repeat this development and multiply by schizogony again ; or a merozoite may grow, without multiplication of its nucleus, into a sporont, which proceeds to sporogony of a typical kind. In Schizocystis sipunculi (Dogiel, 603) the schizont has a principal nucleus near its anterior end, and forms a number of secondary nuclei near the hinder end of the body, apparently from chromidia given off from the principal nucleus, which loses its chromatin. Round the secondary nuclei protoplasm aggregates, and finally about 150 to 200 merozoites are formed, lodged in a cavity in the cyto- plasm of the schizont. The principal nucleus and the maternal body of the schizont now degenerate, and the merozoites are set free. 3. In Porospora gigantea of the lobster, the largest gregarine known, the full-grown individuals round themselves off, become encysted singly, and divide up to form an immense number of so-called " gymnospores " (Fig. 150), each of which consists of a cluster of merozoites grouped round a central mass of residual protoplasm. The subsequent development and the aporogony are unknown ; the schizogony was formerly mistaken for the sporogony (Leger and Duboscq, 621). In the species Porospora legeri, recently described by Beauchamp (592) from the crab Eriphia spinifrons, a similar process of schizogony is recorded ; but in this case an associated couple or syzygy of two trophozoites becomes encysted together, to undergo a similar process of non sexual multiplication. The association is one of two septate trophozoites closely attached, with loss of the protomerite in the posterior individual, as in Didymophyes ; the subse- quent development and sporogony are unknown. Leger and Duboscq (622) THE GREGARINES AND COCCIDIA 337 have described recently a number of new species of Porospora from various Crustacea ; they suggest that the genus Porospora represents the schizogony, the genus CepJudoidophora the sporogony, of the same cycle. 4. In the peculiar genus Ophryocystis (Fig. 151), parasitic in the Malpighian /HO:-Q '&;&>/ ^5£^.Vrt^£?$ ^co9:;.#x w-tf ;$ ttW<^$^§&'>& :/vni '.'•'. • T^-^v'-'C-r^'.: FlG. 148. — Selenidium caulleryi. A, Full-grown intracellular schizont, X 850 ; B, stage in the multiplication of the nuclei of the schizont, x 1,200; C, schi- zogony complete, showing the merozoites, x 1,000; D, young sporont embedded in an epithelial cell, X 700 ; E, free, adult sporont, x 700. After Brasil (596). tubules of certain beetles (Tenebrionidce, Curculionidce, etc.), and formerly regarded as a distinct order of Sporozoa, the Amoebosporidia, a double schizogony takes place ; there are first of all mult i nucleate schizont s which can 22 338 THE PROTOZOA reproduce their like for many generations, but which finally produce mero- zoites which grow up into paucinucleate schizonts, and these produce mero- zoites which grow up into sporonts. The sporogony of this genus is also peculiar. Two sporonts associate, and the nucleus of each sporont divides into three ; the body of each sporont then divides into a smaller cell with one nucleus and a larger cell with two nuclei ; the email cell is a gamete, which is FIG. 149. — General diagram of the life-cycle of Schizocyatis gregarionidea, after Leger (617, ii.). A, Sporozoite escaping from the spore ; B, C, D, E, growth of the sporozoite into the multinucleatc echizont, of which there are two types: the vermiform schizont (a), which attaches itself to the epithelium by its anterior end, and the massive schizont (6), which lies free in the gut of the host ; F, division of the schizont into a number of merozoites, winch may either grow into schizonts again (G1, GP), or may grow into sporonts (GP) ; H, young sporonts ; /, association of two full-grown sporonts ; J, formation of a common cyst by two associated sporonts ; K , division of the nuclei in the sporonts ; L, formation of the gametes by the sporonts ; M , copulation of the gametes ; N, each zygote becomes a sporoblast and forms a spore. enveloped by the larger binucleate cell. The two gametes copulate, and the zygote becomes a single spore with the usual eight sporozoites ; the two binucleate envelope-cells form a protective envelope to the spore during its development, and die off when it is ripe (Leger, 617, i.). (For Schaudinnetta see p. 355.) THE GREGARINES AND COCCIDIA 339 The Gregarinoidea are classified as follows : Suborder I. — Eugregarince (without Schizogony). Tribe 1 : Acephalina. — Without an epimerite and non-septate ; typically, though by no means invariably, " ccelomic " parasites. Example : Monocystis, with several species parasitic in the vesiculae seminales of earthworms, and many allied genera and species ; see especially Hesse. Also many other genera parasitic in various hosts — echinoderms, ascidians, arthropods, etc. Tribe 2 : Cephalina, — With an epimerite in the early stages, at least, of the trophic phase ; in one family, Doliocystidce, non-septate, but all others septate, with protomerite and deutomerite, or with many segments (Tcenio- cystis, Metamera). Typically parasites of the digestive tract, most common in insects. This tribe comprises a great number of families, genera, and species ; see Minchin YIQ. 150. " Gymno- (589). The type-genus Gregarina (Clepsy- spore" of Porospora j . . . , gigantea, consisting of anna) comprises many common species, such a number of spore as O. ovata of the earwig, O. llattarum of zoites arranged radi- the cockroach, O. pdymorpha of the meal- SUTll "3S! worm (Fig. 7, p. 9), etc. Other well-known contains a chromatinic species are — Pterocephalus (Nina) nobilis, from the centipede (Scolopendra spp.) ; Stylo- rhynchus longicottis (Fig. 142), from the cellar-beetle, Blaps mortisaga, and many others. The family Doliocystidce contains species parasitic in marine Annelids. Suborder II. — Schizogregarince (with Schizogony). Various methods of classifying the< Schizogregarines have been proposed. L^ger and Duboscq (645) divide them into Monospora, which produce a single spore in the sporogonic cycle (example : Ophryocystis) ; and Polyspora, which produce many spores. Fantham proposes to divide them into Endoschiza, in which the schizogony takes place in the intracellular phase, as in Selenidium and Ecto- schiza, in which the schizont is a free trophozoite, a» in Ophryo- cystis and Schizocystis ; the aberrant genus SiedlecJna is probably to be referred here also (see Dogiel, 606). The present state of knowledge is hardly ripe, however, for a comprehensive classifica- tion of the schizogregarines, and it may well be doubted whether they are to be considered as a homogeneous and natural suborder ; some of the families of the Schizogregarinae- appear to be more closely allied to particular families of Eugregarinae than to one another. Leger (617, ii.) points out that the family Schizocystidce shows close affinities with the eugregarine family Aotinocephalidce. 340 THE PROTOZOA Pfeffer asserts that the young intracellular stages of the mealwonn- gregarine multiply by fission. Porospora, with its remarkable schizogony, is apparently a septate cephaline gregarine of the FIG. 151. — Diagram of the life-cycle of Ophryocystis, after L6ger (617, i.). A, The spore setting free sporozoitea ; B, the sporozoite attached by its rostrum to the epithelium of the Malpighian tubule ; 0, multiplication of the nucleus of the sporozoite, and growth to form D, the multinucleate or " mycetoid " schizont ; E, division of the multinucleate schizont into a number of mero- zoites (F), each of which may become a multinucleate schizont again, or ({?. H) may become a paucinucleate or " gregarinoid " schizont ; H, division of the paucinucleate schizont to form young sporonts (/, J) ; K, association of two sporonts ; L, formation of a common cyst round the associated sporonts, and division of their nuclei ; M, formation of three nuclei in each sporont ; N, separation of a gamete (g.) within the body of each sporont, while the rest of the body, with two nuclei, becomes an envelope-cell ; 0, the two gametes have fused to form the zygote (z.) or sporo blast ; P, the sporo blast has as- sumed the form of the spore, and its nuclei have divided into four ; ultimately eight nuclei and as many sporozoitea are formed. ordinary type. A character such as the possession of the power of multipb'cation by schizogony is clearly one of great adaptive importance in the life-history of a parasitic organism, and therefore THE GREGARINES AND COCCIDIA 341 not likely to be of classificatory value. The classification of the future will probably be one which divides all gregarines into Cepha- lina and Acephalina, and distributes the "chizogregarines amongst these two divisions. At present the following families of schizogregarines are recog- nized : OphryocystidcB, Schizocystidce , Selenidiidoe, Merogregarinidce, and Porosporidce. For the family Aggregatidue see p. 353. ORDER II. — COCCIDIA. The chief characteristics of the Coccidia are that, with very few exceptions, the parasites are of intracellular habitat during the trophic phase, and that a number of spores or sporozoites are produced within a cyst, all of which are the offspring of a single zygote. Further, there is always an alternation of generations, non-sexual multiplicative schizogony alternating with sexual propagative sporogony. As a general rule the entire life-cycle is confined to a single host, but in one family (Aggregatidce) an alterna- tion of hosts occurs, corresponding with the alternation of genera- tions ; that is to say, the schizogony takes place in one host, the sporogony in another. Coccidia are found as parasites of various groups of the animal kingdom. In contrast to gregarines, they are found sparingly in Insects, and, indeed, in Arthropods generally with the exception of Myriopods ; but they occur commonly in Molluscs, and especially in Vertebrates of all classes. They are found also in Annelids, but not abundantly, and in Flat- Worms (Turbellaria) and Nemertines. A parasite of the gregarine Cystobia chiridotce has been identified by Dogiel (602) as a coccidian, and given the name Hyalosphcera gregarinicola. The intracellular trophozoite is typically a motionless body, spherical, ovoid, or bean-shaped, often with a considerable resem- blance to an ovum ; hence these parasites were formerly spoken of as egg-like psorosperms (" eiformige Psorospermien "), and the same idea is expressed in such a name as Coccidium oviforme, given by Leuckart to the familiar parasite of the rabbit now generally known as C. cuniculi (or C. stiedce) . The same deceptive resemblance extends to the propagative phases, and the eggs of parasitic worms have before now been mistaken for coccidian cysts, or vice versa. The infection of the host takes place in every case, so far as is known at present, by the casual or contaminative method. Resis- tant spores or cysts of the parasite are swallowed accidentally with the food, and germinate in the digestive tract. The sporozoites escape and are actively motile ; in the majority of cases they pene- trate into cells of the intestinal epithelium, but they may under- 342 THE PROTOZOA FIG. 152. — Life-cycle of Coccidium scJiubergi. A — E, Schizogony ; F — 7, gametog- ony ; K, L, syngamy ; L — 0, sporogony. A, Sporozoite liberated from the spore ; B, three epithelial cells to show three stages of the parasite ; in the first (to the left) a sporozoite (or merozoite) is seen in the act of pene- [Conlinued at joot of p. 343. THE GREGARINES AND COCCIDIA 343 take more extensive migrations, and find their way into some other organ of the body, of which they are specific parasites, such' as the liver, fat-body of insects, genital organs, kidneys, and so forth. When they have reached the cell, of whatever tissue it may be, which is their destination, they penetrate as a rule into the cyto- plasm, and come to rest there, but in some cases they are intra- nuclear parasites. The trophozoite grows slowly at the expense of the host-cell, which is at first greatly hypertrophied as a rule, but is ultimately destroyed ; and when full-sized the parasite enters upon the multiplicative phase as a schizont. After several generations of schizogony, a generation of trophozoites is produced ultimately, which become sexually - differentiated sporonts and proceed to sporogony. The great power of endogenous multiplication possessed by these parasites renders them often pathogenic, or even lethal, to their hosts, in contrast to the usually quite harmless gregarines. As a rule, however, the production of a pathological condition in the host reacts on the parasite, and stimulates, apparently, the development of propagative phases, which, by passing out of the host, purge it of the infection. In this way the disease — " coccidiosis," as it is termed generally — may cure itself, and the host recuperates its health, but without acquiring immunity against reinfection. As a typical coccidian life-cycle may be taken that of Coccidium schubergi (Fig. 152), from the common centipede, Lithobiits forficatus, described by Schaudinn (99) in a classical memoir. The complete life-history may be'divided into eight phases, which are described FIG. 52 continued : t rat ing the cell ; the other two cells contain parasites (p.) in different stages of growth (schizonts) : n., nucleus of the host-cell ; 0, D, multiplication of the nuclei of the full-grown schizont; E, the schizont has divided into a number of merozoites (mz.) implanted on a mass of residual proto- plasm ; the merozoites, when set free, may either penetrate into epithelial cells and become schizonts again, as indicated by the long arrow, or may develop into sporonts (gametocytes) ; F, epithelial cell containing two young sporonts, the one male ( 3 ), with fine granules, the other female ( ? ), with coarse plastinoid granules in its cytoplasm : Q more or less widely separated from one another at the time they produce gametes; (3) the female gametocyte does not divide into a number of gametes, but remains undivided to form a single macrogamete, disproportionately large as compared with the male gametes ; (4) the zygote undergoes a process of division, with the result that all the spores produced within the cyst are the offspring of a single zygote, while in gregarines the cyst contains many zygotes and each zygote gives rise to a single spore. When, however, the coccidia are considered as a whole, it is seen at onco that the first two points do not furnish absolute distinctions ; in Selenococ- cidium the trophozoites are motile and extracellular, and in Addeidce the game- tocytes associate together. There remains only the sporogony which stands out as the distinctive feature of each group. It is by no means difficult to understand, however, the manner in whicn the two types of sporogony, different as they may appear, could have arisen from a common source. The common ancestral form, from which the two groups arose by divergent evolution and adaptation to different modes of parasitism, may be supposed to have been a parasitic organism in which the trophozoites that grew into gametocytes were separated from one another, as in coccidia, and consequently, when full-grown, produced their gametes separately ; and each gametocyte produced a number of gametes which differed only slightly from one another, as in gregarines. From such a form the coccidia arose by the acquisition of an intracellular habitat on the part of the trophozoites, whereby the gametocytes remained more or less widely separated when they produced gametes. As a result of this condition the gametes have to seek each other out, and may easily miss one another ; consequently there was a tendency to greater specialization of the gametes. The male gametes became very small and very motile, and were E reduced in large numbers. The female gametocyte, on the other hand, no >nger divided up into a number of gametes, but became a single large macro - gamete. As soon, however, as fertilization is effected, the suppressed divisions of the female gametocyte take place in the zygote, which divides into the sporoblasts produced formerly by the division of the gametocyte. The gregarine-type, on the other hand, arose from the ancestral form by the trophozoites which grow into sporonts being free and motile in the later stages of their growth ; consequently, gametocytes of different sexes were able to come together and produce their gametes in close proximity, and finally to associate intimately and produce their gametes within a common cyst. In such a condition it was impossible that the gametes should miss one another ; consequently there was no tendency to increased specialization of the gametes, but, on the contrary, a tendency for the gametes to lose even the slight degree of specialization inherited from the ancestral form, with the result that a more or less perfect isogamy was developed ; and instead of the microgametes being produced in excess, the numbers of each kind of gamete produced are approximately equal. THE GREGARINES AND COCCIDIA 355 It follows, from the course of evolution sketched in the foregoing paragraphs, that in both gregarines and coccidia the cyst is to be regarded as a secondary acquisition. In the ancestral form there were simply scattered zygotes from which the spore with its contained sporozoites arose ; the spore may, in fact, be regarded as representing the primary form of the encysted parasite, comparable to an encysted zygote of the Flagellata. It is indeed obvious that the cyst of gregarines and coccidia respectively are quite different things. In gregarines the cyst is formed round the two associated gameto- cytes — it is a " copularium," asLeger has termed it ; in coccidia the cyst is a protective membrane formed round the zygote, immediately after fertiliza- tion. In the genus Legerdla among coccidia, however, the cyst is the sole protective membrane formed to enclose the sporozoites, no sporocysts being produced, a condition which is of interest, since it leads on to that found in the Haemospohdia. In both coccidia and gregarines secondary departures from the primary type of the life-cycle occur. In coccidia the gametocytes of certain forms (Adeleidce) have acquired the habit of association prior to gamete-formation ; this has not led, however, to a development in the direction of isogamy, as in gregarines, but merely to a reduction in the number of male gametes formed. In some gregarines, on the other hand, notably in those forms of " coelomic " habitat, or parasitic in the haemocoale, the sporonts in the later stages of growth are inert and motionless ; this condition has led to neogamous association of young sporonts while still motile and capable of coming together proprio motu, Here mention must be made of the remarkable form Schaudinnella de- scribed by Nusbaum (624), parasitic in the gut of an oligochaete worm. The full-grown trophozoites of Schaudinnella are gregarine-like, and may be either free in the lumen of the gut or attached to the epithelium by an epimerite ; the body is non-septate. Temporary associations (syzygies) may be formed which have nothing to do with sexual conjugation, since the associates part again and produce gametes separately and independently. The full-grown sporonts are distinguishable as male and female forms. The female sporonts divide up into eight or ten spherical cells, the macrogametes. The male sporonts divide up into a great number of minute spindle-shaped elements, the microgametes. Copulation takes place between a microgamete and a macrogamete. The zygote may become encysted and cast out with the faeces, or may penetrate into the wall of the intestine. In the first manner infection of new hosts is brought about ; in the second, multiplication of the parasite in the same host. The zygotes in the wall of the intestine grow in size, and divide each into a number of sporozoites. Some doubt may be felt as to whether the life-history of Schaudinnella has been interpreted correctly throughout ; it is unusual for endogenous mult, plication to be preceded by sexual processes, and the development requires further examination. If, however, the account of the gamete-formation be correct, Schaudinnella is a form which in this respect stands very near to the hypothetical ancestral form of gregarines and coccidia. There can be no doub't that the gregarines and coccidia are closely allied in every respect, and that the two groups are distinguished by points of difference which can be referred quite simply to adaptation to slightly different habits in their parasitic life. Bibliography. — For references see p. 494. THE SPOROZOA: II. THE H^EMOSPORIDIA IN the order Hsemosporidia are comprised a number of organisms characterized by the following peculiarities : They are parasites of the blood-corpuscles, red or white, of vertebrates during a part of the life-cycle ; like the Coccidia, they exhibit an alternation of generations, non-sexual schizogony and sexual sporogony ; and, in all cases thoroughly investigated up to the present, the alternation of generations corresponds to an alternation of hosts, the schizogony taking place in the blood or internal organs of a vertebrate, the sporogony in the digestive tract or other organs of an invertebrate ; lastly, resistant spores are not, as a rule, produced in this order, being rendered unnecessary by the fact that the parasite is never, so to speak, in the open, but always sheltered within the body of one or the other of its two hosts during its entire life-cycle. The Hsemosporidia, as the name is generally understood, are a group which comprises a number of forms differing considerably amongst themselves. Some of the types referred at present to this order will, perhaps, when thoroughly investigated, be removed from the order altogether. The existence of these dubious forms renders the precise limits of the group uncertain and ill-defined. All that can be said at present is that the order contains a nucleus of true Haemosporidia presenting very obvious and close affinities with the Coccidia, and, in addition to such forms, certain others, the true affinities of which remain to be determined, but which can be ranked provisionally in the group. Under these circumstances, the occasion is not yet ripe for treating the group in a comprehensive manner, as has been done with Gregarines and Coccidia. The difficulty of dealing with these blood-parasites is enhanced by the fact that there is perhaps no group in the animal kingdom in which the nomenclature-purist has wrought such havoc as in the Haemosporidia. Matters have reached such a pitch that in some cases the popular names of certain forms are more distinctive than their strictly scientific appellations, so that the very raison d'etre of a scientific terminology has been stultified. 356 THE H^JMOSPORIDIA 367 In the sequel, therefore, the Hoemosporidia will be discussed under five principal types, each of which comprises several forms. So far as possible, the " correct " names of these forms will be stated. Finally an attempt will be made to discuss the position and affinities of the group as a whole. The following is a summary of the distinctive characters of the types in question : 1. The Hcemamoeba-Type. — The trophozoites of the schizogonous cycle occur within red blood-corpuscles, and are amoeboid ; they produce a characteristic pigment, termed " melanin." When the blood is drawn and cooled down on a slide, the male sporonts, if present, form filamentous male gametes resembling flagella, and are consequently said to " exflagellate." The invertebrate host, so far as is known, is a mosquito. 2. The Halteridium-Type. — The intracorpuscular trophozoite is a characteristic halter-shaped parasite of red blood-corpuscles, which is amoeboid, and which, like the last, produces melanin-pigment, and " exflagellates " on the slide. Only known from the blood of birds ; the invertebrate host, so far as is known, is a Hippoboscid fly. 3. The Leitcocytozoon-Type. — The full-grown sporonts are found within white blood-cells, which are greatly altered by the parasite. They are not amoeboid, and do not produce pigment, but they " exflagellate " when the blood is drawn. Only known in birds ; the invertebrate host is unknown. 4. The Hcemogregarine-Type. — Parasites usually of red blood-cor- puscles, sometimes of white ; they are not .amoeboid, do not produce pigment, and do not " exflagellate." They occur throughout the whole vertebrate series, but are most abundant in cold-blooded vertebrates. Those of fishes, amphibia, and reptiles, are trans- mitted generally by leeches ; those of mammals and some reptiles apparently by ectoparasitic Arthropods. 5. The Piroplasma - Type. — Parasites of red blood - corpuscles, amoeboid or of definite form ; they do not produce pjgment and do not '' exflagellate "; generally very minute. They are known only in mammals, and the invertebrate host is always a tick. These five typee will now be considered in more detail. 1. The Hcemamcebce. — The characteristic form of parasite in this section is a minute, amoeba-like organism contained within a red blood-corpuscle ; as it grows it gradually exhausts and destroys the corpuscle, and at the same time produces the characteristic melanin- pigment. Such are the well known malarial parasites of mammals and birds. Unfortunately, the accepted rules of nomenclature render it obligatory to use the generic name Plasmodium for these parasites, a most unsuitable name, since they are not plasmodia in any phase except very temporarily, when they are sporulating. They may, however, be termed familiarly " haemamoebae," pro- 358 THE PROTOZOA vided the word be not written in italics or with an initial capital letter ; anything is better than to speak of them as " plasmodia." In human beings three distinct species at least of haemamoebae are recognized — namely, the parasites of tertian, quartan, and pernicious or tropical malaria, now generally named Plasmodium vivax, P. malaria, and P-. fakiparum, respectively ; the last-named is distinguished from the other two by the sporonts being crescent- shaped, and was put formerly in a distinct genus, Laverania, which has been abolished Haemamoebae similar to those causing malaria in man have been described from other mammals — for example, monkeys, several species • bats ; and squirrels. The human malarial parasites go through their sporogony in mosquitoes of the subfamily Anophelinae ; the life-cycle of those of other mammals has not been yet fully investigated. In birds haemamcebse are of very common occurrence. For these Labb6 created the genus Proteosoma, a name still in use unofficially as a distinctive appellation ; but the correct name of the avian malarial parasites, commonly assumed to belong all to one species, is variously stated to be Plasmodium prcecox or P. re- lictum. In contrast with the human malarial parasites, those of birds are transmitted by mosquitoes of the subfamily Culicinae. Lastly, parasites afe known, from certain reptiles, which are intracorpuscular in habitat, amoeboid in form, and produce pig- ment. Hence they appear to be genuine haemamoebae, but they do not ex flagellate when the blood is drawn,* and very little is known of their life-cycle. By some authorities these reptilian forms are referred also to the genus Plasmodium, but it is best for the present to maintain the genus Hcemocystidium, Castel- lani and Willey, for these reptilian forms. Examples are H. metsch- nikovi (Simond), from an Indian tortoise, Trionyx indicus ; H. simondi, Castellani and Willey, from a Ceylon gecko, Hemidactylus leschenaulti ; and various other species. Since the transmission of the malarial parasites by mosquitoes was first discovered by Ross in his experiments on the Proteosoma- parasite of birds, the development of human malarial parasites has been studied in full detail by numerous investigators, amongst whom Grassi and Schaudinn (130) must be specially mentioned. Consequently the life-cycle of these parasites is better known than that of almost any other Protozoa, and is now to be found described in every textbook. It will be sufficient, therefore, to describe the life-cycle of the species parasitic in human beings in brief outline, as typical of this class of parasites (Fig. 156). * Aragao and Neiva have observed in Plasmodium (Hcemocystidium) diploglossi that, in the male gametocytes on the slide, violent streaming movements occur, such as are the prelude, in other hoemamoebse, to exflagellation ; but formation of gametes was not seen. THE H.EMOSPORIDIA 359 The sporozoites introduced into the blood by the proboscis of a mosquito are minute active organisms of slender form (Fig. 156, XIX.). Each sporozoite attacks a red blood-corpuscle and pene- trates into it. Within the corpuscle it becomes a small, amoeboid trophozoite, which grows at the expense of the corpuscle (Fig. 156, I. — V.). A characteristic feature of the young tiophozoite is the possession of a large space — probably a vacuole — in the body, which gives .he parasite an appearance which has been compared to a signet-ring. As the parasite grows, this space disappears and the body becomes compact. The characteristic pigment is formed within the body of the parasite at an early stage of its growth, and as it increases in size the pigment-grains become more numerous. When the parasite is full-grown it is a schizont, and proceeds to multiply by schizogony (Fig. 156, 6 — 10). The body becomes rounded by cessation of the amoeboid movement, and the nucleus, hitherto single, multiplies by repeated division. Then as many small daughter-individuals (merozoites) as there are nuclei are budded off round the whole periphery of the schizont, leaving at the centre a small quantity of residual protoplasm containing the pigment-grams ; this is the characteristic rosette-stage, or corps en rosace. The corpuscle now disintegrates, setting free the merozoites. Th three species of human malarial parasites are distinguished by differ- ences in their amoeboid activity, their effects on the corpuscles, the number of merozoites produced, and other points, but more especially by the time required for a complete schizogonous generation. Thus, in Plaamodium vivax the growth and multiplication of the schizont requires about forty-eight hours ; in P. malaria, seventy-two hours ; in P. falciparum, twenty- four hours or an irregular time. The attacks of fever produced by the parasites occur when the rosettes are breaking up and setting free the merozoites, probably because the disintegration of the body of the parasite sets free toxic substances contained in it. Hence in the tertian ague caused by P. vivax the fever returns every third day ; in quartan ague of P. malaria, every fourth day ; while P. falciparum causes irregular or quotidian fevers, more or less continuous. The schizogony of the tertian and quartan parasites proceeds in the peripheral blood, but that of the pernicious parasite takes place more generally in the internal organs. The amoeboid trophozoites present them- selves under the most varied forms in the corpuscles ; especialy noteworthy in the quartan parasite is the occurrence of haemogregarine-like forms (Billet, 664). There is some doubt as to whether the trophozoites are in all oases within, or merely attached to, the corpuscles. Schaudinn (130) held at first the view that in all cases the parasites were intracellular, and that appearances tending to prove the contrary were the result of alterations due to manipulation in making preparations. It is nevertheless maintained by many authors that some stages, at least, of the parasites are attached to the corpuscles ; Halber- staedter and Prowazek, for example, believe that in P. pitheci the trophozoites which develop into female sporonts are extracellular, whilst thoae which become schizonts are intracellular. Different species of haemamcebse differ also in the effects they produce on 360 THE PROTOZOA FIG. 156. — Life-cycle of a malarial parasite: combined diagram (the figures are not in all cases from the same species, and some of them are schematic). All the figures above the dotted line represent stages passed in human blood ; those below are the stages that are found in the mosquito. I. — V. and 6 — 10, Schizogony of the tertian parasite, Plaamodium vivax, after Schaudinn (130), magnified about 1,500 diameters. I., Youngest intracor- puscular stage, which has arisen either from a sporozoite (XIX.) or a merozoite (10) that has penetrated into, or is attached to, the corpuscle (represented by a circular outline). II. — IV., Further stages of the growth of the para- site ; a vacuole is formed in its body which gives it the characteristic " signete ring1' appearance (IV.). V. and 6, Later stages of growth; the vacuol- [ffontinued at foot of p. 361. THE H^MOSPORIDIA 361 the corpuscles. An effect commonly seen is the so-called " stippling " (Tiipfclung) of the corpuscles, which exhibit a dotted appearance (Schiiffner's dots). The merozoites, when set free, penetrate into other corpuscles, and become in their turn trophozoites, which may either grow into schizonts again and repeat the process of multiplication by schizogony, or may grow into sporonts. As in Coccidia. a number of generations of schizogony. succeed each other before sporonts are produced. At first the parasites are not sufficiently numerous to be perceptible in the blood or to evoke febrile symptoms, and during this, the so-called " incubation-period," schizogony alone occurs, in a.ll probability ; but when the numbers of the parasite are sufficient to affect the health of the host, the reaction of the host against the parasite probably stimulates the production of the propagative phases. The trophozoites which grow into sporonts Fio. 156 continued : disappears ; in 6 the parasite is full-grown and its nucleus is beginning to divide. 7, 8, Progress of the nuclear divisions, complete in 8. 9, Division of the body of the parasite to form the merozoites ; the blood-corpuscle beginning to degenerate. 10, The parasite has divided up intp sixteen mero- zoites, leaving the pigment-grains in a small quantity of residual protoplasm ; the corpuscle has completely disappeared and the merozoites are set free. VI., Vila., VII6., Formation of the gametocytes of pernicious malaria (Plasmodium falciparum) ; the gametocytes arise from the intracorpuscular parasites by a series of stages similar to those represented in II. — V., but without a vacuole in the body. In P. falciparum the ripe gametocytes have the form of crescents, as shown, but in the tertian and quartan parasite*the gametocytes are simply rounded, as Villa, and VIII6. Vila., Male crescent with larger nucleus and scattered pigment ; VII6., female crescent, with a smaller nucleus and the pigment more concentrated round it. (N.B. — Vila, and VII6. are drawn on too small a scale ; the crescent should be as large as XIII.) VIII. — XIII., Stages of the sexual generation of the tertian parasite in the stomach of the mosquito, after Schaudinn. a, Male forms; 6, female forms. (In pernicious malaria the crescents round themselves off, become free from the corpuscle, and assume forms similar to VIII. a and 6.) VIII., Rounded -off parasites free from the corpuscle. IX., Gamete-forma- tion ; in a the nucleus is divided into eight ; in b the nucleus has passed to the surface of the body. X., Further stage ; in a the body of the gametocyte is throwing off the long slender microgametes,' one of which is represented free ; in 6 the nucleus is dividing to throw off a reduction-nucleus. XI., Process of syngamy ; a male gamete is seen penetrating the body of a female gamete. XII., Zygote shortly after fertilization ; the body is growing out and becoming vermiform, with the synkaryon at the hinder end ; male and female chromatic still distinct ; near the zygote is seen a clump of degenerating microgametes. XIII., Motile ookinete formed from the zygote ; the syn- karyon, with male and female chromatin still distinct, is seen near the middle of the body ; the pigment- grains are at the hinder end of the body, whence they are soon rejected. XIV. — XVIII., Sporogony: diagrammatic. The ookinete (XIII.) pene- trates the stomach-wall and becomes encysted (XIV.); its nuclei multiply (XV.), and it forms a number of sporoblasts so called (XVI.) ; in each sporo- blast the nucleus divides to form a great number of small nuclei, which grow out in tongue-like processes from the surface to form the sporozoitcs (XVII.) ; the ripe cyst contains great numbers of sporozoites with a certain amount of residual protoplasm ; the sporozoites when set free (XIX.) pass into the salivary glands, and thence through the proboscis into the blood of the vertebrate again. 362 THE PROTOZOA have, according to Schaudinn (130), no signet-ring stage in their development, but are of compact form, and grow more slowly than the trophozoites which become schizonts. The sporonts are of two types, male and female (Fig. 156, Villa., VIII6.) ; the male forms have a large nucleus and lightly-staining, clearer cytoplasm ; the female forms have a smaller nucleus and more deeply staining cytoplasm. In the tertian and quartan parasites the sporonts are distinguishable from the schizonts by their greater size and more abundant pigment in larger grains. In the parasite of pernicious malaria, the sporonts are further characterized by their sausage- like form (Fig. 166, Vila., VII6.), and are thereby easily dis- tinguishable from the rounded schizonts. The sporonts only undergo further change if taken up by a mos- quito of a species capable of acting as the specific host of the para- site. When human blood containing various stages of the parasite is ingested by a culicine mosquito, all stages of the parasite are digested with the blood ; but if taken up by an anopheline, the ripe sporonts resist the action of the digestive juices of the mosquito, and develop further in its stomach, while all other stages succumb. The sporonts burst the corpuscle in which they are contained, and round themselves off. In the male sporont the nucleus undergoes rapid fragmentation into some four or six nuclei (Fig. 156, IXa.), leaving a residual karyosome at the centre of the body, as in Coccidium (Schaudinn, 99). The daughter-nuclei place themselves at the surface of the body, and grow out with explosive suddenness into fine filaments of chromatin, ensheathed in a scarcely perceptible layer of cytoplasm (Fig. 156, Xa.). Each such filament is a micro- gamete, of slender, spirochaete-like form, without flagella, but endowed with powers of active movement. The microgametes lash about violently, often dragging the body of the sporont after them, and presenting a superficial resemblance to flagella, which, indeed, they were formerly thought to be ; hence the process of microgamete-formation, which can be observed without difficulty in freshly-drawn blood, was thought to represent a flagellated " Polymitus " stage of the parasite, and was termed " exflagella- tion." The microgametes by their movements finally become detached, and swim away from the body of the sporont, which perishes as residual protoplasm. In the female sporont the nucleus divides to give off a reduction- nucleus (Fig. 156, X6.) ; it is then ripe for fertilization by a micro- gamete (Fig. 156, XI.), which penetrates the body and fuses with the female pronucleus. The zygote then changes from a rounded form into an elongated vermicule, termed an " ookinete " (Fig. 156, XII., XIII.), which moves by gliding movements, like a gregarine. The ookinete bores its way through the lining epithelium of the THE H^MOSPORIDIA 363 gnat's stomach, and comes to rest in the subepithelial tissue ; here it rounds itself off and forms an oocyst (Fig. 156, XIV.), becoming surrounded by a delicate membrane, which is not, however, of a tough and impervious naturelike a coccidian oocyst, since the parasite continues to absorb nutriment and to grow in size, bulging out the stomach-wall towards the body-cavity. As it grows, the originally single nucleus of the zygote multiplies by binary fission, and the cytoplasm becomes concentrated round each nucleus to 'form a " sporoblast," so called (Fig. 156, XV., XVI.). In each sporoblast the nucleus divides repeatedly, and then the surface of the sporo- blast grows out into slender tongue-like processes, each carrying out one of the nuclei in it (Fig. 156 XVII.). Thus a vast number of minute sporozoites are formed by a process of multiplication recalling that seen in the schizogony of Aggregate or Porospora. Finally the cyst contains some hundreds, or even thousands, of sporozoites, together with a certain amount of residual protoplasm, in which the melanin-pigment of the macrogamete is contained (Fig. 156, XVIII.). The ripe cysts burst and scatter their contents in the body-cavity (haemocoele) of the mosquito ; the sporozoites pass by means of the blood-currents to the salivary glands, in which they collect in vast numbers. The mosquito is now infective ; at its next feed, which is usually the fourth, counting as the first that by which it first took up the parasites in the infected blood, the tiny sporozoites pass with the salivary secretion down the proboscis into the blood of the man on whom the mosquite feeds, and so produce a new infection. A disputed point in the life-cycle is the manner in which relapses are brought about in malarious persons ; as is well known, persons who have had malaria may have fresh attacks of the disease under conditions which preclude infec- tion by mosquitoes, and leave no doubt but that the parasite has been present in the body in a latent or inconspicuous condition, and has for some reason reacquired the power of multiplication until its presence becomes perceptible again. Two views have been put forward to explain relapses. According to Schaudinn (130), in the healthy intervals all forms of the parasite have died off except the female sporonts, which are the most resistant forms of the parasite, and maintain their existence in a resting state ; when, however, the conditions occur, whatever they may be, which favour a relapse, the female 8}.>oront6 multiply parthenogenetically (Fig. 72, p. 137), and produce a brood of merozoites which are the starting-point of a fresh series of schizogonous generations. Ross, on the other hand, believes that in the healthy intervals the number of parasites in the blood merely falls below that sufficient to pro- duce febrile symptoms, and that a relapse is brought about simply by an increase in the numbers of the parasites present. The number of cysts formed in the stomach of the mosquito may be very Large, 500 or more ; and the cysts themselves vary in size considerably, some developing only a few hundreds of sporozoites, while in others they are to be counted in thousands. Even in mosquitoes of a species susceptible generally to a particular species of malarial parasite, however, the sppronts do not succeed in every case in passing through their sexual stages and developing normally (compare Darling, 669). In many cases also 'the cysta degenerate 364 THE PROTOZOA and form masses of pigment, the so-galled " black spores " of Ross. Similar degeneration- phenomena have been observed by Schaudinn (147) in the oocysts of Cydospora caryolytica, and may be compared to the transformation of chromidia into pigment in the degeneration of Actinosphcerium in cultures (p. 209). The " exflagellation," or formation of microgametes, which takes place, under normal circumstances, in the stomach of the mosquilo, can be seen also in blood freshly drawn and examined on a slide, if ripe sporonts are present. The process is greatly furthered by lowering the density of the blood — for example, by adding to it not more than one-fifth of its volume of ordinary water, or by simply breathing on the blood when drawn (compare Neumann, 677). It is curious that, while so many experimenters have established absolutely beyond all doubt the transmission of haemamoebae by mosquitoes, those of man by anophelines, and those of birds by culicines, no experiments seem to have been performed to determine how long a mosquito, once infected, remains infective without being reinfected. In other cases of similar transmission, such as that of trypanosomes, yellow fever, etc., it is known that the inverte- brate host, once rendered infective, remains so for a very long time, probably for the rest of its life. In the case of malarial parasites this point remains to be tested experimentally. The haemamoebae of Primates have been studied by a number of investigators, and several species distinguished : Plasmodium kochi (Laveran) from the chim- panzee and various African monkeys ; P. pithed from the orang-outang, and P. inui from Macacus spp. (Halberstaedter and Prowazek, Mat his and Leger, 473) ; P. cynomolgi from Macacus cynomolgus (Mayer, A.P.K., xii., p. 314) ; and P. brasilianum from the ouakari, Brachyurus calvus (Berenberg-Gossler). The schizogony appears to be generally similar to that of the species parasitic in man ; ring-stages occur, and the multiplication is in some cases similar to the tertian, in other cases to the quartan parasite. Binucleate trophozoites are of common occurrence, and binary fission also occurs (Flu, A.P.K., xii., p. 323). A striking feature of monkey-malaria is the comparative rarity of multiplicative phases, which may be in relation to the fact that these parasites cause no appreciable symptoms of disease in their hosts ; in both respects they are comparable to non-pathogenic trypanosomes. Transmission is probably effected by anopheline mosquitoes (Mayer). In bats two distinct forms of intracorpuscular parasites have been described under distinct generic names : Polychromophilus, from Vespertilio and Miniop- terus spp., and Achr&maticus, from Vesperugo spp. These two genera are distinguished by the fact that Polychromophilus produces melanin-pigment, and Achromaticus does not. Polychromophilus is apparently an ordinary haemamceba which should be included in the genus Plasmodium. Achro- maticus, on the other hand, appears, from the recent investigation of Yakimoff and others (753), to be a true piroplasm (see below). Plasmodium vassali from squirrels has ring-like young trophozoites, and its schizogony takes place by binary or multiple fission, more commonly the former (Vassal) ; some forms of the parasite figured resemble Piroplasma. The life-history of the Proteosomo-parasite of birds has been studied in detail by Neumann ; the principal phases of the parasite are essentially similar to those of the haemamoebae parasitic in man. Experimenting with canaries, Neumann transmitted the infection by means of Stegomyia fasciata, but this mosquito was found to be less efficient as a host for Proteosoma than the species of Culex. Of Stegomyia only 11 '4 per cent, developed ripe cysts, as against 85 per cent, of Culex ; the development of the parasite is accomplished in nine to eleven days in Culex, in thirteen to fifteen days in Stegomyia • and a far smaller number of the parasites succeed in developing in Stegomyia, in which the maximum number of cysts seen in the stomach of any mosquito was thirty-six, while in Culex much larger numbers, 500 to 1,000, are recorded. But little is known of the life-cycle of the reptilian haemamcebae of the genus Hcemocystidium. Aragao and Neiva have described schizogony of the THE H^EMOSPORIDIA 365 ordinary multiple type, taking place in the blood- corpuscles, in H. tropiduri and H. diploglossi. According to Dobell, however, the schizogony of H. simondi consists simply of binary fission as a rule, sometimes of division into four. The male and female gametocytes, sharply differentiated by their staining properties in this as in other species, are stated also to have the nucleus divided into two when mature ; Woodcock (687), however, disputes the correctness of DobelTs interpretations. In no case as yet is the inverte- brate host of any Hcemocystidium known. 2. The Halteridia. — The characteristic form of parasite in this section, only known to occur in the blood of birds, is an organism which is found within the nucleated red corpuscle, and which does not displace the nucleus of the corpuscle, but grows round it into a halter-like form, whence the name Halteridium given to it by Labbe. Hence the parasite is easily distinguished from Proteosoma, which is more compact in form, and which displaces the nucleus of the corpuscle. Halteridium is amoeboid, but the form-changes are generally slight ; it produces the characteristic melanin-pigment in abundance ; and when the blood is drawn, " exflagellation " of the ripe male sporonts takes place very readily. Not merely the gamete-formation, but the subsequent fertilization and the for- mat on of the ookinete, can be observed on the slide. It is in this form that Macallum first followed out the whole process, and so made clear the true significance of the " Polymitus " stage in the malarial parasites. The correct generic name for the //aftenWmw-parasite is believed to be Hcemoproteus. Labbe considered the halteridia of different birds to be all one species, to which he restricted the specific name danilewskyi (Grassi and Feletti). By other naturalists several species have been distinguished and named after the birds in which they occur, as H. noctuce of the little owl, H. cohimbce of pigeons, etc. The halteridia of different birds show considerable differences in form, structure, and appearance, and there can be no dou bt that there are many species of these parasites ; but it by no means follows that a given species is restricted to a particular host. It is probable that in some cases one and the same species may be capable of infecting several species of avian hosts. The Sergent brothers were unable, however, to infect canaries with H. columbce of pigeons. The life-cycle of these parasites has been the subject of con- flicting statements. We shall consider first the type of develop- ment made known by the Sergent brothers (686) in part, and more fully by Aragao (Fig. 157). The development described by Schau- dinn (132), to which the utmost doubt attaches, will be dealt with later (p. 390). The invertebrate host of H. columbce is a biting fly of the genus Lynchia, of the dipterous family Hippoboscid the pigeon the infection dies out after a time, unless re-infections take place, and the degree to which parasites abound in the blood is related directly to the number of infected flies fed on the bird. This may not be equally true, however, of other species of these parasites. From Aragao's account it would appear that in H. columbce only male and female halteridia (sporonts) occur. In other species, however, indifferent forms occur also, which, it may be supposed, are destined as schizonts to repeat the process of schizogony, and so to maintain the infection in the bird, like the schizonts of the malarial parasites. Anschiitz has described in H. oryzivorce (of Padda oryzivora) a process of schizogony taking place in the circulating blood. The development of the halteridia in the leucocytes may be considered, probably, as equivalent to the schizogony of the malarial parasites. On this interpretation the missing part of the development is that which corresponds to the sporogony of the malarial parasite, and which in this case is either suppressed entirely (" aposporogony," Aragao), or takes place in the verte- brate host, in some manner yet to be described, instead of in the invertebrate. The absence of sporogony, and of any but the sexual phases, in the Lynchia, doubltess explains the short duration of the infectivity of the fly ; according to Aragao, if the flies are fed for three days on clean pigeons, they cease to be infective. Some of the stages in the lung show a certain resemblance to the sporogony of the malarial parasites, especially the formation of sporoblast- like masses, which, however, are probably more comparable to the schizonto- cytes of Caryotropha than to true sporoblasts. THE ILEMOSPORIDIA 360 Lab be described for halteridium a process of multiplication in the red corpuscle which has never been confirmed. He stated that the nucleus of the parasite divided into a number of small nuclei placed at the two ends of the halter- shaped body, which then divided up into two bunches of small merozoites. It is, of course, possible that the development may differ in different species. But it is more probable that the supposed nuclei at the ends of the body are merely metachromatinic grains, possibly the " alkaliphilous " granules described by Mayer (685, p. 234). 3. The Leucocytozoa. — The true leucocytozoa — that is to say, the species of the genus Leucocylozoon of Danilewsky — are only known to occur in the blood of birds, as stated above ; they must be B FIG. 168. — Leucocytozoon ziemanni from the blood of the Little Owl, Athene noctua. A, Male, B, female, C, young form. N., N., nucleus of the parasite ; N1, N1, nucleus of the host-cell. Original ; magnification 2,000. distinguished clearly from the pseudo-leucocytozoa of mammals, which are in reality haemogregarines, and will be dealt with as such below. The leucocytozoa of birds are found in the blood as bodies usually elongated and spindle-shaped, sometimes, however, rounded in form, which represent each a gametocyte, male or female, con- tained in its host-cell (Fig. 158). The exact composition of these bodies is, however, a little doubtful ; it is not quite certain where the host-cell ends and the parasite begins. The centre of the body 24 370 THE PROTOZOA is occupied by an oval, compact mass of cytoplasm containing a nucleus. By some this mass is regarded as the whole parasite, by others as its endoplasmic region alone. In the female forms the cytoplasm is dense and stains deeply, and the nucleus is relatively small, with a distinct karyosome sometimes placed eccentrically. In the male forms the cytoplasm is paler, and the much larger nucleus stains feebly, with a diffuse granular structure and with- out a conspicuous karyosome. Stretched along one side of the body of the parasite is the nucleus of the host-eel] compressed, usually more or less drawn out, and staining deeply. The surface of the body is covered by a thin membrane, which is prolonged usually into two horn-like processes at the two poles of the body. It is doubtful whether these two processes consist solely of the substance of the host-cell, or whether they contain ectoplasmic extensions of the parasite also. In any case it is certain that the parasite modifies the host-cell in a singular manner. It is also disputed whether the host-cell itself is an erythroblast or a mono- nuclear leucocyte. Most recent investigators, however, incline to the latter view ; but Keysselitz and Mayer (A.P.K., xvi., p. 237) state that the host-cell is an erythroblast. No melanin -pigment is formed. The young forms of the parasite are compact, rounded, or haemogregarine-like, contained in white cells with a large nucleus, and without the horn-like processes characteristic of the adult. Fantham (689) has described in L. lovati of the grouse multiplica- tion by schizogony taking place in the spleen. The schizonts pro- duce a number of merozoites which escape into the blood, and doubtless give rise to the j'oung forms of the leucocyte zoa. The periodicity of the sexual forms in the blood observed by Mathis and Leger (473) depends, probably, on successive schizogonous generations occurring in the internal organs, such as Fantham has described. The method of transmission and the invertebrate host are as yet unknown. If blood containing the parasites in the condition of ripe gametocytes be drawn, the sexual phases and fertilization can be studied without difficulty on the slide. The female gametocytes round themselves off, losing their spindle-like form, and burst their envelope. The male gametocytes contract themselves into two or three rounded masses, which give off about eight thread-like microgametes altogether, in a manner similar to the " exflagel- lation " of the malarial parasites. The microgametes become de- tached and fertilize a female. Schaudinn-(132) gave an account of the development of these parasites which cannot be accepted as correct. According to him, L. ziemanni of Athene myetiia is in reality the resting stage of a large trypanosome, which THE HjEMOSPORIDIA 371 when full-grown attaches itself to an erythroblast and develops into the leucocytozoon, losing its locomotor apparatus. The large trypanosomes in question were supposed to be the sexual, propagative phases, male and female, of a very minute spirochsete-like trypanosome, which represented the indifferent, multiplicative form of the parasite. The existence, however, of young forms of the leucocytozoon, no less than the schizogony discovered by Fantham, disprove entirely any such origin from trypanosomes. In correspondence with his ideas upon the nature and orgin of leucocytozoa, Schaudinn regarded the nucleus of the female forms (Fig. 158, B) as con- sisting of a trophonucleus with a kinetonucleus (" blepharoplast ") close beside it ; while the nucleus of the male leucocytozoon (Fig. 158, A) was sup- posed to consist of a cluster of small trophonuclei, each with a small kineto- nucleus beside it, precocious division of the two nuclei of the " male trypano- some " being supposed to have produced a number of couples of nuclei in readi- ness for gamete-formation. These cytological interpretations cannot be upheld. There is nothing in the structure of the nucleus of the male leucocytozoon to support the notion that it is not a single large nucleus, and the " blepharo- plast " of the female form appears to be simply the karyosome, eccentric in position. Schaudinn also described what he believed to be the development of Leucocytozoon (or, as he named it, Spirochceta) ziemanni in Culex pipiens. According to his account, the ookinete became an elongated, worm-like body which divided up to produce an immense, number of spirochaetes, or very slender trypanosomes. The spirochaetes were stated to find their way into the Malpighian tubules, where they multiplied and occurred in vast numbers. The spirochaetes, inoculated by the mosquito into the blood of the owl, there became the " indifferent form of the leucocytozoon." The statements of Schaudinn with regard to the development of Leucocyto- zoon have received no confirmation, in spite of the efforts of the Sergent brothers to find experimental proof for them. These investigators were unable to obtain any development of the leucocytozoon in Culex, or to transmit the parasite from owl to owl by the agency of mosquitoes. They found, however, that mosquitoes were commonly infected with spirochaetes in the Malpighian tubules, but injection of these spirochaetes into the owl produced no infection with Leucocytozoon, and there can be no doubt that the spirochsetes in question were true spirochaetes, not connected in any way with either trypanosomes or leucocytozoa. Mayer (685) obtained only ookinetes, apparently similar to those of hakeridium, but non-pigmented and slightly larger, in mosquitoes fed on owls infected with leucocytozoa, and observed no sign whatever of nuclear multiplication in the ookinetes ; Wood- cock's unpublished results were practically the same as those of Mayer. Mat his and Le'ger (473) obtained no development of L. sabrazesi in mosquitoes, bugs, and leeches, fed on well-infected fowls, nor could they bring about transmission by means of mosquitoes. 4. The Hosmogregarines. — Parasites of this type have been found in the blood of all classes of vertebrates, and are especially common in cold-blooded animals, such as fishes and reptiles. Until quite recently, haemogregarines were not known to occur in birds ; but Aragao (692) has described a number of species para- sitic in the leucocytes of various species of birds in Brazil. It is a ciuious anomaly of the distribution of these parasites that, while common in marine fishes, they are not known in fresh- water fish, with the sole exception of the eel. While in other classes they are parasitic in the red corpuscles, in mammals they are parasitic in either the red or the white corpuscles, but more com- 372 THE PROTOZOA monly in the latter as the so-called " leucocytozoa," not to be con- fused with the true leucocytozoa dealt with in the last section. Haemogregarines present themselves usually as more or less elongated parasites of quite definite form, sausage-shaped or worm- like, nofc amoeboid, lying within the blood-corpuscle. The middle of the body is occupied by a conspicuous nucleus, and there are often numerous metachromatinic grains in addition, but no melanin- pigment is produced. The parasite may be liberated from the corpuscle as a free vermicule, the resemblance of which to a small gregarine is accentuated by its active gliding movements ; liberation of the vermicules may often be seen when the blood is drawn, but no " exflagellation " ever occurs, since, as will be seen when the development is described, the microgametes are formed in a manner totally different from that characteristic of the haemamcebse. In many haemogregarines the body of the parasite, when lodged within the blood-corpuscle, is enclosed in a distinct capsule or mem- brane, which may be of considerable thickness, and often stains deeply. When the parasite is liberated from the corpuscle, the capsule may be left behind as a conspicuous enclosure of the cor- puscle which has puzzled some observers, and has even been described as a distinct form of parasite (compare Sambon and Seligmann) . In H. bicapsulaia the capsule is thickened at the two extremities of the sausage-shaped body to form two caps, plainly visible in the living condition, and staining a bright red colour in preparations made with the Romanowsky-stain (Franca, 712). Different species of haemogregarines differ considerably in their appearance and size relatively to the blood-corpuscle in which they are lodged, and distinct genera have been founded on these differ- ences ; but as yet the complete life-cycle is known in so few cases that it is not possible at present to draw up a classification of these parasites that can have any pretence to be natural. The following are the principal genera that have been suggested for these parasites. Lankesterdla (Drepanidium) is of very small size, the full-grown vermicule being not more than two-thirds of the length of the blood- corpuscle ; type, L. ranarum (minima), parasitic in the blood- corpuscles of the frog. In Karyolysus the parasite is about the same length as the corpuscle, or slightly shorter ; the generic name is derived from the action of the parasite on the nucleus of the host-cell, which is often broken up and " karyolysed," though not invariably. This form of parasite is especially common in Reptilia Squamata, lizards and snakes ; type, K. lacertarum. In the genus Hcemo- gregarina (sens, strict.) the full-grown vermicule is much longer than the corpuscle, within which it is doubled on itself in the form of the letter U, with the nucleus situated at the bend ; type, H. stepanowi of European water- tortoises, Emys lutaria and Cistudo europcea. Finally there are the " leuco- cytozoa " of mammals, for which the generic names Hepatozoon, Miller, and Leucocytogregarina, Porter, have been proposed ; if it becomes necessary to separate them from the genus Hcemogregarina, Miller's name has the priority, as Wenyon (690) has pointed out. The fact, however, of parasitism in a white THE ILEMOSPOREDIA 373 corpuscle, instead of a red, does not of itself supply adequate grounds for a generic, or even for a specific, distinction, since in some species — for example, H. agamce — the parasites may occur either in white or red corpuscles (Laveran and Pettit). For the present, therefore, these leucocytozoa, so called, may remain in the genus Hcemogregarina, until greater knowledge of the life-histories of haemogregarines makes possible a natural classification of these organisms. A haemogregarine of leucocytic habitat has been described also from a frog by Carini (Rev. Soc. Sci., Sao Paulo, 1907, p. 121). As a type of the life-cycle of the haemogregarines may be taken H. stepanotvi (Fig. 159), which has been studied by Reichenow (78). The chief points in'this author's account of the life-history are con- firmed in essential details, but with specific variations, by that given by Robertson (725) for the life-cycle of H. nicorice.* In both cases the developmental cycle in the tortoise comprises two forms of schizogony, the one producing schizonts, the other sporonts ; and the invertebrate host is a leech. (1) The sporozoite penetrates into a blood-corpuscle, and grows into a long vermicule, which is at first doubled on itself (Fig. 159, F). The two limbs of the U-shaped body within the corpuscle fuse together to produce a bean-shaped parasite — the macroschizont. (2) The macroschizont of H. stepanowi, remaining within the blood-corpuscle, goes through its schizogony in the bone-marrow of the tortoise, producing some thirteen to twenty-four macromero- zoites (Fig. 159, B, C). The number produced is larger in the earlier stages of the infection than in older infections (Fig. 159, D — H). In H. nicorice, however, the macroschizont is set free in a capillary of the lung, and there produces about seventy macromerozoites. In the account of the schizogony given by Reichenow, the significance of the recurved vermicules is not clear. In drawn blood they can be observed to be set free from the blood-corpuscles, and then, as free vermicules, to exhibit active powers of movement, which indicate the existence of some sort of locomotor apparatus, probably of myonemes. According to Reichenow, however, liberation from the corpuscle never occurs normally within the body of the tortoise, but the recurved vermicule remains within the blood- corpuscle in which it has grown up, and its two limbs fuse to form the body of the bean-shaped macroschizont. If that is so, it is difficult to understand why the motile vermicule is ever developed. One is inclined to suspect that it becomes free from the corpuscle in which it has developed, and as a " schizo- kinete " (Minchin and Woodcock, 483) finds it way as a motile vermicule to the bone-marrow (or June; in H. nicorice), where it penetrates another corpuscle (or remains free in a capillary vessel, H. nicorice) and becomes the macroschizont. (3) The macromerozoites produced penetrate into blood-cor- puscles, and may (a) repeat the development already described, and become macroschizonts again ; or they may (6) develop into micro- schizonts, which produce micromerozoites in small numbers, * Nothing in the work of these authors confirms in any way the peculiar account of the life-hiatory of H. slepanowi given by Hahn, whose work is criticized by Reichenow. 374 THE PROTOZOA FIG. 169. — Life-cycle of Hcemogrcgarina stepanowi. The figures to the right of the dotted line represent the phases in the blood of the tortoise ; those to the left, the phases in the leech [Contwued at foot of p. 375. THE H^MOSPORIDIA 375 destined to grow into gametocytes (Fig. 1 59, J — L) . In H . stepanowi the microschizont sporulates in the bone-marrow or in the circu- lating blood, and produces six micromerozoites. In H. nicorice it sporulates only in the circulating blood, and produces six to eight micromerozoites. (4) The micromerozoites penetrate into a blood-corpuscle, and may (a) repeat the microschizogony, or (6) develop into sporonts- (gametocytes). (5) The sporonts (Fig. 159, Ml, Mz) are sexually differentiated. They represent the end of the. development in the tortoise, and can only develop further in a leech. H. stepanowi develops in Placob- della catenigera (=Hcementeria costata), H. nicorice in Ozobranchus shipley i. (6) When the leech sucks the blood of an infected tortoise, it may take up every stage of the parasite into its stomach, where, however, all stages are digested except the sporonts, which resist digestion and pass on into the intestine. There they associate in couples, male and female together. The male sporont produces four microgametes, and one of the four penetrates the macrogamete and fertilizes it (Fig. 159, N— R). (7) The zygote forms an oocyst with a thin membrane, and divides within it into eight sporozoites (Fig. 159, S — U], which pass into the blood-spaces and collect in the dorsal blood-vessel of the leech. How they pass from thence into the tortoise is uncertain. The existence of two types of schizogony — macrocysts producing macro- merozoites, and microcysts producing micromerozoites — in the cycle of the same species of haemogregarine, has long been known, but without the significance of this fact being understood. While the life-history described above is very probably typical of the hsemo- gregarines of aquatic cold-blooded vertebrates, where the intermediate host is a leech, that of terrestrial animals, so far as it is known, is of a somewhat FIG. 159 continued : A, Sporozoite ; B, C, early schizogony, in which a large number of mero- zoites are produced ; D, merozoite penetrating a blood-corpuscle ; E — U, later schizogony, in which few merozoites are produced ; in F the reorrvcd vermicule within the corpuscle is seen ; /, free merozoite about to penetrate a corpuscle and recapitulate the stages D — H, or to initiate the next phase ; J, K, the stages of the final schizogonous generation which produces the gametocytes ; L1, L2, sexually-differentiated mer^zoites of the final generation, which grow up into male (If*) or female ( M2) gametocytes respectively ; these are the stages which develop in the leech when taken up by it. N, Association of male and female gametocytes in the gut of the ieech ; 0, formation of four male gametes by the male gametocyte ; P, one of the male gametes has penetrated into the body of the female gamete, and the two pronuclei are undergoing fusion, with formation of a fertilization-spindle ; Q, zygote with synkaryon and the degenerating remains of the male gameto- cyte attached to it, which is seen also in the next four stages ; R, 8, T, succes- sive divisions of the synkaryon ; U, ripe cyst containing eight sporozoites, residual protoplasm, and the remains of the male gametes. After Reichenow (78), modified in arrangement. 376 THE PROTOZOA different type. In these cases the invertebrate host appears to be always an ectoparasitic arthropod. The only life -cycle of such forms which has been described completely is that of the parasite of the leucocytes of rats, which has been described by Miller under the name Hepatozoon pemiciosum. This parasite appears to be identical with that named by Balfour (694) Leucocyto- zoon muris and by Adie L. ratti ; its correct name, therefore, is Hcemogregarina (Hepatozoon) muris. According to Miller, this parasite causes lethal epidemics amongst tame rats, but in London it occurs commonly in the blood of wild sewer- rats, and appears to be quite harmless to them. It is a parasite of world- wide distribution, apparently, having been recorded from rats in the Punjaub (Adie), Khartoum (Balfour), North America (Miller), Brazil (Carini), and various other parts of the world (see Franca and Pinto, A.I.B.C.P., iii., p. 207). The life-cycle of H. muris, according to Miller, is in the main as follows: The sporozoites are liberated in the intestine of the rat, and pass through the wall of the gut into the blood-stream ; they may be found in the circulation twenty-four hours after infection. Ultimately the sporozoites reach the liver .and penetrate into liver-cells ; in this situation they grow into schizonts, which when full-grown sporulate to produce some twelve to twenty, usually about sixteen, merozoites. The merozoites may penetrate into liver- cells again and repeat the schizogony, or they may pass out into the capillaries of the liver ; in the latter event they are taken up by leucocytes, doubtless as an act of phagocytosis. The merozoites are able, however, to resist any digestive action of the leucocytes ; they become encapsuled in the leucocytes, and in this state they are carried into the general circulation. They do not increase in size in the leucocytes, and their further development, so far as the rat is concerned, is at an end. Hence the " leucocytozoon " of the rat is an encapsuled merozoite of a haemogregarine which, strictly speaking, is a para- site of the rat's liver, and not of the blood at all ; in the leucocytes its role is one merely of passive resistance. These merozoites represent at the same time the sporonts, the propagative phase which develops further in the inverte- brate host, in this case a rat-mite, Lcelaps echidninus, which sucks the rat's blood, and so takes up the parasite into its stoim.ch. In the stomach of the mite the haemogregarines are set free as motile vermi- cules which associate in couples. According to Miller, this association is a true copulation of two gametes which fuse into a zygote ; from the analogy of the life-cycle described above, it is more likely that some stages have been overlooked, and that the vermicules are gametocytes which associate, with subsequent production of gametes by the male and fertilization of the female by a microgamete. The zygote, however formed, becomes a motile ookinete which passes through the wall of the gut into the body- cavity of the mite, and there forms an oocyst which, like that of the malarial parasites, has a thin wall, permitting the parasite to absorb nourishment from the surrounding tissues and to grow to a large size. When full-grown, the contents of the oocyst divide up into a large number of sporoblasts, each of which becomes surrounded by a delicate sporocyst. The contents of the spore divide up into some twelve to twenty sporozoites, and then the development of the parasite is at an end so far as the mite is concerned. The cyst and spores are the propa'gative phase, and in order t^hat they may develop the mite must be eaten by a rat ; if this occurs, the sporozoites are liberated in the stomach and the cycle is complete. In the case of other mammalian haemogregarines, fragments of the develop- ment are known which indicate a life-cycle similar in the main to that of H. muris, allowing for specific differences. Forms parasitic in the red blood- corpuscles are H. gerbitti of Gerbittus indicus (Christophers, 699) ; H. bed f our i (jaculi) of the jerboa (Balfour, 693) ; and the three species recently described by Welsh and others (Journ. Path. Boot., xiv.) from marsupials, one of which (H. peramelis) is remarkable for having been found only in the free, extra- corpuscular condition. The schizogony of H. gerbilli has not been described, but that of H. jaculi takes place in the liver, and is of two types, producing in the one case a large number of small merozoites, in the other a small THE H^MOSPORIDIA 377 number of large merozoites (compare H. canis, below). In both H. gerbilli and H . jaculi free vennicules occur, and are set free readily in vitro ; those of H. gerbilli are recurved when contained in the blood-corpuscle. Stages of the development of H. gerbitti were found in a louse, Hcematopinus stephensi ; first free vermicules in the stomach and intestine, later large cysts in the body-cavity containing a great number of spores, each of which encloses six to eight sporozoites. It seems impossible that the parasites encysted in the body- cavity of the louse should get back into the gerbille in any other way than that of being eaten by the gerbille. Christophers found that, though the sporozoites were liberated in the intestinal juice of the gerbille, they soon died in it, but that in the blood- plasma of the gerbille they became extremely active ; this observation may perhaps be interpreted as indicating that the spores germinate in the intestine, and the sporozoites, when liberated, pass at once through the wall of the intestine into the blood- circulation. The crithidial forms seen by Balf our in Pulex cleopatrce can have no connec- tion whatever with the haemogregarine of the jerboa ; the flea is probably not the right host for this parasite. A number of leucocytic gregarines have been described from various mam- mals, amongst which may be mentioned H, canis (Christophers, 700), H. funambuli (Patton, 721), and H. musculi (Porter). The life-cycle of H. canis has been described by Wenyon (84). The schizogony takes place in the bone- marrow and the spleen of the dog, and is of two distinct types. In the one case the schizont divides into a small number of merozoites, usually three, of large size. In the second case the schizogony results in the production cf a large number of small merozoites. The larger merozoites grow up into schizonts again ; the small merozoites pass into the blood, are taken up by the leucocytes, and become the gametocytes, as in H. muris. The sporogony takes place in the tick, Rhipicephalus sanguineus, and is similar throughout to that of H. muris. The sexual phases were not observed by Wenyon, but according to Christophers (701) the vermicules become free in the stomach, and penetrate the epithelial cells, in which they multiply by fission to form gametes ; probably this applies to the male sex alono. The next stage is an oocyst in the tissues of the tick. The oocyst grows in size, its nuclei multiply, some thirty to fifty uninucleate sporoblasts are formed, and each secretes a sporocyst and becomes a spore containing on the average sixteen sporozoites. The oocyst-wall dissolves, and the ripe spores are set free in the body of the tick. Wenyon considers it possible that the dog acquires the infection by eating infected ticks. Free vermicules of H. funambuli were seen in a louse by Patton, and a similar observation was made for H. musculi by Porter. H. musculi also reproduces by schizogony in the bone-marrow of its host. The haemogregarines of birds described by Aragao (692) appear to be very similar to those parasitic in the leucocytes of mammals. The schizogony takes place in the epithelial cells of the gut or in the cells of the liver, lung, or bone-marrow 9 it results in the formation of a number of small, comma- shaped merozoites, which escape from the cell and are taken up by the mono- nuclear leucocytes. They do not, however, remain- in a resting phase in the leucocytes, but grow within them to a fair size. When set free from the leucocyte, they perform active movements. The intermediate host and the mode of transmission remain, however, to be discovered. The schizogony of hsemogregnrines parasitic in snakes has been studied by Sambon and Seligmann, Hartmann and Chagas (89), and Laveran and Pettit (716). It takes place in the capillaries of the liver and lung or in the bone-marrow. The parasite becomes free from the corpuscle in the capillary, and grows to a large size. In H. sebai the number of merozoites formed varies from two or four to over thirty, but is more often from four to eight. The merozoites are larger when a smaller number is produced. Possibly the variation is related to the age of the infection, as in H . stepanowi, or to the destiny of the merozoites, whether to become schizonts or gametocytes, as in H. canis. The sporogony of the haemogregarines of terrestrial reptiles is practically 378 THE PROTOZOA unknown in its details, but the transmission appears to be effected by ticks ; so Karyolysns lacertarum by Ixodes ricinus (Schaudinn, A.P.K., ii., p. 339, footnote), H. mauritanica by Hyalomma cegyptium (Laveran and Pettit, 718), and the hsemogregarines of snakes (Flu, 707). The minute " drepanidia " of frogs and newts appear to stand rather apart from the true haemogregarines ; beyond the fact that they multiply by schizogony in the red blood-corpuscles, but little is known of their develop- ment. According to Hintze, Lankesterdla ranarum has no invertebrate host, but passes from the blood into the wall of the intestine, where it forms re- sistant cysts like a coccidian parasite. The cysts were believed to pass out of the frog with the faeces and infect other frogs by the direct contaminative method. It is, however, very doubtful if the cysts described by Hintze really belong to the cycle of the Lankesterdla ; from other observations it is possible that the drepanidia are not haemogregarines at all, but stages in the life -cycle of a trypanosome (compare Billet, 696). According to Franca (709), " Dacty- losoma " splendens of the frog produces. Leishmania-Mke merozoites, with distinct kinetonuclei (compare also Seitz). Until further researches have been undertaken, the position of the drepanidia must remain uncertain. Neresheimer (720) has described the penetration of the red blood- corpuscles of frogs by Lankesterdla sp., a process in which remarkable phenomena are exhibited. When a Lankesterdla, in approaching a blood- corpuscle, is within a distance from the corpuscle about equal to the length of the parasite, the edge of the corpuscle turned towards the parasite shows distinct amoeboid movements. As the parasite comes still nearer, two long processes are thrown out by the corpuscle, forming a deep bay, into which the parasite enters ; as soon as it does so, the two processes approach each other, fuse and engulf the parasite, just as an amoeba ingests its prey. The parasite, after this point is reached, appears to be drawn into the corpuscle without further exertion on its part ; the protoplasm of the corpuscle closes up behind it, and the corpuscle regains its normal smooth contour, with the parasite lying within it. The whole process of penetration takes one or two minutes. Neresheimer compares the activity of the corpuscle to the " cone of reception " formed by an ovum when approached by a spermatozoon. From the foregoing account of the life-cycles of naemogregarines, it is seen that the sporogony varies greatly, from the production of eight sporozoites in the oocyst of H. stepanowi and H. nicorice, to the condition of H. cants, H. muris, and H. gerbilli, in which a large number of spores are formed with a variable number of sporozoites. It is impossible, therefore, to accept as adequate the diagnosis given by Leger (644) of the " Hcemogregarinidoe " as producing a single octozoic spore (see p. 353, supra). 5. The Piroplasms. — The parasites of this type are minute organisms, capable of amoeboid movement, but generally of a definite form, which is usually pear-shaped or rod-like. They are contained, sometimes as many as a dozen or more together, within a mammalian red blood-corpuscle. They produce no pigment, but destroy the corpuscle in which they are contained, and set free the haemoglobin, which is then excreted by the kidneys of the host. In consequence of this, the diseases produced by these parasites, termed generally " piroplasmoses " (or " babesioses "), are of a very characteristic type, the most striking symptoms being an enormous destruction of blood-corpuscles and a red coloration of the urine by haemoglobin (haemoglobinuria). From this peculiarity are derived popular names, such as " redwater," etc., applied to diseases caused by piroplasms. THE ILEMOSPORIDIA 379 The best-known member of this group of organisms is a parasite of the blood of cattle (Fig. 160), which has been most unfortunate in its nomenclature, and has appeared under a variety of generic names (Hcematococcus, Pyrosoma, Apiosoma, Piroplasma), but of which the correct name is probably Babesia bows (or bigemina). The typical form of this parasite is a pear-shaped body within the blood-corpuscle. It multiplies by binary fission, and is often double in consequence — whence the specific name bigemina, Many other species are now known, parasites of domestic animals in various parts of the world, and of recent years a number of species have been made known from wild animals, but our knowledge of piroplasms in a natural state is not very extensive. No species is known with certainty to be parasitic upon human beings, but a disease known as " spotted fever of the Rocky Mountains " has been stated to be caused by Piroplasma hominis, and it is possible that the organisms 1. FIG. 160 — Piroplasma bigeminum (Babesia bovis) in the blood-corpuscles of the or. a, b, Youngest forms ; c — /, binary fission ; g — ;', various forms of the twin parasites ; k, I, doubly -infected corpuscles. After Laveran and Nicolle. described from the blood of yellow fever patients by Seidelin (757), and named by him Paraplasma fiavigenum, may be allied to the piroplasms. The investigations upon these organisms carried on during the last few years have led to their being divided up into a number of genera based on differences of form and structure. The following enumeration of the genera of " Piroplasmidae " may serve at the same time to indicate the structural varieties exhibited by these parasites (compare Fr -nga, 736). (1) Piroplasma, Patfcon (Babesia, Starcovici). — Pear-shaped forms, dividing by a process of gemmation — hence commonly found in pairs in the corpuscle. Species are known from oxen, sheep, horses (P. cabatti of " biliary fever "), dogs, monkeys, rats, and various wild animals. (2) Theileria, Bettencourt, Franca and Borges. — Bacilliform or rod-shaped parasites arranged in a characteristic figure of a 380 THE PROTOZOA cross.* T. parva is the parasite of " East Coast fever " of cattle in Africa. Other species have been described from the fallow-deer and from Cephalolophus grimmi. (3) Nicollia, Nuttall. — Oval or pear-shaped parasites with peculiar nuclear structure (see below), and with quadruple division, pro- ducing a figure at first like a fan, then like a four-leaved clover. One species, N. quadrigemina from the gondi, Ctenodactylus (Nicolle, 746). (4) Nuttattia, Fran9a. — Parasites oval or pear-shaped (not rod- shaped) ; multiplication-forms like a cross. N. equi, of equine piroplasmosis ; N. herpestidis, of a mongoose (Herpestes ichneumon). (5) Smithia, Fran$a. — Pear-shaped forms, occupying the whole diameter of the corpuscle, not in pairs ; quadruple multiplication in the figure of a cross. 8. microti from Microtus arvalis. Future research will, no doubt, determine the value of these generic distinctions, some of which seem to rest upon a somewhat slender foundation. As is evident from the foregoing classification, the form of the para- site varies considerably in different species, and even in the same species. In many cases the body may show amoeboid changes of shape, and may throw out long pseudopodial processes. The two principal types of form of the full-grown parasite are the pear- shaped and the bacillary forms ; but the smaller parasites may be ring-like, with the nucleus excentric, and placed near the margin of the body in some cases. The relation of these forms to one another, and their significance in the life-cycle, are not clear, but the annular forms appear to be young stages of either the pear-shaped or bacillary forms. Kinoshita claims to be able to distinguish indifferent (schizonts) from sexually-differentiated forms (sporonts) (compare Theileria, p. 382, infra). The minute structure of the body is very simple, since the cyto- plasm has as a rule no enclosures except the nucleus, which is single. In some cases, however, the cytoplasm may be vacuolated to some extent, and in the ring-like forms has a large central vacuole. The nucleus itself appears to be of a simple type of * A confusion has arisen between two parasites very similar as regards the appearances they present in the blood, but differing in every other respect — namely, Theileria parva, the true parasite of " East Coast fever " of cattle, and Bdbesia (Piroplasma) mutatis, also found in cattle. In both parasites alike the charac- teristic cross-forms appear in the blood. In Theileria parva, however, the cross- forms are an aggregation of four distinct gametocytes (see p. 382, infra) which have invaded the same corpuscles, while in Babesia mutans the cross-forms are produced by quadruple fission of an ordinary multiplicative individual ; this difference has the consequence that, since the gametocytes of T. parva are not capable of further development in the blood of the. ox, direct inoculation of blood from an infected to a healthy ox does not produce an infection in the latter, as happens always when a healthy ox is inoculated with blood containing Babesia mutans. The diagnosis of the genus Theileria given by Franca would appear to apply to B. mutans rather than to T. parva. See especially Gonder (739). THE H.EMOSPORIDIA 381 structure, a compact mass of chromatin or karyosome contained in a vacuole-like space — in other words, a protokaryon of the simplest type (compare Brein] and Hindle, 730). The remarkable form Nicollia quadrigemina has an oval nucleus at the blunt end of the body, with two karyosomes, a larger one placed close to the surface, and a smaller one nearly at the centre of the pear-shaped body (Nicolle, 746). With the unreliable method so much in vogue until quite recently, of making preparations by drying blood-smears and staining them with the Romanowsky stain, the nucleus may show various appearances about which much has been written, and which cannot be interpreted with certainty until they have been examined by better cytological methods. In such prepara- tions the appearance is usually presented of a deeply-stained karyosome lying at the edge of, or near to, a diffuse, more or less irregular chromatin- mass ; or the nucleus as a whole may appear as an evenly-stained mass lying usually at one end of the body in bacillary forms, or near the rounded ex- tremity in the pear-shaped forms. In other cases, in addition to the principal chromatinic mass, some specimens may exhibit a grain or dot, which from its staining reactions appears to be chromatin. Many efforts have been made to establish on this slender basis a theory of nuclear dimorphism for piro- plasms, and to interpret the second grain as a kinetonucleua ; but it bears no resemblance to any such body in its structural and cytological relations, and is inconstant in its occurrence, being entirely absent as a general rule. A question much discussed is that of the occurrence of flagellated forms of piroplasms in the blood of the vertebrate host. In a few rare cases, in parasites preserved by the defective method mentioned in the last paragraph, irregular streaks of substance similar to chromatin in its staining properties have been seen extending from the karyosome even some way beyond the body of the parasite (Fantham, 735; Kinoshita, 741), and these appearances have been interpreted as flagella ; but the published figures of these structures do not in the least favour any such interpretation. Kinoshita suggests that the " flagella " figured by him may represent formation of microgametes. Of more value are the observations of Nuttall and Graham-Smith (748) on the living parasites. They observed that a pear-shaped parasite, when free in the blood- plasma, is capable of moving very rapidly, with the blunt end forwards, while the posterior pointed end exhibits active vibrations which they compare to those of a fish's tail. In some cases the hinder end was observed to be prolonged into a flagellum-like process. The authors cited explain the absence of flagellated forms in permanent blood- preparations by supposing that the flagellum becomes retracted when preserved ; if so, it is a structure of a very different kind to a true flagellum, such as that of a trypanosome, and its relations to the progression of the parasite also differ. Breinl and Hindle (730) have figured biflagellate organisms from the blood of dogs dying irom piroplasmosis. The flagellates in question were of transi- tory appearance, and were only found in the blood of the dog the day before its death. The authors interpret these forms as a phase of the piroplasm ; but a consideration of the figures given, and of the circumstances under which the flagellates were found, leave hardly any doubt but that the forms seen were intestinal flagellates, Bodo or Prowazekia sp., which, in the pathological condition of the host, had passed into the blood (see p. 258). The development of the parasite in the vertebrate host appears to consist solely of multiplication by fission (Figs. 160, 161), usually either binary or quadruple, within the corpuscle ; though the presence of the annular forms, apparently representing young 382 THE PROTOZOA individuals, would seem to indicate the existence of some form of schizogony, yet to be discovered, in the tissues or internal organs of the body. When the parasite or parasites have destroyed the corpuscle in which they are lodged, they are set free in the blood- plasma and penetrate other red corpuscles. Theileria parva stands apart from other piroplasms in its developmental cycle in the vertebrate host. According to Gonder (738, 740), the minute sporozoites injected by the tick collect in the spleen and lymphatic glands, where they penetrate into lymphocytes, in which they grow rapidly. The originally single nucleus divides repeatedly, and large mult muck-ate plasmo- dial masses are formed which finally divide up into as many minute mero- zoites, " agamonts," as there are nuclei ; the process recalls strongly the schizogony of Hcemoproteus columbce (Fig. 157, K — R), and leads to the break- up of the lymphocyte. The first schizogonous generation may be repeated several times, but at last a generation of " gamonts " is produced, which are distinguished from the agamonts by characteristic differences in the nuclear structure. The gamonts multiply by a process of schizogony, the final or " gamogenous " generation, ending in the production of gametocytes, minute parasites which do not multiply further, but penetrate into the red blood- corpuscles, where they grow into adult gametocytes of two kinds — male gametocytes, which are long, slender, " bacillary " forms ; and female gameto- cytes, which are plump, rounded, or pear-shaped forms. The gametocytes can only develop further in the tick Rhipicephalus (see below). The forms .found in the red corpuscles in the peripheral blood are either gamonts or gametooytes, incapable of developing beyond the latter stage except in the tick ; this explains a peculiarity of this parasite, namely, that inoculation of infected blood into a healthy animal does not produce an infection. The position of the genus Achromaticus, founded by Dionisi for A. ve&peru- ginis, parasitic in the blood of bats of the genus Vesperugo, is still doubtful. It occurs under a number of different forms, some free in the blood-plasma ; others, more common, within the corpuscles. The free forms are rounded or spindle-shaped ; the intracorpuscular parasites may be also of these two forms, but are more often pear-shaped. Within the corpuscles the rounded and pear-shaped forms divide into two or four by a process of schizogony. According to Gonder (737), the parasite has a double nucleus in all stages, but this is not confirmed by Yakimoff and Co. (753), who regard the parasite as a true Piroplasma. Neumann (745) states that in the bat-mite (Pteroptus vespertilionis) the parasites undergo a transformation into flagellated organisms, and considers Achromaticus allied to trypanosomes. It is not improbable that stages of Achromaticus, both in the vertebrate and invertebrate hosts, have been confused with stages of the trypanosome found in the blood of the same vertebrate hosts. The process of division in Piroplasma cam's (Fig. 161) has been studied in great detail by Nuttall and Graham-Smith (748), and by Christophers (732). The small rounded forms divide by simple binary fission of the ordinary type. In the larger forms the division takes place in a peculiar manner, more akin to gemmation than to ordinary fission. Before division the parasites become amoeboid and irregular in form, and the nucleus has the form of a compact mass. The nucleus then sends out two buds which grow towards the surface of the body, and at this point two protoplasmic buds grow out, into which the nuclear buds pass. The buds increase in size until they become two pear- shaped piroplasms, joined at their pointed ends by the continually-diminishing remains of the body of the original parent-individual The connecting mass dwindles to a mere point, and finally the two daughter-individuals separate. A modification of this method leads to the quadruple fission producing four buds and four daughter-individuals, as in Babesia mutatis. THE ILEMOSPORIDIA 383 The piriform parasites escape from the corpuscle when it is exhausted, and approach other corpuscles, moving with considerable rapidity. The parasite attacks the corpuscle with its blunt extremity foremost, and " rapidly indents its surface. Then violent movement of the thin end of the parasite occurs, and the side of the corpuscle becomes greatly distorted. . . . Gradually the parasite sinks more deeply into the corpuscle, and finally disappears within it, when the movements of the corpuscle cease and it resumes its rounded shape " (Nuttall and Graham-Smith, 748, vi., p. 235 ; compare the penetra- tion of blood- corpuscles by Lankesterella described above). Only piriform or long parasites enter corpuscles, never the round forms • but immediately after its entry into the corpuscle the parasite becomes rounded. If rounded para- sites are set free from a corpuscle by its rupture, they die off, as do also the pear-shaped forms if they do not succeed in penetrating into a corpuscle. FIG. 161. — Diagrams showing the mode of division of Piroplasma cania in the blood-corpuscle. A, Parasite about to divide ; B, the nucleus budding off a smaller mass ; C, the nuclear bud has grown out into a forked strand ; D, the forked ends of the strand are growing out into protoplasmic buds ; E, F, O, growth of the buds at the expense of the main body ; H, I, J, final stages of the division of the body. After Nuttall and Graham -Smith. A peculiar parasite, perhaps allied to the true piropasms, is Anaplasma marginale, which occurs in the blood of cattle, and causes a disease charac- terized by destruction of the red corpuscles and production of high fever, leading to a degeneration of the large parenchymatous organs. The parasite occurs within the red corpuscles, and is described as consisting solely of chromatinic substance, without a cytoplasmic body; hence the parasites were formerly described as " marginal points." The parasite has the form of a round or oval coccus-like body which multiplies by simple fission. It is transmitted by a tick, Rhipicephalus decoloratus. See especially Theiler (752). The transmission of piro plasms was first discovered by the American investigators Smith and Kilborne, who in a classical 384 THE PROTOZOA memoir showed that the parasite of Texas cattle-fever (Babesia boms or bigemina) was transmitted from sick to healthy oxen by the agency of ticks. The method of transmission is of a peculiar type, which finds its explanation in the habits and life-history of ticks. These arachnids have typically three stages in their life- history — (1) the minute six-legged larva hatched from the egg, which, after growing to its full size, sheds its skin and appears as (2) the nymph, eight-legged, but sexually immature ; the nymph after another moult becomes (3) the adult tick, sexually mature and with four pairs of legs. In each of these three stages of the life- history the tick feeds, as a rule, but once. Consequently, if the parasites are taken up by the tick at one stage of its existence, they cannot be re inoculated into another host until a later stage of the tick. Smith and Kilborne found that the parasites taken up by the adult female ticks passed through their ova into the next generation of the ectoparasites, so that the minute larval ticks, progeny of an infected mother, were the infective agents which spread the disease amongst the cattle. Subsequent investigations have confirmed and extended the dis- covery made by Smith and Kilborne, and in every case the in- vertebrate host of any species of piroplasm appears to be a tick. In P. bows (bigeminum) the parasites develop only if taken up by an adult female tick (Koch), but this is not so in other cases. The parasites may be taken up by the tick at various stages, and returned to the vertebrate host at a later one ; for instance, by the larva and returned by the nymph, or by the nymph and returned by the adult, or by the adult and returned by the larva of the next generation. Although the transmission of piroplasms by ticks is well established, the developmental cycle of the parasite in the tick is known only in a fragmentary and incomplete manner. The most complete accounts are those given by Christophers (732) for Piroplasma cants, and Koch (743) for P. bovis, whose observations supplement each other, since Koch studied chiefly the earlier stages, while Christophers' investigations appear to be more complete for later phases of development. Stages in the tick are also described by Dschunkowsky and Luhs (734), but in a disconnected manner, and observa- tions on the development in cultures have been published by Kleine (742) and by Nuttall and Graham-Smith (750). Accounts differ chiefly as to the events at the beginning of the development. So far as it is possible to make a connected story out of the published observations, the development in the tick appears to comprise six principal phases : 1. The piroplasms taken up in the blood pass into the stomach of the tick, and there the pear-shaped forms are se*; free from the corpuscles, these forms alone being capable of further development. After about twelve to eighteen hours they become amoeboid, sending out in all directions slender, stiff, sharply-pointed pseudopodia which are slowly re- tracted and emitted again. Usually the pseudopodia are given off chiefly from the thicker end of the pear-shaped body, but in some cases the form is spherical and the appearance of the parasite strikingly Heliozoon-like (Fig. 162, A — C). The nucleus of the parasite divides into two parts — a larger mass, THE ILEMOSPORIDIA 385 staining more deeply, on which the pseudopodia are centred ; and a smaller, paler body placed more excentrically. In the pear-shaped forms the large, dark nucleus is placed at the blunt end, the small, pale body near the pointed end. Forms similar to these have been obtained in cultures, and evidently H M FlO. 162. — Stages in the development of Piroplasma in the tick A — G, Amoeboid forms (gametes ?) : A, pear shaped, with the pseudopodia given off at the thicker end of the body ; B, C. spherical or Heliozoon-like, with the pseudo- podia radiating out ou all sides ; D — F, fusion of the gametes (?) ; G, result of fusion (?);// — J, globular bodies (zygotes?); K — M, motile vermicides (ookinetes ?). A — J after Koch (P. bovis) ; K — M, after Christophers (P. canis). represent the first stages of the development ; but they appear to have been missed by Christophers, unless it is to be assumed that these forms occur in P. bovis, and not in P. canis. The star-like forms would appear to represent the gametes ; they congregate 25 386 THE PROTOZOA in clusters, and according to Koch they fuse in pairs (Fig. 162, D — 0) ; cytological details of the syngamy, if such it be, are lacking (but compare Theileria, infra). 2. The stellate stage is succeeded by a spherical stage, very possibly repre- senting the zygote. This body grows in size, but its development, as de- scribed by Koch, is difficult to understand, and requires further elucidation. The final result is a globular mass with a single nucleus, found in great numbers on the third day, according to Koch (Fig. 162, J). Whether these bodies have arisen by division of the zygote, or represent simply the zygotes, is not clear, but the latter alternative seems the more probable. 3. The globular stage is succeeded by a club-like or retort-shaped stage. According to Christophers, whose account of the life-cycle appears to begin at this stage, a split appears in the globular body, whereby a portion contain- ing the nucleus is divided off incompletely from a portion which has no nucleus. The non nucleated portion then swings round and forms the tail- piece of the complete club-shaped body, which has a single nucleus at the swollen extremity. The club-shaped bodies appear to represent the ookinetos (Fig. 162, K—M). They are motile and gregarine-Iike, and in some cases have an organ resembling an epimerite, regarded by Christophers as a boring organ, at the anterior extremity. Their size is about four times that of the piroplasons in the blood. 4. The club-shaped bodies pass from the gut of the tick into the ovary and oviduct, and penetrate into the ova. There they become again globular in form, and are found in the yolk of the egg, and later in the cells of the embryo developed from the egg. When, however, the parasites have been taken up by a nymph, as may happen in P. cam's, the globular bodies are found in the tissue-cells of the body. This globular stage, termed " zygote " by Christophers, very probably corresponds to the oocyst of the hsemamosbae. 5. The globular body of the previous stage divides up by multiple fission into a number of " sporoblasts," which do not remain aggregated together, but scatter themselves through the tissues of the tick, larva, nymph, or adult, as the case may be. 6. The sporoblasts divide in their turn into a great number of sporozoites, small bodies with a single nucleus similar in appearance to the piroplasms in the blood. The sporozoites collect in vast numbers in the salivary glands of the tick, and pass into the vertebrate when next the tick feeds. According to Gonder, ticks infected with Theileria parva purge their salivary glands com- pletely of the parasites when they feed, and are only infective for a single meal. The development of Theileria parva in the tick has been described by Gonder (740). Within an hour after passing into the stomach of the tick the parasites become free from the corpuscles. The immature gametocytes die off, but the adult forms proceed to gamete-formation. The free parasites are at first rounded off, but soon send out processes and become amoeboid. The male gametocytes send out a single process, and creep about actively like a h'mar -amoeba ; their nucleus goes through an unequal division, after which the gametocyte becomes a gamete. The female gametes, which are inactive; go through a similar reduction-process. Pairing of two gametes and fusion of the cytoplasmic bodies takes place, but before the nuclei fuse each nucleus goes through a second reduction-division. After copulation of the nuclei the zygote becomes an active ookinete, first retort-shaped and then gregarinifonn, which penetrates into the salivary glands, and there goes through a multiplicative process, very similar to that of H alter idium in the lung of the pigeon (cf. Fig. 157), producing a swarm of sporozoites which are inocuated into the vertebrate host by the tick. Thus in Theileria also there is no flagellated stage at Any part of the life-cycle — a fact which does not, however, prevent Gonder from seeing " blepharopJasts," and even crith- idial forms on every possible occasion ; he seems to consider nuclear reduction and blepharoplast-formation as the same thing. It is a pity that the effect of such excellent work should be marred by so much theoretical bias. Aber wie die Alien sungen . . . ! THE H^MOSPORIDIA 387 From the foregoing it is seen that the development of piroplasms appears to be of a type essentially similar to that of the haem amoebae and hsemogregarines. In the present fragmentary state of our knowledge, however, it would be premature to generalize con- cerning the development of these forms. The most noteworthy feature of the development is the entire absence of flagellated forms from the life-cycle. The alleged flagellate forms of P. cam's in the dog's blood described by Breinl and Hindle have been dealt with above ; it only remains to be mentioned that Miyajima obtained trypanosomes in cultures of the blood of calves suffering from piroplasmosis, an observation which led to the discovery of a trypanosome in calves not previously known to exist (see p. 283). Doubtful Genera of Hoemosporidia. — A certain number of blood- parasites have been described which at present are not sufficiently well known to make it possible to assign to them a definite systematic position. When more thoroughly investigated, many of them may turn out to belong to other groups than the Haemosporidia ; it is even possible that some of these bodies are not parasites at all, but merely some forms of cell-enclosures. The genus Toxoplasma was founded by Nicolle and Manceaux (754) for T. gondii, a parasite of the gondi (Ctenodactylus gondii) ; other species have since been described — namely, T. cuniculi, Carini, from the rabbit, T. canis, Mello, from the dog, and T. talpce, Prowazek, from the mole. The organisms in question are parasites of the white blood- corpuscles, and occur most abundantly in the spleen or liver, causing a disease which is frequently fatal. The parasite is a crescent-shaped body, with one end thicker than the other, and containing a single nucleus ; they multiply by binary or multiple fission. Nicolle and Manceaux regarded them as allied to Leishmania, but their resemblance to this genus appears to be purely superficial, since in Toxoplasma no kinetonucleus is present, and in cultures no flagellated stage is developed. Etteipsisoma thomsoni is the name given by Franca (441) to a parasite of the blood of moles discovered by Thomson (524). It occurs as an amoeboid intracorpuscular parasite with a single nucleus situated at the margin of the body, which contains no melanin-pigment. Multiplication takes place ex- clusively in the lung, and is by binary or multiple fission, according to Fran9a ; the young forms are either vermiform, with the nucleus drawn out, or oval, with a compact nucleus ; they penetrate into the corpuscles and grow there. Franca considers this form to be allied to Toroplasma. The name Toddia bufonis is given by Fran9a (440) to certain bodies in the red blood-corpuscles of batrachia, first described by Todd. The earliest stage in the corpuscle is a small globule of chromatin ; Franca believes that the parasite when it penetrates the corpuscle is reduced to its nucleus alone, and that it gradually forms a cytoplasmic body which becomes substituted for that of the corpuscle. As the cytoplasmic body is formed, crystals appear in it, one large crystal or as many as three smaller ones. Finally the corpuscle is seen with a slightly hypertrophied nucleus pushed to one side, and its contents consisting chiefly of substance which stains intensely blue with the Roman- owsky stain, in which are the crystals and the nucleus of the parasite, now 3 to 3 '5 /i in diameter. No multiplication-stages have been observed. Globidium multifidum is the name given by Neumann (488) to a parasite of the red blood- corpuscles of Oobius minutus and Arnoglossus grohmanni. It was met with in the form of a cluster of some thirty to sixty merozoite-like bodies, each 2'5 /i in length by 1*5 /* in breadth ; similar bodies were seen in 388 THE PROTOZOA blood- corpuscles singly, but their growth and multiplication were not ob- served. The parasite appears to develop in red corpuscles, which it finally fills completely, breaking up the nucleus ; no pigment is formed. The younges't forms show sometimes a grain near the nucleus, possibly a kinetonucleus. With the bodies desciibed by Neumann may bo compared those observed by Mathis and Leger (473, pp. 417-419, Plate XIIL, Figs. 12-16) in a fish, Clarias macrocephalus ; possibly they have some connection with the trypano- some found in the same host. Immanoplasma scyttii, Neumann (488), is a parasite of the red blood- corpuscles of Scyllium canicula. It grows to a size of 30 by 20 , and in life is feebly amoeboid. Its protoplasm stains very deep blue by the Romano wsky stain, and its nucleus appears usually as if separate from the rest of the body of the parasite, lying apparently free from it in the blood- corpuscle. Some forms of the parasite have paler protoplasm with a larger nucleus, others darker protoplasm with a smaller nucleus ; the two forms are possibly male and female. No pigment is produced. The development of the parasite remains at present unknown. Finally mention must be made of the so-called " Kurloff-Demel bodies," found in the leucocytes of the guinea-pig. According to Patella (755) they are true " leucocytozoa," but according to Mathis and Leger (473) they are not of parasitic nature. A memoir will be published shortly by Dr. E. H. Ross, however, in which it will be shown that the Kurloff-bodies are true parasites, representing, apparently, a stage of a motile organism, probably a spirochaete, found free in the. blood. The author proposes for this parasite the name Lymphocytozoon ccibayce. Affinities of the Hcemosporidia. — Two opposed and conflicting theories with regard to the systematic position of the Haemosporidia hold the field at the present time. 1. The older and more generally accepted view is that the Haemosporidia are closely allied to the Coccidia, sufficiently so, in fact, to be classed with them in a single order. Thus,Doflein divides the Telosporidia into two orders, the Gregarinoidea and the Coccidiomorpha, the latter comprising two subdivisions, Coc- cidia and Haemosporidia ; while Mesnil placed the Haemosporidia, together with the genus Legerella, amongst the Coccidia in an order Asporocystea, characterized by the absence of sporocysts in the oocyst, a character that cannot be utilized in this manner now that some haemogregarines have been shown to form sporocysts. 2. Hartmann and others (e.g., Awerinzew) maintain that the Haemosporidia should be removed altogether from the Sporozoa, and should be classed, together with the Haemoflagellates, as an order of the Flagellata, for which the name Binucleata is pro- posed, since the chief structural feature common to all members of the order is supposed to be the possession of two differentiated nuclei, a kinetonucleus and a trophonucleus, distinct from each other. It must be clearly understood that the theory of the Binucleata, as pro- pounded by Hartmann and his school, is not merely one of a general relation- ship between Haemosporidia and Flagellata. This wider point of view will be discussed when the affinities of the Telosporidia as a whole are considered. The question at present under discussion is whether the Haemosporidia, more THE PLEMOSPORIDIA 389 than the other Telosporidia, are allied specially to the Haemoflagellates, more BO than to other Flagellata ; whether, in short, the Haemosppridia should be removed from the Telosporidia altogether, and should be classified, together with the Haemoflagellates, in one natural order, family, or other systematic category. In dealing with the Hsemoflagellates in a previous chapter, cause was shown for believing them to have two distinct lines of ancestry, the one from a Cercomonad, the other from a Bodonid type of Flagellate ; in that case it is the Cercomonad section — that is to say, the trypanosomes and their allies — to which the Haemosporidia must be considered to be specially related on the theory now to be discussed. Leger and Duboscq (646), recognizing distinct Bodonid and Cercomonad stems in the femoflagellates, derive the Gregarincs, Coccidia, and Haemo- gregarines, trom the Bodonid stem (trypanoplasms), the Haemamcebae and Piroplasms from the Cercomonad (trypanosome) type. The close relationship of the Haemosporidia and the Coccidia seems at first sight so obvious, from a general consideration of the life-histories of typical members of each group, that any theory to the contrary must justify itself by convincing and cogent argu- ments. The chief grounds upon which affinities between Haemo- sporidia and Hsemoflagellates are alleged are found, when analyzed, to be of three kinds — namely : first, developmental data ; secondly, structural — that is to say, mainly cytological — peculiarities ; thirdly, resemblances between certain forms which appear to be sufficiently close to link the two groups together by a series of gradual transitions. The evidences of affinity between Haemosporidia and Hsemoflagellates based on these three classes of facts must be considered separately. 1. Developmental Data. — Beginning with the first of the five types of Haemosporidia which have been recognized above — namely, the haemamcebae or malaria] parasites, it is very evident, as Schaudinn (658) first pointed out, that their life-cycle resembles in the closest manner that of the Coccidia. With one exception, every phase in the life-cycle of a malarial parasite has a corresponding phase in that of a coccidian, and the same terminology can be used throughout for describing the stages of the development ; the one ex- ception to this statement — the only phase that requires a special name — is the ookinete-stage of the malarial parasites, which is not known to occur in any coccidian. It is clear, however, that the points in which the life- cycles differ from one another in the two cases are such as can be correlated with the differences in the mode of parasitism — that is to say, with the fact that in Coccidia, speaking generally, there is a single host, and the mode of infection is contaminative, while in the hamamcebae there are two hosts, and the vertebrate is infected by the inoculative method. Corresponding with this difference, the zygote in the Coccidia prepares at once for leaving the body of the host and passing out into the open, and protects itself by a firm envelope ; while that of the haemamoebae, produced in the body of an intermediate host, does not encyst itself, but is actively parasitic, continuing to absorb nourishment from the host and to grow. Further, in the haemamoebee the parasite is always in the body of one or the other of its two hosts, and consequently tough, impervious cysts and spores like those of Coccidia are superfluous and are never formed ; the oocyst is a thin membrane through which soluble foodstuffs can diffuse and sporocysts are not secreted, as is the case also in some Coccidia. The adaptive significance of these differences is so obvious that it docs not require further elucidation or discussion. The development of the halteridium-type, as described by Aragao, can be 390 THE PROTOZOA derived without difficulty from that of the haemamoebae ; and, in spite of the hiatus in what is known of the life-cycle, there is no difficulty in comparing and homologizing the phases of Hoemoproteus cdlumbce with those of a malarial parasite, and consequently with those of a coccidian. The development of Leucocytozoon requires investigation, but the little that is known — namely, the schizogony, sexual phases, and ookinetc -formation — is entirely of the hsemamceba -type. More striking than in any other type of the Haemosporidia are thtt coccidian features of the haemogregarines. In such a form as H. stepanowi the life-cycle is seen to exhibit not merely a. general similarity to that of the Coccidia, but even a special resemblance to particular forms. The mode of gamete -forma- tion is that which characterizes the family Adeleidce among Coccidia, and the many developmental similarities between H. stepanowi and the only known coccidian parasite of a leech, Orchedbius herpobdellas, have led Reichenow to derive them from a common form. In many haemogregarines, apparently, the parasite obtains an entry into the vertebrate host, not by the inoculative method, but by the contaminative, through the vertebrate devouring the invertebrate host. In such cases (H. muris, H. gerbUli) the characteristic coccidian sporocysts reappear in the sporogony. It is not necessary, however, to dilate further on the coccidian affinities of the haemogregarines, since they are recognized by Hartmann and his school, and the latest revisions of the order Binucleata do not comprise the haemogregarines, which are left in the Telosporidia. As regards the piroplasms, it is perhaps unsafe to generalize in the present fragmentary state of our knowledge of the life-cycle, and in particular of the sexual phases ; but so far as it is known, the phases of the development appear to correspond closely with those of the typical Haemosporidia. But at least it can be said that the development of piroplasms does not afford the slightest support to the view that they are in any way allied to Hasmoflagellates ; indeed, it can be affirmed, on the contrary, that, of all the forms included in the Haemosporidia, the piroplasms exhibit the least indications of* flagellate affinity. From a general consideration of the life-cycles of the typical Haemosporidia, such as the haemamcebae and haemogregarines, and omitting doubtful forms, it is very clear that what may be called the nucleus of the group bears a close and unmistakable resemblance to the Coccidia. One section, comprising the haemamcebae, halteridia, and leucocytozoa of birds, are to be derived from an ancestor which formed gametes after the manner of Coccidium, and in these types the phenomena of " exflagellation " can be observed readily. In the other section, comprising at least the haemogregarines, gamete-formation is of the type of that seen in Addeidce, and does not take place until the gameto- cytes have associated ; consequently exflagellation in vitro does not occur, but coupling of the sporonts, as in gregarines, has often been described, but wrongly interpreted as copulation (cf. Sambon and Seligmann). In the face of such profound homologies with Coccidia, what are the argu- ments from the developmental cycle in favour of a contrary opinion ? The case for the alleged Haemoflagellate affinities of the Haemosporidia rests on the famous memoir of Schaudinn (132) on the blood-parasites of the Little Owl, a work which must now be considered briefly. The Little Owl (Athene noctud) harbours the full number of known avian blood-parasites — namely: (1) a proteosoma ; (2) a halteridium ; (3) a small form of trypanosome ; (4) a large form of trypanosome ; (5) a leucocytozoon : (6) a spirochaete. According to Schaudinn, these six forms belong to the life-cycle of three species of parasites. First, the proteosoma (1) is a distinct form, not related to any of the others. Secondly, the halteridium ( 2) and the small trypanosome (3) are alleged to be two phases of the same parasite. Thirdly, the large trypanosome (4), the leucocytozoon (5), and the spirochaete (6), are supposed to represent different phases of one and the same life-cycle. The halteridium (Hamoproteus noctuce) was stated by Schaudinn to be the THE HLEMOSPOREDIA 391 resting intracorpuscular diurnal phase of a trypanosome which at night developed a locomotor apparatus, became free from the blood- corpuscle, and swam freely in the plasma ; in the morning the trypanosome penetrated into a corpuscle, lost its locomotor apparatus again, and became a halteridium. Mate, female and indifferent forms were distinguished. The smallest in- different forms went through a six-day development and growth, in the corpuscle as a halteridium by day, free in the plasma, as a trypanosome by night, until full grown ; then they multiplied rapidly by repeated fission to produce trypanosomes of the smallest size. These young forms might grow up into indifferent forms in their turn, or might become male or female forms ; in the latter event their development was slower, and in its later stages the parasite lost the power of forming a locomotor apparatus or of leaving the corpuscle. Thus arose the adult male and female halteridia, which, in order to continue their development, required to be taken up by a gnat, Cvlev pipiens. In the stomach of the gnat the parasites formed gametes which copulated and produced zygotes in the well-known manner. Each ookinete, according to Schaudinn, formed a locomotor apparatus (see Fig. 30, p. 59) and either became a trypanosome which might be of female or indifferent type, or gave rise to several trypanosomes in the male sex. The trypanosomes of each type multiplied in the digestive tract of the gnat to produce a swarm of trimorphic individuals, but no further copulation of the male and female forms occurred or could occur (Schaudinn, 132, p. 401). Ultimately, after complicated migrations, the trypanosomes were inoculated by the gnat into the owl again ; the male and indifferent forms passed through the proboscis, but the female forms were too bulky to do so, and, as the male forms were stated to die off in the blood, there was effective inoculation of indifferent forms only, which start on the cycle of development already described. These remarkable statements, the origin and significance of which have l>een, for the last seven years, a veritable riddle of the sphinx, have met with general scepticism except from a few devoted partisans, who have been striving continually to find corroborative evidence for Schaudinn'a theories, in spite of the mass of evidence to the contrary that has been steadily accu- mulating. Recently Mayer (685) has affirmed that in owl's blood containing only halteridia, kept under observation in hanging drops under the micro- scope, trypanosomes make their appearance which could only have come there by transformation of halteridia. These experiments are supposed to prove conclusively one part, at least, of Schaudinn's statements — namely, that the halteridia are merely intracorpuscular stages of trypanosomes. Against Schaudinn's views, on the other hand, two principal objections, out of many, may be urged : First, that the development of Hcemoproteus coluntbce, as made known by the Sergent brothers and by Aragao, is of a totally different type to that described by Schaudinn ; it comprises no trypanosome-phases at any point of the life-cycle, and the invertebrate host is not a gnat, but a biting fly ef an altogether different kind. To meet this objection, Mayer proposes to restrict the name Hcemoproteus to forms which develop after the manner of H. columbce, and to revive the name Halteridium (in italics and with an initial capital letter) for parasites that, on the Schaudinnian theory, are really trypanosomes. Secondly, that the small trypanosomes of Athene noctua are connected by every possible transitional form with the fargest found in the same bird, and there is every reason to suppose that in this case, as in other birds or vertebrates of all classes, they are all merely forms of one polymorphic try- panosome (Minchin and Woodcock, 42). It may be added that the whole mystery receives a complete solution on a simple supposition — namely, that the trypanosome of the Little Owl, like other known species of trypanosomes (see p. 308 ), has intracorpuscular forma which have been confused with the true halterida ; on such an assumption, so eminent an investigator as Schaudinn can be acquitted of having made what would appear at first sight to bo a gross error of observation, and Mayer's observations are easily explained. Mayer seems, in fact, to have figured 392 THE PROTOZOA guch forms on his Plate XXII., Figs. 2-4 — small intracorpuscular forms, more or less Leishmania-like, without pigment, and with, apparently, distinct tro phonucleus and kinetonucleus. ; It is not necessary to deal with Schaudinn's statements concerning Leuco- cytozoon further than has been done above (p. 370). It is now as certain as anything can ever be in such matters that Lcucocylozoon has nothing whatever to do with either trypanosomes or spirochaetes. The six forms of blood- parasites of the Little Owl may be regarded as belonging to five species, namely: A proteosoma (1), a halteridium (2), a trypanosome (3 and 4), a leucocytozoon (5),- and a spirochaete (6). Of these five, it is probable that only the proteosoma, the trypanosome, and possibly the spirochaete, can develop in, and be transmitted by, a gnat ; the halteridium and the leucocytozoon require, probably, quite different intermediate hosts. If, therefore, a Culex were fed on an owl containing in its blood halteridia and leucocytozoa abun- dantly, and trypanosomes and spirochsetes in scanty numbers, the first two parasites might be expected to die out after the ookinete-stage, while the trypanosomes, and possibly the spirochaetes, would multiply, and thus produce very easily the impression that they were derived from the intracorpuscular parasites. Even less cogent for the theory of Hsemoflagellate affinities than the argu- ments deduced from the development of Haemosporidia are those based on the development of HaemoflageUates. Thus the schizogony of Schizotry- panum discovered by Chagas has been compared to that of a malarial parasite, and has been adduced seriously as an additional proof of the alleged affinities between trypanosomes and haemamcebae. But " schizogony :' — that is, repro- duction by simple or multiple fission without concomitant sexual phenomena, — occurs throughout the whole range of the Protozoa, and affords no proof whatever of genetic affinities. Those who bring forward such an argument must surely have forgotten that the word "schizogony" was originally coined by Sehaudinn for the non-sexual multiplication of Trichosphcerium sieboldi, a marine Rhizopod (p. 181). 2. Cytological Data. — The theory of the Haemoflagellate affinities of the Haemosporidia has led to the most laborious and painstaking efforts to discover in the body of each and every Haemosporidian parasite, in at least some of ite phases, a second nucleus, the homologue of the kinetonucleus ; and any little granule, however minute, that can be coloured like chromatin is pro- claimed triumphantly to be the inevitable kinetonueleus, or any streak of similar staining properties to be a flagcllum. Consider first by itself the case of a cell in which, in addition to the nucleus, there is seen a grain which, by some particular dye, is stained in a manner similar, or nearly so, to the chromatin of the nucleus. This is not by itself a decisive proof that the grain in question is chromatin, since, as pointed out above, other grains may take up so-called " chromatin-stains " ; the body in question may therefore be chromatin or some other substance. If it be chromatin, it may be a chromidial granule extruded from the nucleus ; or it may be a body of the nature of a karyosome, situated close to the edge of the nucleus, or possibly, in some cases, where the nucleus has no limiting mem- brane, a little way from the main mass of the nucleus ; or it may be a true kinetonucleus. If it be not chromatin, it may be a centrosome or blepharo- plast ; or a grain of metachromati nic substance, such as volutln ; or, lastly, some other kind of metaplastic body. There are therefore many possible alterna- tives before a grain that stains like chromatin can be identified definitely as being a kinetonucleus and nothing else. What are; the criteria by which a grain that stains like chromatin can be identified as a kinetonucleus, to the exclusion of other possible interpretations of its nature ? In the first place, according to modern views (see p. 288, supra, and compare especially Rosenbusch, 505), a kinetonucleus is not a simple granule, mass or lump of chromatin, but it is a true nucleus with centriole, karyosome, and a nuclear cavity, actual or virtual, containing nuclear sap at least, if not peripheral chromatin also. Secondly, a kineto- THE ILEMOSPORIDIA 393 nucleus when present is a permanent cell-element which, like the principal nucleus, divides when the cell divides, and is propagated by fission equally with the cell itself. Thirdly, and this is the most important criterion of all, the kinetonucleus is in relation with a flagellum during at least some phases of the development, though for a time the locomotor apparatus may be temporarily absent, its existence indicated only by the kinetonucleus during resting phases. The smaller chromatinic body of Leishmania may be cited as an example of a body which fulfils these conditions, and which can be identified unhesita- tingly as a true kinetonucleus, homologous in every way with that of a try- panosome. But with the alleged kinetonuclei of Haemosporidia the matter stands quite otherwise. It is not possible to discuss fully here every separate instance, but a few typical examples of such bodies may be dealt with briefly. In female halteridia and leucocytozoa (Fig. 158), a large grain is seen by the side of the nucleus, and often interpreted as a kinetonucleus. Until this body has been shown conclusively to be related in some phase of the life- history to a flagellum, it is far simpler to regard it as a karyosome which, like that of the merozoites of Adelea (Fig. 153, F), is excentric, or possibly extranuclear in position ; assuming, that is, that the body in question is a true chromatinic nuclear element. In the merozoites of Proteosoma, Hartmann (675) has discovered a flagellum- likef process at the anterior end, arising from a grain which he regards as a kinetonucleus (" blepharoplast " in the German use of the term), thus con- firming certain obiter dicta of Schaudinn (132, p. 436) with regard to the mero- zoites and'sporozoites of the tertian parasite. It may be pointed out that the rostrum of the sporozoites of Gregarines appears to be a perfectly similar structure, which very possibly represents a rudimentary flagellum arising from a true blepharoplast of centrosomic nature. Hartmann's discovery is therefore more proof of the affinities of proteosoma with other Telosporidia thai; with Haemoflagellates. The supposed kinetonuclei of piroplasms have been mentioned above ; the entire absence (pace Hartmann) of flagellated stages throughout the life- cycle make it impossible to accept any such interpretation of the nature of these granules so highly inconstant in their occurrence. Lastly it should be mentioned that Schaudinn, and recently Hartmann, have maintained that the microgametes of halteridia and other Haemosporidia have the structure of a trypanosome. Inasmuch as Schaudinn also pointed out the great structural similarity between trypanosomes and spermatozoa, this point might not count for much, even if it were true ; unless the Metazoa also are to be classified amongst the Binucleata, a conclusion which, indeed, seems to follow from the nuclear theory of Hartmann and Prowazek (63). In objects of such extreme minuteness, however, statements ascribing to them complicated details of structure must be regarded with great scepticism until thoroughly substantiated. It is a sufficient warning of the need of caution to bear in mind the controversy that has raged over the question of the minute structure of spirochaetes, with regard to which Schaudinn was obliged to retire from the position he took up at first — namely, that their structure was similar to that of a trypanosome. 3. Possible Transitional Forms, — The parasite of kala-azar was originally described by Laveran under the name Piroplasma donovani,* in the belief that it was a true piroplasm ; and many writers have been struck by the external similarity of the two parasites, in spite of the difficulty in finding in Piroplasma a satisfactory representative of the constant and definite kinetonucleus of Leishmania. In fact these two genera are often cited as the connecting link between Haemoflagellates and Haemosporidia, and are supposed to indicate the course of evolution whereby serum-parasites of the first type became * On the other hand, the parasite of Oriental Sore was first described by Wright under the name Helcosoma tropicum, and referred to the Microsporidia. 394 THE PROTOZOA cell- parasites of the second (compare L6ger and Duboscq, 646). However enticing such a view may seem when only the forms parasitic in the verte- brate hosts are taken into consideration, the facts of the development in the invertebrate hosts must dispel completely any notion of affinity between the two types. Nothing could be imagined more different than the develop- ment of Leishmania, with its typical leptomonad forms (Fig. 140), and that of Piroplasma (Fig. 162), with no flagellated stages at all in its life-cycle. It becomes evident at once that any apparent resemblance between the two genera is due to convergent adaptation induced by a similar mode of parasitism, and that the two forms are in reality poles apart, with no more real affinity than porpoises and fishes, or bats and birds. It is certainly not at this point that any transition from one group to the other is to be sought.* In the foregoing paragraphs an attempt has been made to sum up the arguments for and against the theory tnat the Haemosporidia are to be removed from the vicinity of the Coccidia, and classified with the trypanosomes and allied forms in an order of the Flagellata. When the evidence on each side is weighed in the balance, in one scale must be placed the complete similarity of the life-cycleo of typical Coccidia and Haemosporidia, a similarity seen in every phase of the life-cycle, and extending even to minor developmental details ; and in the other scale certain cell-granules of doubtful significance. It is almost inconceivable that more importance should be attached to cytological details, the genetic and classifi- catory value of which is at present quite uncertain, than to the homologies of the life-cycle as a whole, in estimating the affinities of the orders of Protozoa ; the more so since even in the Hoemo- flagellates themselves the possession of the binucleatc type of structure does not, apparently, indicate a common ancestry for all members of the group. The conclusion reached is, then, that the Haemosporidia as a group, excluding doubtful forms insufficiently investigated at present, are closely allied to the Coccidia. It is, indeed, probable that there are two lines of evolution in the group — the one repre- sented by the haemamoebae, halteridia, and true leucocytozoa, descended from a Coccidium-\ike ancestor ; the other represented by the haemogregarines, from an ancestral form similar to Adelea or Orcheobius. Leger (644) has classified the haemogregarines in the section Adeleidea of the Coccidia, and one may regret that the distinguished French naturalist did not go one step farther and place the haemamo3bae in his section Eimeridea (see p. 352, supra). On the other hand, any resemblances which the Haemosporidia exhibit to trypanosomes and allied forms are due to convergent adaptation on the part of the Flagellates themselves, and more especially to the secondary acquisition by the latter of intracellular * Leger and Duboscq (046), who derive Leishmania and Babesia directly from Crithidia as a common ancestor, do not seem to have taken the development of Babesia (Piroplasma) into consideration at all ; they neither refer to it in their text nor cite any of the relevant memoirs in their bibliography. THE H^MOSPORIDIA 395 parasitism, and consequent temporary loss of the locomotor apparatus. It may well be, therefore, that some forms now generally included amongst the Haemosporidia (e.g., possibly the drepanidia) may prove, when better known, to be stages of Haemo- flagellates, and to have in reality nothing to do with the true Haemosporidia. Affinities of the Telosporidia. — From the foregoing discussion, the conclusion has been drawn that the Coccidia and the typical Haemosporidia are closely allied, sufficiently so to be grouped together in a single order, for which the name " Coccidiomorpha " may be used. In a former chapter (p. 354) the relationship of the Gregarines and Coccidia was discussed, and it was pointed out that there was no difficulty in assuming a common ancestral origin for the two groups — a conclusion which, indeed, has never been called in question. The Telosporidia, taken as a whole, may be regarded, therefore, as a homogeneous and natural group, in which the close affinity existing between its constituent members may be regarded as indicating a common phylogenetic origin. If this conclusion be accepted, it remains to discuss the affinities of the Telosporidia as a whole to other groups of Protozoa. It is not unreasonable to suppose that a parasitic group of this kind has been evolved from free-living non-parasitic ancestors, and the question to be discussed is to which of the groups of Protozoa the ancestral form of the Telosporidia belonged. Of the three great classes of the Protozoa, the Infusoria may almost certainly be excluded from consideration in regard to this question, since, in view of the very specialized and definite features of this group, there are no grounds whatever for connecting them with the Telosporidia. There remain, therefore, only the Sarcodina and Mastigophora to be considered. At different times two opposed theories have been put forward with regard to the affinities and ancestry of the Sporozoa. One view sees in them the descendants of typical forms of Sarcodina, such as Amoeba (Awerinzew, 890) ; the other derives them from flagellate ancestors such as are represented at the present day by Euglena or Astasia. It is no longer possible, however, to regard the Sporozoa as a whole as a homogeneous group, and the two so-called " subclasses," Telosporidia and Neosporidia, must be considered separately, each on its own merits. The Nedsporidia are considered at the end of the next chapter. The question here is of the Telo- sporidia alone. For this group opinion is practically unanimous at the present day in favour of a flagellate ancestry, a theory which must be considered critically. One of the main arguments generally put forward for the theory of the flagellate origin of the Telosporidia is the existence of flagel- 396 THE PROTOZOA lated stages in the life-history. In the first "place, the micro- gametes are very often flagellated, as has been stated frequently in the two foregoing chapters. In the second place, the youngest stages in the development — the merozoites or sporozoites — exhibit structural features which are either those of a flagellate swarm- spore (Hartmann, 675 ; Schaudinn, 132), or can readily be derived from a flagellula in which the flagellar apparatus has become rudi- mentary, as in the sporozoites of gregarines, where the rostrum may be interpreted, with a high degree of probability, as representing a rudimentary flagellum. The existence of flagellated stages of the kinds mentioned in the development of the Telosporidia is by no means, however, a cogent argument for a flagellate ancestry for the group, since quite typical Sarcodina of all orders exhibit flagel- late swarm-spores and gametes. It may be urged that in the case of these types of Sarcodina, also, the existence of flagellate stages indicates a flagellate ancestry ; but such an argument merely evades the question at issue, which is not whether the Telosporidia are derived from Flagellata indirectly through Sarcodine ancestors, but whether or not they are descended directly from ancestors that were typical Flagellata. The existence of flagellated swarm- spores and of gametes representing a modification of such swarm- spores is not sufficient of itself to prove a flagellate ancestry for the Telosporidia. Far more cogent arguments for the flagellate affinities of the Telosporidia may be drawn from the characters of the adult forms, especially from the gregarine-type of body, elongated and ver- micular in character, and perfectly definite and constant in form, which occurs in every group of the Telosporidia at one point or another in the life-history. Such a type of body can be readily derived, as Butschli (2) pointed out, from an organism similar to Astasia or Euglena, in which the flagellar apparatus has been lost, and all special organs of nutrition, whether holozoic or holophytic, have disappeared in relation with the parasitic mode of life. On the other hand, the gregarine-type of body cannot be derived from the adult forms of the Sarcodina, which are typically amoeboid, and without any definite body-form other than that imposed by the physical nature of their body-substance. We may therefore consider the ancestral form of the Telosporidia to have been a flagellate organism with an elongated form of body, with a definite form, owing to the presence of a cuticle of a certain degree of thickness and toughness, and with a flagellar apparatus at the anterior end. Such a form would have been not unlike the leptomonads now found commonly as parasites of insect-guts ; but there is no reason to suppose the ancestral form to have had a kinetonucleus and the third type of flagellar insertion. Such a THE ILEMOSPORIDIA 397 form probably used its flagellum for the purpose of attaching itself to the epithelium of the digestive tract, as leptomonads do now (compare Figs. 136, 137) ; and from this primitive type of attach- ment the epimerite of the gregarines may have been derived by secretion of chitin round the attaching flagellum, just as the primitive tuft of fixing cilia, the " scapula," of the primitive Vorticellids appears to become converted into the chitinous stalks of such forms as Epistylis (p. 441). The conclusion drawn from these various considerations is, therefore, that the Telosporidia may be regarded as a group de- scended from flagellate ancestors modified in adaptation to a para- sitic mode of life ; not, however, specially from flagellates of the " binucleate " type of structure. Bibliography. — For references see p. 496. CHAPTER XVI THE SFOROZOA : III. THE NEOSPORIDIA A TYPICAL member of the subclass Neosporidia is a parasite of which the life-cycle is initiated by the liberation from the spore of one or more amcebulae within the body of the host, in the digestive tract in all known cases. For this initial amoebula-phase Stempell's term, planont (i.e., " wanderer "), may be employed conveniently, since in no case does it remain in the lumen of the digestive tract, but penetrates into the wall of the gut, and in most cases migrates thence into some organ or tissue of the host, where it lives and multiplies actively, being usually at this stage an intracellular parasite, in some cases, however, occurring free in the blood or lymph. The planont-phase is succeeded typically by a plasmodial phase, which arises in some cases by simple growth of the amcebula (probably then a zygote), accompanied by multiplication of its nuclei ; in other cases by association together and cytoplasmic fusion of at least two distinct amoebulae, of which the nuclei remain separate. The plasmodial stage is very characteristic of this sub- class ; it represents the principal or " adult " trophic phase of the parasite, and is also the spore-forming phase ; and, as the name Neosporidia implies, the production of spores begins, as a rule, when the plasmodium is still young, and continues during its growth. In some cases, however, no plasmodium is formed, but the planont-phase is succeeded by uninucleate " meronts " or schizonts, which multiply by fission and give rise ultimately to sporonts in which spore-formation sets a limit to the growth. In such forms the general course of the life-cycle is not essentially different in any way from that of a member of the Telosporidia, such as Coc- cidium. The tendency, therefore, of many Neosporidia to form spores during the trophic phase cannot be used to frame a rigorously- exact definition of the group. A more distinctive characteristic of the subclass is the complete absence of flagellated phases in any part of the life-cycle, and more especially the fact that 398 THE NEOSPOR1DIA 399 the sporozoites are always, apparently, amoebulae. and never gregarinulae.* The Neosporidia are divisible into two sections, known re- spectively as the Cnidosporidia and the Haplosporidia. The Cnidosporidia are distinguished by the possession in the spore of peculiar structures termed polar capsules, which are lacking in the Haplosporidia. A polar capsule (Fig. 163) is a hollow, pear-shaped body, with a tough envelope, probably chitinoid in nature. It is situated at one pole of the spore, with its pointed end immediately below the surface, in continuity with a minute pore in the sporocyst. Coiled up within the capsule is a delicate filament, often of great length, probably of the same nature as the capsule, and continuous with it. Under suitable stimu- lation the polar filament is shot out through the pore in the sporocyst. In their structure the polar capsules resemble the nematocysts of the Coelentera. Each polar capsule is formed within a capulogenous cell. The Cnidosporidia comprise four orders — the Myxosporidia, Actinomyxidia, Micro- sporidia, and Sarcosporidia. The Haplo- sporidia constitute an order apart. Order I. : Myxosporidia. — This order is characterized chiefly by the following points : The principal trophic phase is a multinucleate plasmodium of relatively large size, resembling an amoeba in its appearance and movements. The spores are also relatively large, and exhibit typically a binary symmetry, having a sporocyst composed of two valves and usually two polar capsules, sometimes increased in number to four, rarely reduced to one. The Myxosporidia comprise a great number of genera and specie*, parasitic for the most part in cold-blooded vertebrates, especially fishes, in which they are found very commonly. They are not as yet known as parasites of birds or mammals, but a few species are known from invertebrate hosts. Myxosporidia are typically tissue-parasites, occurring in various tissues of the body, by preference muscular or connective, but also * A possible exception to this statement is furnished by the family Codospor- idiidai of the Haplosppridia (p. 424). But the position of all the forms in this order is more or less questionable, and their attachment to the typical Neosporidia is still probationary. FIG. 163. — Polar cap- sules of the spores of Myxosporidia. a, Polar capsule with the filament coiled within it ; b, with the filament partly ex- truded ; c, d, with the filament completely extruded. After Balbiani. 400 THE PROTOZOA end. — , other classes of tissue. A few species are known to attack the nervous system — for instance, Lentospora (Myxdbolus) cerebralis, cause of " Drehkrankheit " in Salmonidce (Plehn), and Myxdbolus neurobius of trout (Schuberg and Schroder). In the tissue attacked the parasite may be concentrated at one spot, so as to form a dis- tinct cyst visible to the naked eye ; or parasite and tissue may be mixed up together in a state of " diffuse infiltration " such that microscopic examination is required to detect the parasite, and as its body becomes used up, to form spores, the tissue becomes in- filtrated with vast numbers of spores lying singly or in groups between the cells. In many species of Myxosporidia, on the other hand, the spore- forming plasmodial phase is found in cavities of the body — not in any known instance in the lumen of the digestive tract, but frequently in the gall-bladderor urinary bladder of the host. In such cases the parasite may lie quite freely in the cavity it inhabits, or may be attached by its pseudopodia to the lining epi- thelium ; in the latter case the attachment is purely mechanical, and does not involve injury to the epithelial cells. As might be expected, the Myxo- sporidia parasitic in tissues are often very deadly to their hosts, and are sometimes the cause of severe epidemics among fishes. Those species, on the other hand, which inhabit cavities with natural means of exit from the body appear to be as harmless to their hosts as are the majority of parasitic Protozoa in nature. The adult trophic phase is usually a large amoeba-like organism with a distinct ectoplasm and endoplasm. >In some species the ectoplasm, which appears to be purely protective in function, ex- hibits vertical striations, or is covered by a fur of short, bristle-like processes, the nature and significance of which are uncertain — as, for example, Myxidium lieberkuhni, the common parasite of the urinary bladder of the pike (Esox lucius). The form of the body changes constantly, with extrusion of pseudopodia, which are used for locomotion to a limited extent, more often for fixation, but never for food-capture. They may, however, by increasing the body-surface, increase also the power of absorption of food- FIG. 164. — OMoromyxum leydigi, parasite of the gall- bladder of the dogfish, skate, etc. ; trophozoite (plasmodium) in an active state. ect., Ectoplasm ; end., endoplasm ; y., yellow globules in the endo- plasm ; sp., spores, each with four polar capsules. After Thelohan, from Minchin, magnified 525. THE NEOSPORIDIA 401 stuffs by diffusion, the method by which the organism, like other sporozoan parasites, obtains the required nourishment. The pseu- dopodia vary in form in different species, from coarsely lobose and blunt to fine filaments ending in sharp points. In some species the formation of pseudopodia is localized at one pole of the body, termed " anterior," and in such cases a peculiar propulsive pseudo- podium (" Stemm-pseudopodium ") may be developed at the posterior pole like a tail, which by its' elongation pushes the body forward.* The endoplasm is distinguished from the ectoplasm by its coarsely granular appearance. In addition to numerous nuclei and stages of spore-formation, the endoplasm may contain various metaplastic products, such as crystals, pig- ment-grains, fat-globules, etc. ; but never food-vacuoles or solid ingested food-particles. The plasmodial trophozoite forms spores in its endoplasm, as a rule, during the whole period of growth, but may also multiply by plasmotomy. In Myxidium lieberkiihm, for example, plas- motomy proceeds actively during the summer months, and leads to the wall of the pike's bladder being carpeted with the slimy, orange -coloured plasmodia, the presence of which can generally be detected at a glance ; spore- formation, on the other hand, takes place almost exclusively during the colder months of the year. Spore-formation in the Myxosporidia is a somewhat complicated process, and is accompanied by sexual phenomena, which are commonly stated to be autogamous, but which are probably nothing of the sort. There is a slight difference between the mode of spore-formation in the Disporea, in which each trophozoite produces but two spores, and the Polysporea, which produce many. * Auerbach (758, p. 11) seems to have mistaken altogether the significance of Doflein's " Stemm-pseudopodium," and applies the term to the anterior pseudo- podia, which appear to be rather tactile in function in such cases. 26 ,, FIG. 165.— young which plasmodial trophozoites m the spore-formation has not begun. A, Individual moving forward by means of the " Stemm-pseudopp- diurn " (st. ps.) ; B, individual in which only the anterior pseudopodia are developed. After Doflein. 402 THE PROTOZOA FIG 166. — For description see foot of opposite page. THE NEOSPORIDIA 403 An example of the Disporea is Ceratomyxa drepanopsettce, of which the spore-formation is described by Awerinzew (759). The trophozoite has at first only two nuclei, which are considered by Awerinzew to be derived, " beyond all doubt," from division of a single nucleus ; it seems far more probable, on the contrary, that the binucleate trophozoite is to be derived from the association and fusion of two distinct planonts. In the binu- cleate trophozoite each nucleus divides by karyokinesis into two nuclei, a larger and a smaller (Fig. 166, A). The two smaller nuclei are vegetative, the two larger generative, in function. Round each of the two generative nuclei the protoplasm becomes concentrated so as to form two cells which lie embedded in the endoplasm of the trophozoite. These two cells are usually of distinctly different sizes, and represent a microgametocyte and a macro- gametocyte respectively. Each gametocyte next divides into two gametes (Fig. 166, B, C, D), and in each gamete a certain amount of chromatin is extruded from the nucleus, first into the cytoplasm of the gamete, and then into the endoplasm of the mother-trophozoite. Then each microgamete copulates with one of the two macrogametes (Fig. 166, E, F). The two zygotes thus formed represent the sporoblasts, each of which forms a spore independently of the other. Each sporoblast divides into two cells, a larger and a smaller (Fig. 166, togastris, studied by Schroder (781), for example, the sporonts are distinguished from the meronts by being enclosed in a delicate cyst, within which the sporont multiplies by successive divisions into eight uninucleate sporoblasts (Fig. 173), connected at first by a central mass of protoplasm like a rosette ; but as soon as the sporocyst is formed the sporoblasts become separate. The nucleus of each sporoblast divides until there are five, two for the amoebula, one for the polar capsule, and two for the sporocyst, and the development is similar to that of the spore* of Nosema bombycis already described. A noteworthy feature of many Microsporidia is- that the spores formed are of two sizes, microspores and macrospores, which may differ considerably in their dimensions. In Pleistophora longifilis the macrospores are 12 n in length by 6 /* in breadth, while the microspores are 2 or 3 M in length and broad in proportion (Schuberg). It is very probable that these differences are related to differences in sex of the contained amcebulae, and that the two kinds of spores produce macrogametes and microgametes respectively. H FIG. 173. — Stages in the spore-formation of Thdohania chcetogastris. A, Uni- nucleate sporont ; B, G, division of its nucleus into two ; D, E, F, O, division of the nucleus and body into four ; H, division into eight sporoblasts ; 7, eight sporoblasts, each with the nucleus dividing again ; J, two sporoblasts from a clump, showing further divisions of the nuclei ; K, young spore showing two parietal and three central nuclei (nucleus of the capsulogenous cell and two nuclei of the amoebula). After Schroder (781). In Pleistophora periplanetce, according to Shiwago, several planonts (" amneboids ") fuse into a plasmodium ; their nuclei become resolved into chromidia which become mixed together — a process interpreted by Shiwago as chromidiogamy. Prom the chromidia secondary nuclei are formed, which become the nuclei of the sporonts (" daughter-amoeboids "). The sporonts become free from the plasmodium and form spores. If this account be con- firmed, it is clear that the alleged autogamy of the Microsporidia, if it occurs, is not necessarily an autogamy without amphimixis. In Thdohania mcenadis, according to Perez (778), the nucleus of the sporont becomes resolved into a cloud of chromidia, from which the eight nuclei of the sporoblasts are recon- structed. The greatest difference in the vegetative phase from the condition described for Nosema bombycis is seen in the genus Olugea, where the multiplication of the meront leads to -the formation of a multinucleate plasmodium — a result easily explained on the supposition that the nucleus of the meront divides repeatedly, but the body as a whole does not do so. In this way a relatively large plasmodial trophozoite, comparable to that of the Myxosporidia, is pro- THE NEOSPORIDIA 417 duced, which may form a conspicuous cyst. From the plasmodial stage sporonts arise by separation of a mass of protoplasm round a nucleus within the body of the parasite, and thus distinct cells are formed lying in vacuoles in the plasmodium. Such cells are commonly termed " pansporoblasts," but the use of this term is best avoided, since the cells La question are in no way equivalent to the pansporoblasts of Myxosporidia, which are associa- tions of two gamonts ; but they correspond exactly to the sporonts of Nosema and other genera, and proceed to the formation of spores in the manner that has been described already, dividing first into several sporoblasts. The plasmodia of the Olugea-typo lead, as already stated, to the forma- tion of conspicuous cysts, visible to the naked eye, in the tissues of the host ; but the composition and nature of these cysts are at present a matter of dispute. According to Stempell (784), in Qlugea anomala, the body of the parasite is sharply defined and marked off from the tissues of the host by a thick membrane or autocyst (" Eigencyst ") formed by the parasite itself (Fig. 174, e). Within the autocyst is contained the plasmodium, consisting of € FIG. 174. — Qlugea anomala, Honiez : part of a section of a cyst, e., Envelope (autocyst) ; bn, vegetative nuclei ; sp., spores ; pap, sporont lying in a space in the protoplasm. After Stempell. protoplasm containing many nuclei, amongst which the most conspicuous are large — indeed, relatively gigantic — vegetative nuclei, which multiply by direct division. From the vegetative nuclei the minute nuclei of the sporonts are stated to arise, while in other case vegetative nuclei break up and de- generate. Schroder (781) and Schuberg, on the other hand, maintain that the large vegetative nuclei of Stempell are in reality tissue-nuclei of the host, greatly hypertrophied and mixed up with the plasmodium of the parasite. Schuberg found that Pleistophora longifilis, from the testis of the barbel, causes a hyper- trophy, not only of the host-cell in which it is contained, but also of neigh- bouring cells, the effect of which is to produce a sort of host-plasmodium, as it were, containing gigantic host-nuclei of irregular form (Fig. 171), amongst which the sporonts and spores of the parasite are scattered. Mrazek also interprets the supposed vegetative nuclei of Myxocystis as hypertrophied host-nuclei (see below). This interpretation of the composition of the plas- modium greatly diminishes, or even abolishes, the principal distinction between Qlugea and the other genera of Microsporidia. In opposition to this view, 27 418 THE PROTOZOA Stem i*U (78G) brings forward a number of arguments, the most cogent of which is the existence of the autocj'st separating the plasmodium of tho parasite, containing the nuclei of disputed nature, from the tissues of the host. The most recent investigations of Awerinzew and Fennor confirm com- pletely StempeU's interpretation of the cysts of Qlugea anomala ; compare also Weissenberg. These authors find nuclei of various sizes in the protoplasm of tho cyst, larger or smaller. The larger nuclei are found in the outer, non- vacuolated protoplasmic layer of the Qlugea ; they grow in length and become sausage -shaped, and are ultimately segmented into smaller nuclei, which may form chains at their first origin, like the meronts of Nosema and other forms. In this way arise the smaller nuclei, which either become sporonts, or remain as Vegetative nuclei in the protoplasmic walls of the vacuoles containing the spores, where they ultimately degenerate and break up. The sporonts are stated to arise tn toto from nuclei, without visible participation of tho protoplasm of the cyst ; they become enclosed separately in vacuoles, within which each sporont forms a cluster of spores. Thus,, in older cysts the central part of the body becomes divided by tine protoplasmic partitions into a mass of separate chambers or vacuoles, each containing ripe spores. Glugea anomala is to be regarded, therefore, as a colonial organism in which meronts and sporonts, homologous with those of Nosema, etc., lie embedded in the protoplasm of their own cyst — the meronts in the peripheral zone of growth, the sporonts and spores in the central protoplasmic region of the cyst. Classification. — The two types of the trophic phase that have been do- scribed in the foregoing paragraphs have been utilized by Perez (779) to sub- divide the Microsporidia into two suborders, as given below. Stempell (785), on the other hand, divides the group into three families ; the un- certainty that prevails at present with regard to the exact structure of the trophic phases in some forms is a hindrance to finality in the classification of this order. SUBORDER I. : SCHIZOGENEA (sou Oligosporea). — The principal trophic phase is a uni nucleate meront which multiplies by fission, and from which the sporont finally arises. Several genera, characterized by the number of spores produced by the sporont : One spore, Nosema ; two spores, Perezia ; four spores, Ourleya ; eight spores, Thelohania ; sixteen spores, Duboscqia (see below) ; n spores, Pleistophora ; but Stempellia (Leger and Hesse, 776), for 8. mutabilis, parasite of the fat-body of Ephemerid larvae, produces spores to the number of eight, four, two, or one indifferently ; Ociosporea, the species of which are parasitic in Muscidoe, produces eight spores in one species, one in another. These anomalies indicate that the classification by the number of spores produced by the sporont is purely artificial (Chatton and Krempf). Telomyxa glugci- formis (Leger and Hesse), also from the fat-body of Ephemerid larva;, pro- duces «ight, sixteen, or n spores, and stands apart from all other known Microsporidia in possessing two polar capsules in the spore. SuBOBDEBlI. i BLASTOGENEA (seu Polysporea). — The principal trophic phase is a multinucleate plasmodium producing sporonts by internal cleavage ; example : Qlugea, To this section, also, the peculiar form Myxocystis has been referred, which was discovered by Mrazek in the body-cavity of Oligo- chaetes. Myxocystis occurs in the form of large masses floating freely in the body-cavity, each mass remarkable for an envelope composed of a fur of vertical filaments, not unlike stiff cilia, and enclosing nuclei and spores in various stages of development. According to the most recent investigations of Mrazek, however, each of these masses represents in reality a lymphocyte containing numerous parasites, which multiply and form spores, and provoke a great hypertrophy of the host-cell, accompanied by multiplication of its nucleus. Hence the true Myxocystis is an intracellular parasite referable, apparently, to the order Schizogenea, and characterized chiefly by the peculiar form of its,, spores. Diiboscqia legeri, Perez (780), from the body-cavity of Termes lucifugus, is perhaps an organism of similar nature ; it is described THE NEOSPORIDIA 419 as a floating plasmodium in which sporonts arise, each of which produces sixteen spores ; it has, however, been referred by its discoverer to the Blasto- genea. Order IV. : Sarcosporidia. — The parasites of this order are con- sidered at present to constitute a single genus, Sarcocystis, with numerous species. In contrast to the three orders of Cnidosporidia dealt with in the foregoing pages, the Sarcosporidia are pre-eminently parasites of the higher vertebrates, more especially of mammals, occurring occasionally, though rarely, in man (see Darling) ; but they are known also to occur in avian and reptilian hosts, though sparingly. On the other hand, no Sarcosporidia are known to be parasitic in invertebrate hosts of any kind. In their hosts the Sarcosporidia are tissue-parasites, occurring principally in the striped muscles, but occasionally in unstriped. In a few oases they are found in connective tissue, but this appears to be a secondary condition in which a parasite living first in the muscle-fibres becomes free from them at a later period. As a general rule the Sarco- sporidia appear to be harmless parasites, which do not make their presence known by any symptoms of disease, and can only be detected by post-mortem examination. Some species, however, are an exception to this rule, and are extremely pathogenic to their host — for example, Sarcocystis muris of the mouse. The extent to which the health of the host is impaired appears to be directly pro- portional to the numbers of the parasite in the body, and conse- quently to the power which a given species may possess of multiplying and overrunning the host. In most species the capacity for endogenous multiplication appears to be extremely limited. In spite of the fact that Sarcosporidia are very common parasites of domestic animals, and have been found frequently in man, our knowledge of their structure and life-history is in. a very backward state. As a rule Sarcosporidia present themselves as opaque, whitish bodies, usually elongated and cylindrical in form, encysted in the muscle-fibres of the infected animal, and known commonly as " Miescher's tubes." They are distinctly visible to the naked eye, and often very large. Sarcocystis tenella of the sheep reaches a length of 16 millimetres, while in the roebuck (Cervus capreolus) cysts of 50 millimetres in length are recorded. The Miescher's tube, when examined microscopically, is seen to be a body of complex structure, and consists chiefly of vast numbers of sickle- shaped spores — " Rainey's corpuscles " — lying in clumps or bunches contained in chambers separated off from one another by partitions. The whole organism is enclosed by a distinct envelope, often ex- hibiting vertical striations, and the partitions between the chambers containing the sporos are continuations of the envelope. The exact 420 THE PROTOZOA structure of the spores is still a matter of dispute, and it is possible that there is more than one kind of spore even in the same species of parasite. A remarkable feature of the spores — in some species, at least — is that they are motile when set free : for example, in 8. muris. They are also extremely delicate structures, easily injured by external media, in marked contrast to the spores of the other orders of Cnidosporidia. The spores of 8. muris, 8. bertrami (of the horse), and 8. tenella, reproduce them- selves by division (Negri, Fiebiger, Teichmann). Finally it must be mentioned that the spores of Sarcosporidia contain a true toxin, which was named by Laveran and Mesnil " sarcocystine." Its properties have been investigated recently by Teichmann (25) and Teichmann and Braun (26). The natural mode of transmission of the Sarcosporidia remains to be discovered. It was found by Theobald Smith that mice could be infected experimentally with 8. muris by feeding them with the flesh of other infected mice ; but it is extremely unlikely that cannibalism is the method whereby sheep and other ruminants become infected with these parasites. All experiments indicate that the spores germinate in the digestive tract of the new host ; but the delicate nature of the spores seems to preclude any possi- bility of the occurrence of ordinary coiitaminative infection, as in other Cnidosporidia. In this connection attention should be drawn to the statement of Watson, that the spores are to be found in the circulating blood, indicating the possibility of transmission by an intermediate host. In spite of several recent investigations upon the structure and develop- ment of the Sarcosporidia, the subject is in a confused state, even the structure of the spores being still disputed. It is therefore difficult to obtain a clear notion of the course of the life-cycle in these organisms. According to Laveran and Mesnil, the spores of S. tenella (Fig. 175) are sausage-shaped bodies, curved, with one end more pointed than the other. At the pointed end is a striated structure representing a polar capsule, and at the blunt end is a nucleus, while the middle of the body is occupied by coarse, deeply-staining, metachromatinic grains. Watson also figures a large nucleus near the blunt end of the spore, and places the polar capsule at the pointed end. Negri also describes the spores of 8. muris and 8. bertrami as having the nucleus near tha blunt end, while the opposite extremity appears hyaline and homogeneous for a certain distance. Betegh, again, describes a nucleus at the blunt end of the spore, and one or two " centrosomes " in the middle region. Erdmann (790), on the other hand, places the nucleus in the middle of the body amongst the metachromatinic grains, and describes it as consisting of a large dense karyosome lodged in a small vacuole ; she does not seem to be decided, however, whether the polar capsule is at the pointed or the blunt end of the spore. Teichmann describes a large nucleus at the blunt end of the body, and is doubtful as to the existence of a polar capsule. So far as it is possible to draw any conclusions from so many contradictory statements, the clear description given by Laveran and Mesnil seems to be, on the whole, confirmed. But' according to Crawley, the spores of 8. rileyi are binucleate ; compare those of Gatirocyatis (Pig. 179, p. 428). It is not THE NEOSPORIDIA 421 clear which part of the spore contains the amoebula which is liberated from it, as presently to be described. In addition to spores having the complicated structure described for those of S. tenetta, there appear to be also spores of much simpler structure, as, for example, in S. muris. Apparently the more complicated spore is propa- gative in function, serving to infect new hosts, while the simpler form, which should perhaps be regarded rather as a sporo blast, as a simple cell not differ- entiated as a spore, serves for spreading the infection in the same host. The occurrence of the simpler type of spore in S. muris would account for the manner in which this parasite overruns its host, and is usually lethal to it, while 8. tenella, which appears to produce chiefly propagative spores, is a harmless parasite. How far these suggestions are true must be determined by future investigations. The discovery made by Smith, mentioned above, that mice could be infected with S. muris by feeding them with the flesh of other infected mice, has been confirmed and extended by other observers. According to Negre, the faeces of mice which have been fed with infected muscular tissue are infective to other mice, if ingested by them ; they possess this power about fifteen to sixty days after the mouse was fed with muscle containing Sarcosporidia, and retain their infectivity even if kept dry in an open bottle for a month, or heated to 65° C. for fifteen minutes. Negri was able to infect guinea-pigs with S. muris by feeding them with the flesh of infected mice, and found that in the guinea- pig the parasite appeared with quite different char- acters from those which it presents in the mouse, so that it might be taken easily for a distinct species. Darling also infected guinea-pigs with S. muris in the same way, and points out the resemblance between the experimental sarcosporidiosis of the guinea-pig and a case of human sarcosporidiosis observed by him; it is suggested that the sarco- Fl°- 175. — Spores of sporidia occasionally observed in the human subject "T th i h* are those of some domestic animal undergoing a conditfon . \ 3JJ modified or abortive development in a host that is staining with iron- not their usual one. Erdmann also infected mice hsematoxylin: N., with S. tenetta in a similar manner. It is remarkable nucleus ; c, striated' that parasites generally so harmless should be so body (polar capsule?), little specific to particular hosts, and the results of After Lave ran and Negri render the value of the characters used for Mesnil. distinguishing species of Sarcosporidia as doubtful in their validity as the distinctions founded on their occurrence in certain hosts. According to Erdmann (791), the spore germinates in the intestine of the new host, and the first act in the process is the liberation from the spore of its toxin, sarcocystine, which causes the adjacent epithelium of the intestine to be thrown off. At the same time an amoebula is set free from the spore ; and, owing to the intestine boing denuded of its lining epithelium, the amcebula is able to penetrate into the lymph-spaces of the submucous coat and establish itself there. Before this happens, however, the metachromatinic grains of the spore disappear, and it is suggested that this disappearance is related to the secretion of the sarcocystine, and that the toxin is contained in the metachro- matinic grains. If, however, a polar capsule be discharged during the germina- tion of the spore, as in other Cnidosporidia, it might well bo that the toxin is contained in the polar capsule, and is set free by its discharge, like the poison in the nematocysts of the Ccelentera. However that may be, it would appear as if the sarcocystine were a weapon, as it were, the function of which is to facilitate the invasion of the germ, the amoebula, by destroying the lining epithelium of the gut. The liberation of the amoebula from the spore initiates the first period of the development, which is passed in the lymph-spaces of the intestine, and which lasts, according to Erdmann, some twenty-eight to thirty days. 422 THE PROTOZOA Analogy with other Neosporidia would lead us to identify this with the planont-phase, initiated, possibly, by sexual processes between different amcebula^ and subsequent active multiplication. The second period of the development begins with the penetration of the amoebula into a muscle-fibre, in which the parasite grows into a Miescher's tube and forms spores. The intramuscular development of the parasite begins by multiplication of the nuclei to about twelve, forming a plasmodium (Fig. 176, A). This next becomes divided up, in parasites about thirty- three days old, into separate cells, pansporo blasts or sporonts, which multiply actively by division. The form of the para- site now becomes elongated; this stage is reached in from forty-eight to sixty days (Fig. 176, 5). At this point the para- site may disintegrate, setting free the sporonts, or may develop into a Miescher's tube. In the first case the sporonts wander out and establish themselves in other muscle-fibres, where each sporont initiates a fresh development, thus spread- ing the infection in the tissues of the host. In the second case a membrane is secreted round the body, which forms the striated envelope prolonged inwards to form the chambers. The striated envelope of the Miescher's tube has generally been com- pared to the striated ectoplasm of some Myxosporidia — e.g., Myxidium liebtr- kuhni ; but according to Fiebiger it is not ectoplasm, but altered muscular tissue. The nuclei of the muscle-fibres are stimulated by the parasite to multi- plication and migration. The body then consists of a peripheral zone of sporonts, multiplying actively, and a central region in which spores are differentiated. In the development of the spore, the sporont becomes sausage-shaped, and multiplies by division. Finally the sausage-shaped bodies become spores, and are stated to be at first binucleate ; probably one nucleus is that of the amcebula, the other that of the capsu- logenous cell, parietal cells being absent ; but these statements are at present hypo- thetical andrcquire substantiation. Fufiy- formed spores are found in parasites eighty to ninety days after the infection of the host. In old infections the parasites may have destroyed the muscle-fibre completely, so that the Miescher's tube lies in the connective tissue. In such forms the centre of the body may consist of granular debris, derived from the disintegration of spores which are past their prime and have degenerated. So far as it is possible to draw 'conclusions in the present state of knowledge, the Sarcosporidia would appear to be true Cnido- sporidia, with spores which contain each a single polar capsule, Via. 176. — Four stages in the de- velopment of a " Miescher's tube ' ' of Sarcocystis muris in the pectoral muscles of white rats infected ex- perimentally. A, Parasite 25 p in length, fifty days after infection ; the contents of the body beginning to divide into separate cells ; B, parasite of the same age, 35 /t in length, division of the contents further advanced ; C, parasite of the same age, 60 (n. in length, con- taining separate cells ; at the centre the division of a sppront into two sickle-shaped bodies is seen to be taking place ; D, middle portion of a tube about 450 p in length, seventy days after in- fection, showing, two couples of sickle-shaped bodies formed by division of a sporont. After Negri. THE NEOSPORIDIA 423 and from which an amcebula is liberated, as in other Neosporidia (Amoebogenise). Order V. : Haplosporidia. — The distinctive features of this order are for the most part of negative character, and, as the name im- plies, the tendency is towards simplicity in structure and develop- ment. The spores are without the polar capsules which are so marked a peculiarity in the four previous orders, and have the form of simple cells, each with a single nucleus, and with or without a sporocyst, which, however, when present, is not forined by distinct parietal cells. In organisms of such simple structure, the absence of distinctive peculiarities renders the limits of the group indefinite, and the affinities of its members vague and undecided, and it is possible that the order Haplosporidia, as generally understood, is a hetero- geneous assemblage, many members of which present only develop- mental analogies to the true Neosporidia — that is to say, a simi- larity in the life-history which is an adaptation to a similar mode of life, and not a true indication of genetic affinity. L6ger and Duboscq (646) point out that the characters — " peu limitatifs " — of the Haplosporidia would suit Protista of the most diverse affini- ties, and scarcely mark them off from yeasts or Chytridinese. With the exception of the family Haplosporidiidce, they regard the group Haplosporidia as purely provisional, and comprising heterogeneous forms with undecided affinities. The life-cycle of a typical Haplosporidian parasite is very simple. The initial phase is an amcebula or planont, which multiplies by fission, division of the nucleus being followed by division of the body to form two planonts, which may continue to divide for many generations. From a planont arises ultimately a plasmodial phase, the result of divisions of the nucleus without corresponding divisions of the body, which grows to a relatively large size. The plasmodium is the principal trophic phase. It may multiply by plasmotomy or by schizogony, or may proceed to spore-formation, and then it divides into as many cells as there are nuclei. The cells formed in this way are either sporoblasts, each of which becomes a single spore (Oligosporulea), or they represent sjyronts (" pansporo- blasts "), which give rise each to a cluster of spores (Polysporulea). The spores are usually simple rounded bodies . invested by a more or less distinct protective membrane, which in rare instances becomes a definite sporocyst prolonged even into tails or spikes. The Haplosporidia were divided by Caullery and Mesnil (802) into three families. In order to include forms more recently dis- covered, Ridewood and Fantham have extended the classification, and recognize two suborders : 424 THE PROTOZOA SUBORDER L : OUOOSPOKITLEA. — The plasmodium divides at onoe into sporoblasts, each of which becomes a single spore. Family Haplosporidttdoe. — Spores with a double envelope, the outer some- times prolonged into tails or processes. Genera : Haplosporidium, Urospor- idium, and Anurosporidium ; all the known species are parasitic in Annelids. Family Bertramiidae. — Spores with a simple envelope, or with none. Bertramia, with several species : B. capitellce* parasite of the ccelome of Capitdla capitata ; B. asperospora, a common parasite of the body-cavity of Rotifers. B. kirkmanni, described by Warren from Rotifers in Natal, is stated to have several nuclei and a vacuole in the spore, and appears to belong to a distinct genus. In this family the genus Iclthyosporidium is ranked provisionally, as the mode of spore-formation is unknown as yet. Ichthyosporidium is a common parasite of fishes, often lethal to an extreme degree. It occurs in the form of plasmodia, sometimes irregular, sometimes more or less spherical in form, scattered in various organs, but usually in the muscles or the connective tissue ; the plasmodium contains numerous vesicular nuclei with distinct karyosomes, and may be naked at the surface, or marked off from the sur- rounding tissues by a membrane or envelope, often of considerable thick- ness. The plasmodia multiply actively by plasmotomy, and an intense infection is produced. Parasites with a single nucleus are also found, which may either represent the planbnt stage, or may be derived from the division of a plasmodium ; from them the plasmodial stage arises by multiplication of the nuclei. No other stage of the parasite is known, and the method of transmission remains to be discovered. Bertramia bufoni*, described by King (Proc. Acad. Sci. Philad., 59, p. 273), is possibly a species of Ichthyosporidium or allied to this genus. Family Ccdosporidiidce, for the genera Codosporidium, Mesnil and Marchoux and Polycaryum, Stempell : All the species known are parasites of Crustacea (Phyllopoda and Cladocera). The plasmodium forms globules of fatty substance in the interior ; it becomes encysted as a whole, and breaks up into sporozoite-like bodies within the cyst. Caullerya mesnili, Chatton (803), parasite of the epithelium of the mid-gub of Daphnia spp., produces, by fragmentation of the plasmodium, spores with resistant envelopes containing each about thirty nuclei. Chatton considers it to be intermediate between the Haplosporidiidce and Ccdo- sporidiidce. ; possibly it should be referred to the next suborder. Blastulidium pcedopJitJiorum, Perez, referred to this family, is, according to Chatton (804), a Chytridinian. Codosporidium Uatettce, Crawley, is referred by Leger (C.R.A.S., cxlix., p. 239) to the genus Pdtomyces (Myce- tozoa, p. 243). STJBOEDEB II. : POLYSPOEULEA. — The plasmodium divides into sporonts, each of which produces a cluster of spores. Two genera, each with a single species : Neurosporidium cephalodisci, from the nervous system of Cephalodiscus nigrescent (Ridewood and Fantham) ; and Rhinosporidium kinealyi, from the septum nasi of human beings in India (Minchin and Fantham ; Boattie) ; a case has also been observed in America (Wright). Rhinosporidium causes vascular pedunculated growths or tumours, resembling raspberries, in the septum nasi or floor of the nose. In sections of the growth, great numbers of the parasite are found embedded in the connective tissue, while the mature cysts may be in the stratified epithelium (Wright). The youngest parasites are rounded cells with a single nucleus and a distinct envelope (Beattic). By division of the nucleus the parasite becomes a multinucleate plasmodium, the so-called " granular stage," often of irregular form, but this may be due to the action of the preserving reagents. Older parasites are spherical, with the envelope thickened to form a thick transparent cyst, external to which a nucleated envelope is formed by cells of the connective tissue (Beattie). The contents of the cyst (Fig. 177, A) become divided up into numerous THE NEOSPORIDIA 425 uninucleate sporonts (" pansporoblasts ") towards the centre or at one pole, while the peripheral zone or the opposite pole remains in the plasmodial condition. The sporonts grow in size, and at the same time multiply by repeated fission to form a cluster of about sixteen spores, a " spore-morula " (Fig. 177, B), enclosed by a membrane. Between the spore-morulae an indefinite framework is formed by the residual protoplasm in which the sporonts have developed (Beattie). Hence the full-grown parasite exhibits three zones, which may be concentric or polar in arrangement : a plasmodial region, peripheral or polar ; an intermediate zone of spore-formation; and a central or polar region containing ripe spore-morulae. The process of spore -formation continues until the whole cyst is full of spore-morulae. The ripe cysts burst and scatter their contents in the tissues. It is possible that spores set free in this way may germinate in the tissues and give rise to fresh cysts ; but it is more probable that the spores, if they escape the phagocytes, are dis- charged from the surface of the epithelium. Prom the analogy of other Neosporidia, it is reasonable to suppose that the youngest uni- nucleate forms of the parasite are the multiplicative phase in the tissues, and that the spore-morulae represent the propagative phase. Nothing is known, however, of the mode of transmission of the para- site or of the manner in which the infection is acquired. A parasite is described by Laveran and Pettit from Salmo irideus, which in the opinion of the authors presents affinities with Rhinosporidium and Neuro- sporidium. It causes a disease in the fish, termed in German " Taumelkrankheit." B Fio. 177. — Rhinosporidium kinealyi. A, Segment of a section through a cyst : e., hyaline envelope ; p.z., peripheral zone of pansporo blasts ; t'.z., inter- mediate zone of pansporo blasts contain- ing a few spores ; c.z., central zone of ripe spore-morulse; B, ripe spore-morula ; m., membrane ; ap., spores. After Minchin e.nd Fantham. In addition to the more or less typical genera of Haplosporidia mentioned in the foregoing para- graphs, a number of other forms have been described, of which the affinities and systematic position re- main for the present uncertain. Such are the " Serumsporidia " of Pfeiffer, and other forms, for a review of which the reader must be referred to the comprehen- sive memoir of Caullery and Mesnil (802) or to the original descriptions. The remarkable form, Schewiakovetta schmeili, however, presents peculiarities which deserve special mention. It is a parasite of the body-cavity of Cyclops spp., and was the subject of detailed study by Schewiakoff. In the active con- dition it occurs as an amoeba with a single nucleus and a contractile vacuole, or as a plasmodium formed by fusion of such amoebae. Encystment of either the amoebae or the plasmodia occurs, and within the cyst a number of simple, uninucleate spores are formed, which, although possessing a distinct envelope, multiply further by fission, with mitosis of the nucleus. Germination of the spores sets free small amoebulae. In many points this form is unique amongst the Sporozoa, and should perhaps be classed rather with the parasitic amoebae. Incertce parasite of African relapsing fever; S. gattinarum of fowls; S. anserina of geese ; and numerous other species from various hosts. In structure the body of these species appears to be little, if any- thing, more than a flexible thread of chromatin ; but the develop- ment indicates rather that, as in the genus Cristispira, the interior of the body is divided into minute segments or chambers. The species parasitic in blood are transmitted by the agency of blood-sucking arthropods. S. duttoni, for example, is transmitted by a tick — Ornithodoros moubata — which lives in the mud-floors of huts or in the soil in spots where caravans camp habitually. The spirochaetes are taken up from human blood by the adult ticks, and pass through the egg into the next generation of nymphs,* which transmit the infection to human beings. 5. Treponema, the name proposed by Schaudinn for T. pattidum, the spirochaete of syphilis discovered by him- A second species — T. pertenue, the parasite of yaws (framboesia) — is also recognized. Structurally this type is very similar to the last. Some authors — for instance, Gross (899) and Dobell (895) — consider that there is " no valid reason for drawing a generic distinction between Treponema pattidum and such forms as ' Spirochceta ' recwnrentis, etc.5' Gross combines Types 4 and 5 under the name Spironema proposed by Vuillemin ; but since this name is preoccupied, Dobell places them together in Schaudinn's genus Treponema. The forms parasitic in the blood of human beings and other vertebrates were generally regarded as Bacteria of the genus Spirillum, or at least of the- section Spirillacea, until quite recent years, and the diseases caused by them were spoken of as spirilloses. The chief points of difference between tho spirilla of relapsing fevers and those of the ordinary type were the flexibility of the body in the former and the failure to grow them in cultures. Tho con- fusion prevailing at present originated with Schaudinn's famous memoir on the blood-parasites of the Little Owl (132). While, on the one hand, it is to Schaudinn's credit to have recognized the affinities of the parasitic " spirilla " to Ehrenberg's free-living genus Spirochata he was, on the other hand, misled by the superficial resemblance between spirochaetes and certain small, slender forms of trypanosomes, which again he connected, quite erroneously, with the life-cycle of Leucocylozoon (see p. 370). Schaudinn therefore regarded the spirochaetes as Protozoa allied to trypanosomes, and endeavoured to prove a similar type of organization in both classes of organisms : a nuclear * The six-legged larval stage ia suppressed — that is to say, passed through in the egg — in this species of tick. 468 THE PROTOZOA apparatus with kinetonucleus and trophonucleus, and a locomotor apparatus with flagellum and undulating membrane. Schaudinn further constructed a hypothetical form of " Urhaemoflagellat " connecting the spirochaeto and trypanosome type of organization ; he put forward the suggestion (903) that " as the general structural plan of a trypanosome (nuclear and locomotor apparatus) may be found realized in various groups of Protozoa as a transitory developmental condition (comparable somewhat to the gastrula -condition in the Metazoa), so also the spirochaete may crop up occasionally as a morpho- logical type in the development of Protozoa, and as a developmental stage may indicate to us phylogenetic relations." Schaudinn lived long enough, fortunately, to retract many of his state ments with regard to the structure of spirochaetes, and acknowledged that the trypanosome -type of structure was not to be made out in the minute parasitic spirochaetes^ Nevertheless, since his time the investigators of these organisms have been divided into two camps — those who hold fast to Schaudinn's theorj' of the spirochaetes as Protozoa, and those who class them with Bacteria, respectively ; it being generally assumed, for some unknown reason, that ii they are not Protozoa they must be Bacteria, or vice versa. A third set of authorities compromise by placing the spirochaetes in an intermediate position between the two groups. In considering the question of the affinities of the spirochaetes, attention has been directed not only to their structure, but also to their life-history ; and a hot controversy has raged with regard to their mode of fission, whether it takes place longitudinally, as in a trypanosome, or transversely, as in a bacterium. Investigators contradict each other flatly with regard to this point ; but from the most recent investigations it seems probable, at least, that the division is always transverse, and that the appearance of longitudinal division is due to the peculiar method of " incurvation :' described by Gross (Fig. 194). A spirochaete about to divide grows greatly in length, and one end of the body doubles back on itself, continuing to do so until the recurved limb of the body is of the same length as the remainder ; the two halves twist round each other and produce an appearance which may be mistaken easily for longitudinal fission ; but the actual division of the body takes place at the point where it is bent over, and is transverse. With regard to the development, nothing has been found in the least con- firmatory of Schaudinn's statements with regard to " Spirochceta ziemanni" with the sole exception of the statements of Krzysztalowicz and Siedlecki (901), who profess to have seen trypanosome-stages in the development of Treponema pallidum ; but their statements are entirely unconfirmed by other investi- gators. Of a very opposite type are the statements of Leishman (902) with regard to the development of S. duttoni in the tick. The spirochaete appears to break up into minute masses of chromatin, " coccoid granules," in the ova and tissue-cells of the tick. The coccoid granules appear to develop into spirochaetes again. The observations of Leishman have recently been fully confirmed by the in- vestigations on the development of Spiroschaudinnia gallinarum published by Hindle(900) who gives a useful diagram of the entire life-history. Bosanquet (894) also observed the formation of coccoid bodies in Cristispira anodontce by the segmentation of the elongated body into a number of coccoid bodies like a string of beads. A development of this type suggests very strongly affinities with bacteria, but none whatever with Protozoa of any class. The coccoid grains may be compared with the spore -formation in bacteria, and with that described by Gross (898) in Saprospira grandis. In all cases, through- out the series of living beings, wherever an organism exhibits in its fully- developed " adult " stage peculiarities of a special kind, it is above all to the early developmental forms that the naturalist turns for indications of the true affinities of the organism in question. Recently the structure of spirochaetes has been studied carefully by Gross (897, 898), Zuelzer (904), and Dobell (895), by means of proper cytological methods of technique. The results show a complete difference in every CLASSIFICATION OF THE MAIN SUBDIVISIONS 469 respect between spirochsetes and trypanosomes and other flagellates. In the words of Dobell, " the nuclear and cytoplasmic structures are wholly different ; a trypanosome has a flagellnm, a spirochaete has none ; the crista is not an undulating membrane ; the cell-membranes are not similar ; and, moreover, the method of division is quite different in the two organisms." Doflein (7) places the spiroohaetes as a group named the Pro- flagellata, supposed to be transitional from bacteria to flagellates. Zuelzer (904) takes a similar view, rejecting, however, any affinity between spirochaetes and Hartmann's " Binucleata." Awerinzew (890) puts forward the remarkable suggestion that the Flagellata " pass on into different Binucleata, and end with the Spirochceta FIG. 194. — Stages in the division of Cristispira pectinis. A, B, Two successive stages of the incurvation ; G, incurvation complete ; D, division of the body at the point where it is bent back ; E, F, separation of the two daughter- spiroohffites. After Gross (897). (sic)" from which it would appear that he regards the spirochaetes as the last product of the line of evolution that produced the trypanosomes and allied forms. For the various reasons that have been set forth above, it appears impossible to include the spirochaetes any longer in the Protozoa. Dobell regards them as " an independent group of unicellular organisms which show very little affinity to any other group/' Gross, on the other hand, considers that the Spironemacea — i.e., the genera Cristispira, Saprospira, and Spironema, in the sense in which this genus is understood by him (see above) — form a family which can be ranked in the bacteria, but which is related to the Cyanophyceae, especially the Oscillatoriae. 470 THE PROTOZOA THE CHLAMYDOZOA. The name Chlamydozoa of Prowazek (Strongyloplasmata, Lip- schiitz) was proposed in order to include in the first place a class of highly problematic organisms believed to be the causes of certain diseases of man or animals. It is not yet certain exactly what diseases are to be referred to Chlamydozoa. According to Hart- mann (909), undoubted chlamydoToal diseases are vaccinia and variola, trachoma, and molluscum contagiosum, amongst human beings, and in birds epithelioma contagiosum and diphtheria. Further diseases probably attributable to Chlamydozoa are hydro- phobia, scarlet fever, measles, foot-and-mouth disease of animals, and " Gelbsucht " of silkworms. In all these diseases the virus has certain common properties, while exhibiting specific peculiarities in each case. It can pass through ordinary bacterial filters without losing its virulence, and it produces characteristic reaction-products or cell-inclusions in the infected cell. In order to understand why these organisms should be men- tioned in a book dealing with Protozoa, the subject is best dealt with in an historical manner. The advances in the knowledge of the diseases mentioned may be summarized briefly in four principal stages : 1 . Various investigators at different times have made known the existence of peculiar cell-inclusions in the infected cells in a certain class of diseases, inclusions which have been known by the names of their discoverers — for instance, in trachoma (Prowazek's bodies), vaccinia. (Guarnieri's bodies), scarlet fever (Mallory's bodies), hydrophobia (Negri's bodies), etc. 2. By many investigators the characteristic cell-inclusions were identified as the actual parasitic organisms causing the disease. They received zoological names, were referred to a definite position in the ranks of the Protozoa, and attempts were made to work out and construct a developmental cycle for them. The supposed parasites of molluscum contagiosum were referred to the coccidia ; those of vaccinia and variola were given the name Cytoryctea ; of hydrophobia, Neurorycies ; of scarlet fever, Cydasterium. Calkins (908) studied in great detail the cell-inclusions of vaccine and smallpox, and described a complete developmental cycle, in its main outlines as follows : The primary infection is brought about, probably, at some spot on the mucous membrane of the respiratory or buccal passages by air- borne germs (spores). After active pro- liferation at the seat of the primary infection, the parasites are carried to all parts of the body in the circulation, probably during the initial fever. These two early phases are hypothetical. The third phase is the appearance of the parasites in the cells of the CLASSIFICATION OF THE MAIN SUBDIVISIONS 471 stratified epithelium of the epidermis. In this situation they run through two cycles — the one cytoplasmic, the other intranuclear. The first is the vaccine-cycle, and is the only part of the develop- ment of which the harmless vaccine-organism is capable ; the variola-organism, however, after passing through a vaccine-cycle, proceeds to the extremely pathogenic intranuclear cycle. The vaccine-cycle, according to Calkins, begins with the appear- ance of " gemmules " in the cytoplasm of the cells affected. Each gemmule is a minute grain of chromatin without cytoplasm of its own at first, but as it grows a cytoplasmic body is formed. When full-grown, the parasite sporulates by fragmentation of its nucleus into a great number of grains, which, as gemmules, pass into other cells and repeat the development already described. Several generations of this type may succeed each other before giving rise to the next type. The intranuclear variola-cycle begins in the same way with gemmules, which, however, penetrate into the nucleus, and develop a cytoplasmic body. According to Calkins, they become sexually differentiated, and produce gametes which conjugate. The final result is the production of numerous spores, which are probably the means of spreading the infection. Calkins referred Cytoryctes to the Microsporidia. Now, however, he inclines to the opinion that the genus should be placed amongst the Rhizopods (4). Negri (910) also describes a developmental cycle for Neuroryctes hydrophobia, which he regards as a true Protozoon, and which Calkins refers also to the Rhizopoda. Siegel (914) describes under the name Cytorhycles organisms of a type perfectly different from those described by Calkins. He distinguishes four species — Cyto- rhyctes vaccinias of vaccine and smallpox, C. luis of syphilis, C. scarla- tinas of scarlet fever, and C. aphiharum of foot-and-mouth disease. 3. The parasitic life-cycles described by Calkins and others have been criticized by a number of investigators, .who have maintained that the bodies in question are not Protozoa, nor even independent living organisms at all, but merely degeneration-products of the cell itself, provoked by a virus yet to be found. Thus, with regard to Guarnieri's bodies (Cytoryctes) of vaccine, it is maintained by Foa, Prowazek, and others, that they consist of nucleolar substance (plastin) extruded from the nucleus ; that they have no definite developmental cycle ; and that infection can be produced by lymph in which Guarnieri's bodies have been destroyed, or by tissue in which they are not present. With regard to the Negri bodies, Acton and Harvey (906) come to the same conclusions, and state that similar nucleolar extrusions can be brought about also by other stimuli than the rabies- virus. 472 THE PROTOZOA 4. The foregoing sceptical phase has been succeeded by the positive belief that the true parasitic organism in these diseases consists of certain minute bodies — the Chlamydozoa or strongyloplasms.* The chief characteristics of the Chlamydozoa, according to Prowazek and Lipschiitz (913) are, first, their minute size, smaller than any bacteria hitherto known, enabling them to pass the ordinary bacterial filters ; secondly, that they develop within cells, in the cytoplasm or nucleus, and produce characteristic reaction- products and enclosures of the cell (their position within the cell is not the result of phagocytosis) ; thirdly, that they pass through a series of developmental stages, and are specially characterized by their mode of division, which is not a simple process of splitting, as in bacteria, but is effected with formation of a dumb-bell-shaped figure, as in tbe division of a centriole. Two dots are seen con- nected by a fine line like a centrodesmose, which becomes drawn out until it snaps across the middle, and its two halves are then re- tracted into the body. Chlamydozoa have not yet been grown successfully in cultures, but infections can be produced with pure colloid-filtrates, free from bacteria, but containing the minute bodies theicselves. They are characteristically parasites of epi- blastic cells and tissues. As an example of the development of a chlamydozoon may be taken that of the vaccine- virus, which, according to Prowazek (913) and Hartmann (909), is briefly as follows : 1. The infection begins and ends with numerous " elementary corpuscles " (gemmules of Calkins ?), which occur both within and amongst the cells. They are very minute, and can pass bacterial filters. 2. Within the cells the elementary corpuscles grow into the larger " initial bodies." ' 3. The infected cell extrudes nucleolar substance — plastin — from its nucleus, which envelops the parasites as in a mantle (hence the name Chlamydozoa, from x\afj.v^, a mantle), thus producing in the case of vaccine the characteristic Guarnieri's bodies, in which the paiasites multiply. It is this mantle of nucleolar substance, apparently, which represents the " cytoplasm " of Cytoryctes, as described by Calkins. * The name Chlamydozoa, as denoting a clasi of microscopic organisms, must on no account be confused with the names Cytoryctes, Neuroryctes, etc., which represent the generic names of the supposed parasites of variola and rabies re- spectively. To those who regard Cytoryctes, etc., as true organisms, the Chlamydo- zoa are merely chromidia or dots of chromatin in the body of the parasite ; to those who believe in the Chlamydozoa as complete organisms, Cytoryctes, etc., are cell-inclusions or degeneration-products of the nucleus. The conceptions implied in the words Chlamydozoa and Cytoryctes respectively are antagonistic and mutually destructive ; if the one is a reality, the other is non-existent. It is altogether incorrect to speak of Cytoryctes, Neuroryctes, etc., as genera of Chlamy- dozoa. CLASSIFICATION OF THE MAIN SUBDIVISIONS 473 4. Finally, the Guarnieri's body breaks up, and the cell becomes full of initial corpuscles, which divide up in their turn into numerous elementary corpuscles, and the cycle is complete. An interesting problem, from both the medical and biological points of view, is that of the relation of the organism of vaccinia (cow-pox) to that of variola (small-pox). It is well known that an inoculation with vaccine-lymph (vaccination) produces a transitory local disturbance which confers partial immunity against infection with variola. It does not seem to be quite clear whether the or nanisms, of vaccinia and variola are to be regarded as two distinct species or as two phases or conditions of the same species of or- ganism; the latter is the view of Calkins, as stated above. Manson has suggested (Brit. Med. Journ., 1905, ii., p. 1263) that the relationship between the organisms of vaccinia And variola may be similar to that, between Leishmania iropica, of Oriental Sore, and L. donovani, of Eala-azar. No evidence has been brought forward as yet, however, to show that an infection with Oriental Sore confers any immunity against Eala-azar. The Chlamydozoa have been most studied in those cases where their power of producing disease has forced them upon the atten- tion of medical investigators, but it is not to be supposed that as a group of organisms they occur solely as parasites of higher animals. It is probable that they are of widespread occurrence, and that the peculiar nuclear parasite of Amoeba known as Nucleophaga, Dan- geard, for instance, should be referred to the Chlamydozoa (com- pare Schepotieff, 269), and perhaps also the similar parasite of Paramecium described by Calkins under the name Caryoryctes. No Chlamydozoa are known, however, to occur as free-li ving, non- parasitic organisms, but this circumstance may be due to theii extreme minuteness ; the species "mown owe their detection to the disturbances they cause in their hosts. Finally, it must be men- tioned that the parasitic theory of cancer, sometimes thought to be long since defunct, has been revived recently by Awerinzew (907), who is of opinion that cancer is caused by intranuclear parasites of the nature of Chlamydozoa. Such, briefly summarized, is the present position of the problem. Future research must decide the truth or falsity of one or the other of the solutions that have been advocated. It only remains to discuss briefly the nature of the Chlamydozoa, if the interpreta- tion of Prowazek and his adherents be accepted. According to Prowazek and Lipschiitz (913), the Chlamydozoa belong neither to the Bacteria nor to the Protozoa. Hartmann (909), however, seems to consider that their development and their characteristic mode of division are Protozoan characteristics. The " development," how- ever, seems to consist of little, if anything, more than growth in size As "elementary corpuscles" they are smaller, as "initial bodies" larger. The dumb-bell-shaped figure seen in division may mean 474 THE PROTOZOA simply that their substance is of a viscid or semifluid nature, and that their bodies are Hot limited by a membrane ; consequently, when the two halves travel apart in the process of division, the substance of the body is drawn out into a connecting thread until its surface tension overcomes its cohesion. On the other hand, they exhibit nothing of cell- structure or of any other characteristics which indicate any affinity to the Protozoa. Their type of organiza- tion seems to be the simplest possible in a living body — a mere grain of chromatin without cytoplasm, and without a membrane or envelope of any kind. In the latter respect they appear to be of a simpler type of organization than any bacterium, and perhaps represent more nearly than any other known organism the simplest possible form of living being. Bibliography. — For references see p. 504 lie domum, saturce, venit Hesperus, ite capettx. BIBLIOGRAPHY The references to literature are numbered consecutively, but are grouped according to the chapters. An asterisk (*) attached to a reference' indicates that the work in question contains full references to the previous literature of the subject. Memoirs in which only new species are described are not cited, unless there is some special reason for doing so. All new species are recorded in the " Zoological Aecofd," published annually by the Zoological Society of London ; the last volume published up to date if that for 1910 ; the volume for 1911 wiU appear towards the end of 1912. The titles of the subject-matter of articles are in many cases not given verbatim, but in abbreviated form. The abbreviations employed for the titles of periodicals are given below. (In other cases the titles of periodicals are abbreviated in a manner which does not require special explanation.) A.I.C.P. Archives do Institute Bacteriologipo Camara Pestana (Lisbon). A.I.P. Annalee de 1'Institut Pasteur (Paris). A.K.G.A. Arbeiten aus dem kaiserlichen Gesundheitsamte (Berlin). A.P.K. Arohiv fiir Protistenkunde (Jena). A.S.T.H. Archiv fur Schiffs- und Tropenhygiene (Leipzig). A.T.M.P. Annals of Tropical Medicine and Parasitology (Liverpool). A.Z.E. Archives de Zoologie experiinentale et generale (Paris). B. A.S.C. Bulletin Internationale de 1' Academic des Sciences a Cracovie. B.B. Biological Bulletin (Woods Holl, Mass.). B.C. Biologisches Centralblatt (Leipzig). B.I.P. Bulletin de 1'Institut Pasteur (Paris). B.S.P.E. — de la Societe" de Pathologie Exotique (Paris). B.S.Z.F. — de la Societe Zoologique de France (Paris). C.B.B.P.K. Centralblatt fur Bakteriologie, Parasitenkunde und Infections- kr&nkheiten (Jena). C.B.A.S. Comptes-rendus hebdomadaires des Seances de 1' Academic des Sciences (Paris). C.R.S.B. des Seances et Memoires de la Societ6 de Biologie (Paris). J.E.M. Journal of Experimental Medicine (Baltimore). J.E.Z, — of Experimental Zoology (Baltimore). J.H. — of Hygiene (Cambridge). J.L.S. — of the Linnean Society : Zoology (London). M.I.O.C. Memorias do Institute Oswaldo Cruz (Rio de Janeiro). P.R.S. Proceedings of the Royal Society of London. Py. Parasitology (Cambridge). P.Z.S. Proceedings of the Zoological Society of London. Q.J.M.S. Quarterly Journal of Microscopical Science (London). S.B.A.B. Sitzungsberichte der koniglich-preussischen Akademie der Wissen- schaftcn zu Berlin. S.B.G.B. — der Gesellschaft naturforschender Freunde zu Berlin. S.B.G.M.P. — der Gesellschaft fiir Morphologic und Physiologic in Miinchcn. S.M.I. Scientific Memoirs by Officers of the Medical and Sanitary Depart- ments of the Government of India (Calcutta). V.D.Z.G. Vorhandlungen der deutschcn zoologischen Gesellschaft (Leipzig). Z.A. Zoologischer Anzeiger (Leipzig). Z.a.-P. Zeitschrift fiir allgemeine Physiologic (Jona). Z.H. — fiir Hygiene und Infectionskrankheiten (Leipzig). Z.w.Z. — fiir wissenschaftliche Zoologie (Leipzig). 475 476 THE PROTOZOA CHAPTER I General Works on Protozoa. (1) BRUMPT, E. (1910). Precis de Parasitologie. Paris : Masson et Cie. *(2) BiJTSCHLi, O. (1882-1889). Protozoa. Bronn's Klassen und Ordnungen die TUer-Reichs, I. (3) — (1910). Vorlesungen iiber vergleichende Anatomic, 1. Leipzig: W. Engelmann. (4) CALKINS, G. N. (1901). The Protozoa. New York : Macmillan and Co. *(5) — (1909). Protozoology. New York and Philadelphia : Lea and Fiebiger. *(6) DELAQE, Y., and HEROTTARD, E. (1896). Traite de Zoologie Concrete, I. Paris : Schleicher Frere&. *(7) DOFLEIN, F. (1911). Lehrbuch der Protozoenkunde. Third edition. Jena: Gustav Fischer. (8) HARTOG, M. (1906). Protozoa. Cambridge Natural History, vol. i. London: Macmillan and Co. (9) KENT, W. S. (1880-1882). A Manual of the Infusoria. London : David Bogue. *(10) LANG, A. (1901). Lehrbuch der vergleichenden Anatomic der wirbellosen Thiere, 2te Auflage. Jena : Gustav Fischer. (11) LANKESTEB, E. R. (1891) Protozoa. Encyclopaedia Sritannica, ninth edition ; reprinted in Zoological Articles. London : A. and C. Black. (12) — (1903 and 1909). A Treatise on Zoology. Part I., Fascs. 1 and 2. London : A. and C. Black. (13) MINCHIN, E. A. (1907). Protozoa. Allbutt and Rolleston : A System of Medicine, vol. ii., part ii., p. 9. (14) PROWAZEK S. V., and others (1911). Handbuch der Pathogenen Protozoen. Leipzig : J. A. Barth. Lief. 1 and 2. (15) ROLLESTON, G., and JACKSON, W. H. (1888). Forms of Animal Life. Second edition. Oxford : Clarendon Press. CHAPTER II In addition to the general works cited under the previous chapter, see especially : (16) GOODEY, T. (1911). A Contribution to our Knowledge of the Protozoa of the Soil. P.R.8. (B.), Ixxxiv., p. 165. (17) LATJTERBORN, R. (1901), Die " sapropelische " Lebewelt. Z.A., xxiv., p. 50. (18) LAVERAN, A., and MESNIL, F. (1899). De la Sarcocystine, toxine des Sarco- sporidies. C.R.S.B., Ii., p. 311. (19) — and PETTTT, A. (1911). Les trypanotoxines. B.S.P.E. iv. p. 42. (20) MESNIL, F. (1905). L'Heredite dans les Maladies a Protozoaires. B.I.P., iii., p. 401. (21) MINCHIN, E. A. (1910). Phenomena of Parasitism amongst Protozoa. Journ. Quekett Microscop. Club (2), xi., p. 1. (22) ROTTDSKY, D. (1910). Le Trypanosoma leurisi Kent renforce. C.B.8.B., brix., p. 384. (23) — (1911). La possibility de rendre le Trypanosoma leurisi virulent pour d autres rongeurs que le rat. C.R.A.S., clii., p. 56. (See also Bulletin of the Sleeping Sickness Bureau, vol. iii., pp. 81 and 265 for further references on this subject.) (24) RUSSELL, E. J., and HTTTCHINSON, H. B. (1909). The Effect of Partial Sterilization of Soil on the Production of Plant Food. Journ. Agric. Sci., iii., p. 111. (25) TEICHMANN, E. (1910). Das Gift der Sarcosporidien. A.P.K., xx., p. 97. (26) — and BRAXTN, H. (1911). Ein Protozoentoxin (Sarcosporidiotoxin). A.P.K., xxii., p. 351. (27) WENDELSTADT and FELLMER, T. (1910). Einwirkung von Kaltbliiterpaa- sagen auf Nagana- und Lewisi-Trypanosomen. Zeitschr. f. Immunitdts- forschung, v., p. 337. (28) WINTER, F. W. (1907). yntersuchung fiber Peneroplis pertusus (Forskal). A.P.K., x., p. 1. BIBLIOGRAPHY 477 CHAPTER in In addition to the general works cited under Chapter I., see especially : (29) HERON-ALLEN, E., and EABLAND, A. (1909). A New Species of Technitella. Journ. Quekett Microsc. Club (2), x., p. 403. (30) KOLTZOFF, N. K. (1903). Formbestimmende elastische Gebilde in Zellen. B.C., xxiii., p. 680. (31) — (1906). Die Gestalt der Zelle. Arch. mikr. Anal., Ixvii., p. 364. (32) PBOWAZEK, S. v. (1908). Biologic der Zellen, I. B.C., xxviii., p. 782. (33) — (1909). Theorie der Cytomorphe. Z.A., xxxiv., p. 712. (34) RHTIMBLER, L. (1898). Physikalische Analyse von Lebenserscheinungen der Zelle, I. Arch. Entwicklungsmech., vii., p. 103. (35) — (1902). Die Doppelschalen von Orbitdites. A.P.K., L, p. 193. (36) VEBWORN, M. (1888). Biologische Protisten-Studien. Z.w.Z., xlvi., p. 455. CHAPTER IV In addition to Nos. 34 and 35, see : *(37) BUTSCHJJ, 0. (1894). Microscopic Foams and Protoplasm. (Translation by E. A. Minchin.) London : A. and C. Black. (38) FATTRE'-FREMIET, E. (1908). La Structure des Matieres Vivantes. B.S.Z.F., xxxiii., p. 104. *(38'5) — (1910). Les Mitochondries des Protozoaires et des Cellules sexuelles. Arch. cTAnat. Microsc., xi., p. 457. *(39) FISCHER, A. (1899). Fixirung, Farbung und Bau des Protoplasmas. Jena : Gustav Fischer. (40) RHTJMBLER, L. (1902). Der Aggregatzustand und die physikalischen Beson- derheiten des lebenden Zellinhalts. Z.a.P., ii., p. 183. CHAPTER V In addition to the references cited above for Chapters I. and III., and those cited below for Chapter X., see : (41) GOLDSCHMIDT, R. (1907). Lebensgeschichte der Mastigamoben. A.P.K., Suppl. I., p. 83. (42) MINCHIN, E. A., and WOODCOCK, H. M. (1911). The Trypanosome of the Little Owl (Athene noctua). Q.J.M.S., Ivii., p. 141. (43) SCHAUDINN, F. (1894). Camptonema nutans. 8.B.A.B., lii., p. 1227. Re- printed, Schaudinn's Arbeiten, 1911, p. 50. (44) SCHTJBERG, A. (1905). Cilien und Trichocysten einiger Infusorien. A.P.K., vi., p. 61. CHAPTER VI In addition to the works cited here, see also the bibliographical references for Chapter VII: (45) ARAGAO, H. DE B. (1910). Ueber Polytometta agilis. M.I.O.C., ii., p. 42. (46) AWBRINZEW, S. (1907). Struktur des Protoplasma und des Kerns von Amoeba proteus (Pall.). Z.A., xxxii., p. 45. (47) — (1909). Entwicklungsgeschichte von Coccidien aus dem Darme von Cerebratulus sp. (Barrouxia spiralis). A.P.K., xviii., p. 11. (48) CALKINS, G. N. (1903). The Protozoan Nucleus. A.P.K., ii., p. 213. (48'5) CHAGAS, C. (1911). Die zyklischen Variationen des Caryosoms bei zwei Arten parasitischer Ciliaten. M.I.O.C., iii., p. 136. *(49) CHATTON, E. (1910). La structure du Noyau et la Mitose chez les Amoebiens. A.Z.E. (5), v., p. 267. (50) COLLIN, B. (1909). La Conjugaison d' Anoplophrya branchiarum (Stein) (A. circvlans, Balbiani). A.Z.E. (5), i., p. 345. *(51) DOBELL, C. C. (1909). Chromidia and the Binuclearity Hypothesis. Q.J.M.S., liii., p. 279. 478 THE PROTOZOA *(52) DOBELL, C. C. (1911). Contributions to the Cytology of the Bacteria. Q.J.M.8., Ivi., p. 395. " Autorreferat " ia A.P.K., xxiv., p. 84. (53) ENTZ, G. (1909). Organisation und Biologic der Tintinniden. A.P.K., xv., p. 93. (54) ERHABD, H. (1911). Die Henneguy-Lenhosseksche Theorie. Ergebn. Anat. Enttoick., xix. (second half), p. 893. (55) FAURE'-FKEMIET, E. (1910). Appareil nucleaire, Chromidies, Mitoohondries. A.P.K., xxi., p. 186. (56) FRAN9A, C., and ATHIAS, M. (1907).' Les Trypanosomes des Amphibiens, II. Le Trypanoaoma rototorium de Hyla arborea. A.I.C.P., i., p. 289. (57) GOLDSCHMIDT, R. (1904). DieChromidienderProtozoen. A.P.K., v.,p- 126. (58). — and POPOFF, M. (1907). Die Karyokinese der Protozoen und der Chromi- dialapparat der Protozoen- und Metazoenzelle. A.P.K., viii., p. 321. (59) GUUXERMOND, A. (1910). Corpuscules metachromatiques ou Grains de Volu- tine. A.P.K., xix., p. 289. (60) HARTMANN, M. (1909). Pplyenergide Kerne. B.C., xxix., pp. 481 and 491. (61) — (1911). Die Konstitution der Protistenkerne. Jena : Gustav Fischer. (62) — and CHAOAS, C. (1910). Flagellatenstudien. M.I.O.C., ii., p. 64. (63) — and PROWAZEK, S. v. (1907). Blepharoplast, Caryosom und Centrosom. A.P.K., x., p. 306. (64) HERTWIO, R. (1898). Kerntheilung, Richtungskdrperbildung und Befruch- tung von Actinoaphcerium Eichhorni. Abhandl. bayer. Akad. (II. Cl.) xix., p. 631. (65) — (1899). Encystierung und Kernvermehrung bei Arcetta vulgaria. Kup- ffer's Festschrift, p. 567. (66) — (1902). Die Protozoen und die Zelltheorie. A.P.K., i., p. 1. (67) — (1903). Das Wechselverhaltnis von Kern und Protoplasma. S.B.Q.M.P., xviii. p ;T7. (68) — (1907). Der Chromidialapparat und der Dualismus der Kernsubstanzen. Ibid., xxiii., p. 19. (69) JAHN, E. (1904). Kernteilung und Geisselbildung bei den Schwannern von Stemdnitis flaccida. Her. Deutech. Bot. Oes., xxii., p. 84. (70) JAMCKI, C. (1910). Parasitische Flagellaten, I. Lophomonaa Uattarum, L. striata. Z.w.Z., xcv., p. 243. (71) — (1911). Der Parabasalapparat bei parasitischen Flagellaten. B.C., xxxi., p. 321. (71*5) — (1912). Parasitische Arten der Gattung Paramoeba. Verh. Natur- forach. Gea. Basel, xxiii. (72) L»GER, L., and DUBOSCQ, O. (1911). Deux Gregarines des Crustaces. A.Z.E. (5), vi., " Notes et Revue," p. lix. (73) MAIBR, H. N. (1903). Der feinere Bau der Wimperapparate der Infusorien. A.P.K., ii., p. 73. (74) MESNIL, F. (1905). Chromidies et Questions connexes. B.I.P., iii., p. 313. (75) MINCHIN, E. A. (1911). Some Problems of Evolution in the Simplest Forms of Life. Journ. Quekett Microsc. Club (2), xi., p. 166. (76) NA'GLER, K. (1911). Protozoen aus einem Almtumpel, I. Amoeba hartmanni, n. sp. Anhang : Zur Centriolfrage. A.P.K., xxii, p. 56» 77) POPOFF, M. (1909). Die Zellgrosse, ihre Fixierung und Vererbung. Arch. Zell/orschung, iii., p. 124. (78) RBICHENOW, E. (1910). Hoemogregari^a stepanowi. Die Entwicklungsge- sohichte einer Hamogregarme. A.P.K., xx., p. 251. (79) ROBERTSON, M. (1911). The Division of the Collar-Cell* of the Calcarea Heterocada. Q.J.M.S., Ivii., p. 129. (80) — and MINCHIN, E. A. (1910). The Division of the Collar-Cells of Clathrina coriacea. Q.J.M.8., lv., p. 611. (81) SCHAUDINN, F. (1896). Der Zeugungskreis von Paramoeba eilhardi. S.B.A.B., p. 31. Reprinted, Schaudinn's Arbeiten, 1911, p. 116. (82) — (1896). Das Centralkorn der Heliozoen. V.D.Z.O., vi., p. 113. (With discussion by Lauterborn and Biitschli.) (83) SIEDLECKI, M. (1905). Die Bedeutung des Karyosoms. B.A.S.C.,f. 559. (84) WENYON, C. M. (1911). Oriental Sore in Baghdad, together with Observa- tions on a Gregarine in Stegomyia fasciata, the Hsemogregarines of Dogs, and the Flagellates of House Flies. Py., iv., p. 273. (86) ZUELZER, M. (1904). Difflugia urceolata. A.P.K., iv., p. 240. (86) — (1909). Wagnerella boreali*. A.P.K., xvii.. p. 135. BIBLIOGRAPHY 479 CHAPTER VII In addition to the works cited here, see also Nos. 45, 48, 49, 60, 56, 58, 60, 62, 64i 66, 69, 70, 71, 71'5, 78, 79, 80, 81, 82, and 86 above. (87) ARAQAO, H. DB B. (1904). Amoeba diplomitotica. M.I.O.C., L, p. 33. (88) AWERINZEW, S. (1904). TeUung von Amoeba proteus. Z.A., xxvii., p. 399. (89) HAKTMANH, M., and CHAQAS, C. (1910). Schlangcnhamogregarinen. A.P.K., xx., p. 361. (90) (1910). Die Kernteilung von Amoeba hyalina. M.I.O.C., ii., p. 159. (91) HEKTWIO, R. (1903). Korrelation von Zell- und Kerngrosse. B.C., xxiii., pp. 49 and 108. (92) — (1908). Neue Probleme der Zellenlehre. Arch. f. Zettforschung, i., p. 1. (93) LEBEDEW, W. (1908). Trachelocerca phcenicopterus. A.P.K. xiii., p. 70. (94) MOROFF, T. (1908). Die bei den Cephalopoden vorkommenden Aggregata- Arten. A.P.K., xi., p. 1. (95) NAQLEB, K. (1909). Entwicklungsgeschichtliohe Studien iiber Amoben. A.P.K., xv., p. 1. (90) — (1911). Caryosom und Centriol beim Teilungsvorgang von Chilodon uncinatw. A.P.K. , xxiv., p. 142. (97) PROWAZEK, S. v. (1903). Die Kernteilung des Entoaiphon. A.P.K., ii., p. 325. (97'5) REICHENOW, E. (1909). Hosmatococcua pluvialis. A.K.O.A., xxxiii., p. 1. (98) SCHAUDINN, F. (1894). Kerntheilung mit nachfolgender Korpertheilung bei Amoeba crystattigera. S.B.A.B., 1894, p. 1029. Reprinted, Schaudinn's Arbeiten, 1911, p. 95. (99) — (1900). Der Generationswechsel bei Coccidien. Zool. Jahrbiicher (Abth. /. Anat.), xiii., p. 197. Schaudinn's Ar' eiten, 1911, p. 208. (100) SCHEWIAKOFF, W. (1887). Die karyokinetische Kerntheilung der Euglypha alveolata. Morph. Jahrbuch, xiii., p. 193 (101) SWABCZEWSKY, B. (1908). Die Fortpffonzungserscheinungen bei Arcella vulgaris. A.P.K., xii., p. 173. CHAPTER VIII In addition to the works cited here, see also Nos. 41, 47, 50, 51, 57, 64, 67, 68, 74, 75, 81, 85, 92, 93, 99, and-101. (102) BAITSELL, G. A. (1911). Conjugation of Closely Related Individuals of Stylonychia. Proc. Soc. Exper. Biol. Med., viii., p. 122. (103) BOTT, M. (1907). Fortpflanzung von Pdomyxa. A.P.K.. viii., p. 120. (104) CALKINS G. N. (1904). Studies on the Life-History of Protozoa, IV. J.E.Z., L, p. 423. (105) — (1906). The Protozoan Life-Cycle. B.B., xi., p. 229. (106) — and CULL, S. W. (1907). The Conjugation of Paramecium aurdia (eaudatum). A.P.K.. x., p. 375. (107) DANQEAKD, P. A. (1911). La Conjug&ison des Infusoires cilies. C.B.A.S., clii., p. 1032. (108) — (1911). La Feoondation des Infusoires cilies. C.E.A.8., clii., p. 1703. (109) DEHORNK, A. (1911). Permutation nucleaire dans la Conjugaison de Col- pidium cclpoda. C.B.A.S., clii., p. 1354. (110> DOBELL, C. C. (1911). The Principles of Protistology. A.P.K., xxiii., p. 269. (111) DOFLBIN, F. (1907). Die Konjugation der Infusorien. 8.B.O.M.P., xxiii., p. 107. (112) ENBIQUES, P. (1907). La Coohigazione e il Differenzi&mento sossuale negli Infusori. A.P.K., ix., p. 195. ^ (113) — (1908). Die Conjugation \md-aexuelle Difterenzierung der Infusorien. A.P.K., xii., p. 213. (114) GEDDES. P., and THOMSON J. A. (1901). The Evolution of Sex. Revised edition London. (115) HAMBURGER, C. (1908). Die Conjugation von Stentor cnsrvicus. Z.w.Z., xc., p. 423. (116) HARTMANN, M. (1909). Autogamie bei Protisten. A.P.K., xiv., p. 264. 480 THE PROTOZOA (117) HARTOO, M. (1910). Apropos of Dr. Hartmann's " Autogamie bei Proto- zoen." A.P.K., xviii., p. 111. (118) HERTWIG, R. (1902). Wesen und Bedeutung der Befruchtung. Sitzber. k. Akad. Wise. Miinchen., xxxii., p. 57. (119) — (1905). Das Problem der sexuellen Differenzierung. V.D.Z.O., 1905. p. 186. (120) HIOKSON, S. J. (1910). The Origin of Sex. Ann. Rep. Trans. Manchester Microsc. Soc., 1909, p. 34. (121) JENNINGS, H. S. (1910). What Conditions induce Conjugation in Para- mecium ? J.E.Z., ix., p. 279. (122) MAUPAS, E. (1889). Le Rajeunissemcnt karyogamique chez les Cilies. A.Z.E., (2) vii., p. 149. (123) MULSOW, K. (1911). Fortpflanzungserscheinungen bei Monocystis roatrata. A.P.K., xxii., p. 20. (124) PEARL, R. (1907). A Biometrical Study of Conjugation in Param&ium. Biometrika, v., p. 213. (125) POPOFF, M. (1908). Die Gametenbildung und die Conjugation von Car- chesium polypinum. Z.w.Z., Ixxxix., p. 478. (126) PRANDTL, H. (1906). Die Konjugation von Didinium nasutum. A.P.K., vii., p. 229. (127) PROWAZEK, S. v. (1905). Der Erreger der Kohlhernie, Plasmodiophora brassicos. A.K.O.A., xxii., p. 396. (128) — (1907). Die Sexualitat bei den Protisten. A.P.K., ix., p. 22. (129) SCHAUDINN, F. (1896). Copulation von Actinophrys. S.B.A.B., p. 83. (130) — (1902). Krankheitserregende Protozoen, II. Plaamodium vivax. A.K.G.A., xix., p. 169. (131) — (1903). Die Fortpflanzung einiger Rhizopoden. A.K.G.A., xix., p. 547. (132) — (1904). Generations- und Wirtswechsel bei Trypanoeoma und Spiro- chcete. A.K.G.A., xx., p. 387. Reprinted, with " Nachtrag," in Fritz Schaudinn's Arbeiten, 1911. (133) — (1905). Die Befruchtung bei Protozoen. V.D.Z.G., xv., p. 16. (134) SCHILLING, C. (1910). Autogamie bei Trypanosoma lewisi. A.P.K., xix., p. 119. (135) STEMPELL, W. (1906). Die neuere Protozoenforechung und die Zellenlehre. S. B. Med.-naturwiss. Ges. Munster i. W., June 13. (136) STEVENS, N. M. (1910). The Chromosomes and Conjugation in Boveria aubcylindrica, var. concharum. A.P.K., xx., p. 126. (137) VERSLUYS, J. (1906). Die Konjugation der Infusorien. B.C., xxvi., p. 46. (138) WOODRUFF, L. L. (1905). Life- History of Hypotrichous Infusoria. J.E.Z., ii., p. 585. (139) _ (1908). Life-Cycle of Paramecium. Amer. Nat., xlii., p. 520. (140) — (1909). Further Studies on the Life-Cycle of Paramecium. B.B., xvii., p. 287. (141) — (1911). Two Thousand Generations of Paramecium. A.P.K., xxi., p. 263. (142) — (1911). The Adaptation of Paramcecia to Different Environments. B.B., xxii., p. 60. (143) — and BAITSELL, G. A. (1911). Rhythms in the Reproductive Activity of Infusoria. J.E.Z., xi., p. 339. CHAPTER IX In addition to the works cited here, see also Nos. 41, 65, 78, 85, 86, 99, 101, 130, and 131. (144) ELPATIEWSK,*, W. (1907). Fortpflanzung von Arcetta wlgaris. A.P.K., x., p. 441. (145) KHAINSKY, A. (1910). TJber Arcellen. A.P.K., xxi., p. 165. (146) SCHAXJDINN, F. (1899). Der Generationswechsel von Trichoaphcerium sieboldi. Anhang. Abhandl. Preuss. Akad. Wies. (147) _ (1902). Cydospora caryolyttca. 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Parasitisohe Protozoen aus dem inneren der Rotatorien. B.A.S.C., 1901, p. 358. *(305) SCHAUDINN, F. (1896). Heliozoa. Das Tierreich., Berlin, 1896. (306) SOHOTJTEDEK, H. (1907). Quelques Flagelles. A.P.K., ix., p. 108. (307) WELDON, W. F. R., and HICKSON, S. J. (1909). The Heliozoa. Lankester's Treatise on Zoology, i., fasc. 1, p. 14. 486 THE PROTOZOA (g) Radiolaria. See also No. 152. (308) BOBGEKT, A. (1911). Fremdkorperskelete bei tripyleen Radiolarien. A.P.K., xxiiL, p. 126. (309) BRANDT, K. (1902). Die Colliden. A.P.K., i., p. 59. (310) BTJTSCHLI, O. (1906). Die chemiache Natur der Sk^etsubstanz der Acan- tharia. Z.A., zxx., p. 784. (311) GAMBLE, F. W. (1909). The Eadiolaria. Lankester's Treatise on Zoology, i., faso. 1, p. 94. (312) HARTMANN, M., and HAMMER, E. (1909). Die Fortpflanzung von Radio- larion. 8.B.G.B., 1909, p. 228. (313) HAKTOO, M. (1910). Radiolaria. Encyclop. Brit., eleventh edition, xxii., p. 802. (314) HTTTH, W. (1911). Fortpflanzung von Thalassicotta. S.B.O.B., 1911, p. 1. (315) MOROFF, T. (1910). Vegetative und reproduktive Erseheinungen bei Thalassicolla. Hertung s Festschrift, i., p. 73. (316) — and STIASNY, G. (1909). Bau und Entwicklung von Acanthomelron peUueidum. A.P.K., xvi., p. 209. (317) SCOTT, R. (1911). On Traquairia. Ann. Botany, xxv., p. 459. (318) STIASNY, G. (1910). Die Beziehung der sog., " gelben Zellen " zu den kolonie-bildenden Radiolarien. A.P.K., xix., p. 144. CHAPTER XH MASTTGOPHORA (a) General Works. (319) HARTOG, M. (1910). Flagellata. Encyclop. Brit., eleventh edition, x., p. 44. *(320) SBNN, G. (1900). Flagellata. Engler and Fraud, " Die natiirlichen Pflan- zenfamilien," I. Teil, 1. Abth., a. p. 93. (321) WILLEY, A., and HICKSON, S. J. (1909). The Mastigophora. Lankester's Treatise on Zoology, i., fasc. 1, p. 154. (b) Flagellata. See also Nos. 41, 45, 62, 70, 71, 97, 97-5, 153, 160, 213, 223, 236, and 277. (322) ALEXEIEFF, A. (1909). Les Flagelles Parasites de 1'Intestin des Batraciens Indigenes. C.R.8.B., Ixvii., p. 199. (323) — (1909). Trichomonas a Quatre Flagellea Anterieurs. C.R.S.B., Ixvii., p. 712. (324) — (1910). Les FlageHes Intestinaux des Poissons Marins. A.Z.E. (5), vi., Notes et Revue, p. i. (325) — (1911). Notes sur les Flagelles. Ibid., p. 491. (326) — (1911). " Kystesde Trichomonas intestinalis." C.R.S.B., Ixxi., p. 296. (327) — (1911). . La Position des Monadides dans la Systematique des Flagelles, etc. B.8.Z.F., xxxvi., p. 96. (328) BBNSEH, W. (1909). Trichomonas intestinalis und vaginalis des Menschen. A.P.K., xyiii., p. 115. (329) BERLINER, E. (1909). Flagellaten-Studien. A.P.K., xv., p. 297. (380) BOHNE, A., and PROWAZEK, S. v. (1908). Zur Frage der Flagellatendysen- terie. A.P.K., xii., p. 1. (331) CHATTON, E. (1911). Pleodorina californica a Banyuls-sur-Mer. Bull. Sci. Franc. Belg. (7). xliv. p. 309. (332) COMES, S. (1910). Lophophora vacuolata. Boll. Ace. Oioen. Catania (2), xiii., p. 11. (333) — (1910). A Proposito del Dimorfismo sessuale riscontrato in Dinenympha gracilis. Ibid., p. 20. (334) DANILEWSKY, W. B. (1886). Une Monade (Hezamitus), Parasite du Sang. Arch. Slav. Biol., i., p. 85. (335) DOBELL, C. C. (1908). Structure and Life-History of Copromonas subtilia. Q.J.M.8., Hi., p. 75. BIBLIOGRAPHY 487 (336) DOBEIX, C. C. (1908). The " Autogamy " of Bodolacertce. B.C.. xxviii , p. 548. (337) FOA, A. (1905). Due nuovi Flagellati parassiti (Calonympha grassii and Devescovina striata). Rend. Ace. Lineei, xiv. (2), p. 542. (338) GBASSI, B., and FOA, A. (1904). Processo di Divisione delle Joenie e Forme affini. Ibid., xiii. (ii.), p. 241. (339) HAASB, G. (1910). Euglena sanguinea. A.P.K., xx,, p. 47. (340) HAMBURGER, C. (1905). Dunaliella salina und eine Amobe aus Salinen- wasaer von Cagliari. A.P.K., vi., p. 111. (341) — (1911). Euglena ehrenbergii, insbesondere die Korperhulle. Sitz-ber. Heidelberg. Ak. Wiss., 1911. (342) HABTMANN, M. (1910). Bau und Entwicklung der Triohonymphiden (Trichcnympha hertwigi). Hertwig's Festschrift, i., p. 349. (343) HASWELL, W. A. (1907). Parasitic Euglena). Z.A.. xxxi., p. 296. (344) KEYSSELITZ, G. (1908). Studien iiber Protozoen. A.P.K., »., p. 334. (345) LATJTERBORN, R. (1895). Eine Siisswasserart der Gattung Mvlticttia (M. lacustris). Z.w.Z., lx., p. 236. (345-5) — (1911). Pseudopodien bei Chrysopyxis. Z.A., xxxviii., p. 46. (346) LIEBETANZ, E. (1910). Die parasitische Protozoen des Wiederkauermagens. A.P.K., xix., p. 19. (347) LOHMANN, H. (1902), Die Coccolithophoridse. A.P.K., i., p. 89. (348) MAKTET, C. H., and ROBERTSON, M. (1911). Caecal Parasites of Fowls, etc. Q.J.M.8., Ivii., p. 53. (349) MOBOFF, T. (1903). Einige Flagellaten. A.P.K., iii., p. 39. (350) NEBESHEIMEB, E. (1911). Costia necatrix. Vide PROWAZEK (14), p. 98. (351) Noo, F. (1909). Le Cycle evolutif de Lamblia intestinalis. B.8.P.E., ii., p. 93. (352) PASCHEB, A. (1910). Chrysomonaden aus dem Hirsohberger Grossteiche. Leipzig : Werner Klinkhardt. (353) PLIMMER, H. G. (1909). Report on Death* at the Zoological Gardens during 1908. P.Z.8., 1909, p. 125. (354) PROWAZEK, S. v. (1903). Flagellatenstudien. A.P.K., ii., p. 195. (355) — (1904). Einige parasitisohe Flagellaten. A.K.Q.A., xxi., p. 1. (356) RODENWALDT, E. (1911). Trichomonaa, Lamblia. Vide PROVTAZEK (14), p. 78. (357) SCHERFFEL, A. (1911). Die Chrysomonadineen. A.P.K., xxii., p. 299. (358) SENN, G. (1911). Oxyrrhis, Nephroselmis und einige Euflagellaten. Z.w.Z., xcvii., p. 605. (359) STEIK, F. (1878, 1883). Der Organismus der Infusion sthiere. III. Leipzig: Wilhelm Engelmann. (360) STEVENSON, A. C. (1911), The Protozoa parasitic in Bufo regvlaris. in Khartoum. Rep. Wellcome Lab. Khartoum, iv., p. 359. (361) WENYON, C. M. (1910). A Flagellate of the Genus Cercomonas. Q.J.M.8., Iv., p. 241. (362) — (1910). Macrostoma mesntfifrom the Human Intestine. Py., iii., p. 210. (c) Dinoflagellata and Cystoflagellata. (363) BOBQEBT, A. (1910). Kern- und Zellteilung bei marinen Ceratium-Aiten. A.P.K., xx., p. 1. (364) CAULLEBY, M. (1910). Ettdbiopsis chattoni, Parasite de Calanus helgolandicus Butt. Sci. Fran<;. Belg. (7), xliv., p. 201. (365) CounfcRE, H. (1911). Les EUobiopsis des Crevettes bathypelagiques. C.R.A.S., clii.. p. 409. (366) CHATTON, E. (1906). Les Blastodinidds. C.R.A.8., cxliii., p. 981. (367) — (1907). Nouvel Aper?u sur les Blastodinid£s (Apodinium mycetoides). O.R.A.S., oxliv., p. 282. (368) — (1910). Sur 1'Existence de Dinoflagell^s parasites coelomiques. Les Syndinium chez les Cop^podes p^lagiques. U.R.A.S., cli., p. 654. (369) — (1910). Paradinium poucheti, Flagell6 parasite d Acartia dausi. C.R.8.B., Ixix., p. 341. (370) DOGIEL, V. (1906). Die Peridinien. Mitth. Zool. Slat. Neapd, xviii., p. 1. (371) DUBOSCQ, O., and COLLIN, B. (1910). La Reproduction sexuee d'un Pro- tiste parasite des Tintinnides. G.R.A.8., cli., p. 340. (372) JOLLOS, V. (1910). DinoflageUatenstudien. A.P.K., xix., p. 178. (373) KOFOID, C. A. (1905). Craspedotella, a New Genus of the Cystoflageilata. Bull. Mus. Harvard, xlvi., p. 163. 488 THE PROTOZOA (374) KOFOID, C. A. (1906). Asymmetry in Triposolenia. Univ. California Ptibl. Zool., iii., p. 127. (375) — (1906). Structure of Oonyaulax triacantha. Z.A., xxx., p. 102. (376) — (1907). Structure and Systematic Position of Polykrikos. Ibid., xxxi., p. 291. (377) — (1907). The Plates of Ceratium. Hid., xxxii., p. 177. (378) — (1908). Exuviation, Autotomy, and Regeneration, in Ceratium. Univ. California Publ. Zool., iv., p. 345. (379) — (1909). On Peridiniur.i steini. A.P.K., xvi., p. 25. (380) — (1909). Morphology of the Skeleton of Podolampas. Ibid. p. 48. (381) — (1909). Mutations in Ceratium. Butt. Mus. Harvard, Iii., p. 211. (382) — (1910). A Revision of the Genus Geratocorys. Univ. California Publ. Zool., vi., p. 177. (383) — (1910). Forms of Asymmetry of the Dinoflagellates. Proc. Internal. Congr. Zool., vii. (384) KttSTER, E. (1908). Eine kultivierbare Peridinee. A.P.K., xi., p. 351. (385) PLATE, L. (1906). Pyrodinium bahamense. A.P.K., vii., p. 411. (386) SCHUTT, F. (1895). Die Peridineen der Plankton-Expedition. Ergebn. Plankton- Exped., iv. (387) STBIK, F. (1883). Der Organismus der Infusorien. III. (ii.) Die Natur- gesohichte der Arthrodelen Flagellaten. Leipzig : W. Engelmann. CHAPTER XIII H^5MOFLAGELLATES (a) General Works. See also the Bulletin of the Sleeping Sickness Bureau, London, for abstracts and reviews of literature. (388) ALEXEIEFF, A. (1911). La Structure des " Binucldates " de Hartmann. C.R.8.B., Ixix., p. 532. (389) BRUMPT, E. (1908). L'Origine des He'moflageltes du Sang des Vertebres. C.R.8.B., Ixiv., p. 1046. (390) HARTMANN, M., and JOLLOS, V. (1910). Die Flagellatenordnung " Binu- cleata." A.P.K., xix., p. 81. *(391) LAVEBAN, A., MESNH,, F., and NABARRO, D. (1907). Trypanosomes and Trypanosomiases. London : Bailliere, Tindall and Cox. *(392) LUHE, M. (1906). Die im Blute schmarotzenden Protozoen. JMense's Handbuch der Tropenkrankheiten, iii., p. 69. *(393) PATTON, W. 8. (1909). Our Present Knowledge of the Huemoflagellates and Allied Forms. Py., ii., p. 91. *(394) THIMM, C. A. (1909). Bibliography of Trypanosomiasis. London : Sleep- ing Sickness Bureau. *(395) WOODCOCK, H. M. (1909). The Hsemoflagellates and Allied Forms. Lan- kester s Treatise on Zoology, i., iasc. 1, p. 193. (b) Trypanosoma and Trypanoplasma. See also Nos. 19, 22, 23, 27, 42, 56, 132, 134, 686, and 696. (396) BALDREY, F. S. H. (1909). Die Entwicklung von Trypanosoma lewisiin der Rattenlaus Harniatopinus apinulosus. A.P.K., xv., p. 326. (397) — (1911 Evolution of T. evanai through the Fly : Tobanus and Stomoxys. Journ. Trap. Veterin. Sci., vi., p. 271. (398) Bosc, F. J. (1904). La Structure etl'Appareil Nucleaire des Trypanosomes. A.P.K., v., p. 40. (399) BOUET, G. (1906). Culture du Trypanosome de la Grenouille (T. rotatorium). A.I. P., xx., p. 564. (400) — and RotrBAtrD, E. (1910). Transmission des Trypanosomes par les Glossines, I. and II. A.I.P., xxiv., p. 658. III., B.S.P.E., iii., p. 699. IV., Ibid., p. 722. (401) BOUFFARD, O. (1910). Qlossina palpalis et T. Cazalboui. A.I. P., xxiv. p. 276. BIBLIOGRAPHY 489 (402) BRADFORD, J. E., and PLIMMER, H. G. (1902). The T. l.rucii found in Nagana, or Tse-tse Fly Disease. Q.J.M.8., xlv., p. 449. (403) BREINL, A., and HINDUS, E. (1910). Life-History of T. lewisi in the Rat- Louse. A.T.M.P., iii., p. 553. (404) BRUCE, D. (1911). Morphology of T. evansi. P.R.S. (B), Ixxxiv., p. 181. (405) — - (1911). Morphology of T. gambienae. Ibid., p. 327. (406) — and BATEMAN, H. R. (1908). Have Trypanosomes an Ultramioroscopical Stage in their Life-History ? (No /) P.R.8., (B), Ixxx., p. 394. (407) — HAMERTON, A. E., BATEMAN, H. R., and MACKIE, F. F. (1909). T. ingens, n. sp. P.R.8. (B), Ixxxi., p. 323. (408) (1909). Development of T. gambienae in Olosaina palpalis. Ibid., p. 405. (409) (1909). A Trypanosome in the African Elephant. Ibid., p. 414. (410) (1910). Development of Trypanosomes in Tsetse Flies. Ibid., Ixxxii., p. 368. (411) (1910, 1911). Trypanosome Diseases of Domestic Animals in Uganda, I-V. Ibid., Ixxxii., p. 468 ; Ixxxiii., pp. 1, 15, 176, and 180. (412) (1910). The Natural Food of Olossina palpalis. Ibid., Ixxxii., p. 490. (413) (1910). Mechanical Transmission of Sleeping Sickness by the Tsetse Fly. Ibid., p. 498. (414) (1911). Experiments to Ascertain if T. gambiense during its Development within Olossina palpalis is Infective. Ibid., Ixxxiii., p. 345. (416) (1911). Further Researches on the Development of T. gam- biense in Olossina palpalis. Ibid., p. 513. (416) and BRUCE (LADY) (1911). T. gattinarum. Rep. Sleeping Sickness Comm., xi., No. 32, p. 170. (417) (1911). A Trypanosome found in the Blood of a Crocodile. Ibid., No. 36, p. 184 (418) BRUMPT, E. (1906). Le Mode de Transmission des Trypanosomes et des Trypanoplasmes par les Hirudinees. C.R.S.B., Ixi., p. 77. (419) — (1907). L'Heredite' des Infections a Trypanosomes et a Trypanoplasmes chez les Hotes intermediates. Ibid., Ixiii., p. 176. (420) BUCHANAN, G. (1911). Developmental Forms of T. brucei (pecaudi) in the Internal Organs of the Gerbil. P.R.S. (B), Ixxxiv., p. 161. (421) CARINI, A. (1910). Stades Endoglobulaires des Trypanosomes. A.I. P., xxiv., p. 143. (422) — (1910). Formas de Eschizogonia do T. lewisi. Soc. de Med. e Cir. de Sao Paulo, August 16, 1910 (quoted from B.I.P., ix., p. 937). (423) — (1911). Presence de Trypanosomes chez les bovid£s, a Sao Paulo. B.8.P.E., iv., p. 191. (424) — (1911). Schizogonien bei Trypanosomen. A.P.K., xxiv., p. 80. (425) CHAQAS, C. (1909). Eine neue Trypanosomiasis des Menschen. M.I.O C , i., p. 159. (426) — (1911). Le Cycle de " Schizotrypanum cruzi" chez 1'Homme et les Animaux de Laboratoire. B.8.P.E., iv., p. 467, (427) CRAWLEY, H. (1910). T. americanum from the Blood of American Cattle. Journ. Comp. Path. Therap., xxiii., p. 17. (428) DARLING, S. T. (1911). Murrina. Journ. Infect. Diseases, viii., p. 467. (429) — (1911). Mode of Infection and Methods of Controlling an Outbreak of Equine Trypanosomiasis in the Panama Canal Zone. Py., iv., p. 83. (430) DOFLEIN, F. (1909). Problem der Protistenkunde. I. Die Trypanosomen. Jena: G. Fischer. (431) — (1910). Experimentelle Studien iiber die Trypanosomen des Frosches. A.P.K., xix., p. 207. (432) DuTTpN, J. E., TODD, J. L., and TOBEY, E. N. (1906, 1907). Certain Para- sitic Protozoa observed in Africa. Part L, Liverpool Trop. Med. Memoirs, xx., p. 87. Part II., A.T.M.P., i., p. 287. (433) ELDERS, C. (1909). TrypanosomiasiB beim Menschen auf Sumatra. C.B.B.P.K. (I Abth. Orig.), liii., p. 42. (434) FANTHAM, H. B. (1911). life-History of T. gambiense and T. rhode- siense as seen in Rats and Guinea-pigs. P.R.8. (B), Ixxxiii., p. 212. 490 THE PROTOZOA (435) FISCHER, W. (1911). Zur Kenntnis der Trypanosomen. Z.H., Ixx., p. 93. (436) FRANCA, C. (1908). La Biologie des Trypanosomes. AJ.C.P., ii., p. 43. (437) — (1908). Le Cycle Evolutif des Trypanosomes de la Grenouille. lbid>t p. 89. (438) — .(1908). Le Trypanosome de 1'Anguille (T. granulosum). Ibid., p. 113. (439) — (1910). Un Trypanosome du Lerot (T. eiyomis). Ibid., iii., p. 41. (440) — (1911). Hematozoaires de la Guinee Portuguaise. Ibid., pp. 201, 229. (441) — (1911). Les Hematozoaires des Taupes. Ibid., p. 271. (442) (1911). Relation autogenetique entre les grands et les petits Trypano- somes de la Grenouille. C.R.8.B., Ixx., p. 978. (443) — (1911). La Transformation "in vitro des Formes crithidiennes de " T. rotatorium " en Formes trypanosomiques. B.S.P.E., iv., p. 634. (444) FRIEDRICH, L. (1909). Bau und Naturgeschichte des Trypanoplaama helicis. A.P.K., xiv., p. 363. (445) FRY..W. B. (1911). The Extrusion of Granules by Trypanosomes. P.R.S. (B), Ixxxiv., p. 79. (445-5) GONDEB, B. (1911). Arzneifeste Mikroorganismen. I. T. lewisi. G.B.B.P.K. (I Abth. Orig.), Ixi., p. 102. (446) HAMBURGER, C. (1911). Einige paraaitische Flagellaten. Verh. Heiddberg. Naturhi«t.-Med. Ver. (n. F.), xi., p. 211. (447) HARTMANN, M. (1910). Eine weitere Art der Schizogonie bei Schizotrypanum cruzi. A.P.K., xx., p. 361. (448) HINDLE, E. (1909). Life-History of T. dimorphon. Univ. California Publ. Zod., vi., p. 127. (449) — (1910). Degeneration Phenomena of T. gambiense. Py., iii., p. 423. (450) — (1910). A Biometrio Study of T. gambiense. Ibid., p. 455. (451) — (1911). The Passage of T. gambiense through Mucous Membranes and Skin. Ibid., iv., p. 25. (452) JOLLOS, V. (1910). Bau und Vermehrung von Trypanoplaama helicis. A.P.K., xxi., p. 103. (453) KEYSSELITZ, G. (1904). Trypanopkis grobbeni. A.P.K., iii., p. 367. (454) — (1906). Generations- und "Wirtswechsel von Trypanoplasma borreli. A.P.K., vii., p. 1. (455) — (1907). Die unduliercnde Mem bran bei Trypanosomen und Spiroohaten. A.P.K., x., p. 127. (456) — and MAYER, M. (1908). Die Entwicklung von T. britcei in Glossina fusca. A.S.T.H., xii.. p. 532. (457) KLEINE, F. (1909). Positive Infectionsversuche mit T. brucei durch Olossi-nn palpalis. Dtutach. Med. Wochenschr., xxxv., p. 469. Die Entwicklung von Trypanosomen in Glossinen. Ibid., p. 924. Die Aetiologie der SchlafkrankLeit. Ibid., p. 1257. Tsetsefliegen und Try- panosomen. Ibid., p.' 1956. (458) — (1910). Trypanosomenbefunde am Tanganyika. Ibid., xxxvi., p. 1400. (459) — and TAUTE, M. (1911). Erganzungen zu unseren Trypanosomenstudien. A. K.O.A., xxxi., p. 321. Reprinted as " Trypansomenstudien." (460) KOCH, R., BECK, M., and KLEINE, F. (1909). Die Tatigkeit der zur Erfor- schung der Schlafkrankheit im Jahre 1906-07 nach Ostafrika entsandten Kommission. A.K.O.A., xxxi., p. 1. (461) LAVERAK, A. (1911). Identification et Classification des Trypanosomes des Mammif&res. A.I.P., xxv., p. 497. (462) — (1911). Les Trypanosomes, ont-ils des Formes latentes chez leursHStes vert^bres ? C.R.A.8., cliii., p. 649. (464) — and MESNIL, F. (1902). Des Trypanosomes des Poissona. A.P.K., i. p. 475. (465) — and PETTTT, A. (1910). DCP Trypanosomes du Mulot et du Campagnol (T. grosi et T. microti). C.R.S.B., Ixviii., p. 571. (466) (1910). Le Trypanosome du Lerot (Myoxua nitela) et la Puce qui parait le propager (T. blanchardi). Ibid., p. 950. (467) LEBAILLY, C. (1906). Les Hematozoaires parasites des Teleost6ens marins. Arch. Parasitol., x., p. 348. (468) LEBEDEFF, W. (1910). T. rotatorium, Gruby. Hertwig's Festschrift, i., p. 397. (469) MACHADO, A. (1911). Zytologiuche Untersuohungen iiber T. rotatorium, Gruby. M.I.O.C., iii., p. 108. (470) MANTETTFEL (1909). Studien fiber die Trypanosomiasis der Ratten. A.K.G.A., xxxiii., p. 46. w BIBLIOGRAPHY 491 (471) MARTIN, C. H. (1910). Trypanoplasma congeri — I. The Division of the Active Form. Q.J.M.S., lv., p. 485. (472) MARTIN, G., LEBCETTF, A., and ROTTBAUD, E. (1908). Transmission du " Nagana " par Ics Stomoxes et les Moustiques. B.S.P.E., i., p. 355. (473) MATHIS, C., and LBGER, M. (1911). Parasitologie et Pathologic humaines et animates au Tookin. Paris : Masson et Cie. (474) MESNIL, F. (1910). L'Identification de quelques Trypanosomes pathogenes. B.S.P.E., iii., p. 376. (475) — and BRIMONT, E. (1908). Un Hematozoaire nouveau (Endotrypanum) d'un Edente de Guyane. C.R.S.B., Ixv., p. 581. (476) MINCHIN, E. A. (1908). The Development of Trypanosomes in Tsetse-Flies and Other Diptera. Q.J.M.S., Hi., p. 159. (477) — (1908). Polymorphism of T. gambiense. Py., i., p. 236. (478) — (1909). The Flagellates parasitic in the Blood of Freshwater Fishes. P.Z.S., 1909, p. 2. (479) — (1909). Structure of T. lewisi in Relation to Microscopical Technique. Q.J.M.S., liii., p. 755. (480) — and THOMSON, J. D. (1910). Transmission of T. lewisi by the Rat-Flea (Ceratophyttus fasciatus). P.R.S. (B.), Ixxxii., p. 273. (481) (1911). Transmission of T. lewisi by the Rat-Flea. Brit. M ed. Journ. , 1911, i., p. 1309. (482) (1911). An Intracellular Stage in the Development of T. lewisi in the Rat-Flea. Ibid., ii. (August 19), pp. 361-364. (483) — and WOODCOCK, H. M. (1910). Blood-Parasites of Fishes occurring at Rovigno. Q.J.M.8., lv., p. 113. (484) MOORB, J. E. S., and BREINL, A. (1907). Cytology of the Trypanosomes, part i. A.T.M.P., i., p. 441. (485) (1908). T. equiperdum. P.R.S. (B.), Ixxx., p. 288. (486) and HINDLB, E. (1908). Life-History of T. lewisi. A.T.M.P., ii., p. 197. *(487) NERESHEIMER, E. (1911). Die Gattung Trypanoplasma. Vide PROWAZEK (14), p. 101. (488) NEUMANN, R. O. (1909). Protozoische Parasiten im Blut von Meeresfischen. Z.H., Ixiv., p. 1. (489) NOVY, F. G., and McNBAL, W. J. (1905). Trypanosomes of Birds. Journ. Infect. Diseases, ii., p. 266. (490) and TORREY, H. N. (1907). Trypanosomes of Mosquitoes and Other Insects. Ibid., iv., p. 223. (491) OTTOLBNQHI, D.( 1908). T. Iruceiund T. equinum. C.B.B.P.K. (I. Abth. Orig.), xlvii., p. 473. (492) — (1909). Die Entwicklung einigor pathogencr Trypanosomen im Sau- getierorganismus. A.P.K., xviii., p. 48. (493) PATTON, W. S., and STRICKLAND, C. (1908). The Relation of Blood-sucking Invertebrates to the Life-Cycles of Trypanosomes. Py., i., p. 322. (494) PETRIE, G. F. (1905). The Structure and Geographical Distribution of Certain Trvpanosomes. J.H., v., p. 191. (495) — and AVAKI, C. R. (1909), On the Seasonal Prevalence of T. lewisi in Mus rattua and in Mus decumanua. Py., ii., p. 305. (496) PoLicARD,A.(1910). Sur la Coloration vitale des Trypanosomes. C.R.8.B., Ixviii., p. 606. (497) PROWAZEK, S. v. (1905). Studien iiber Saugetiertrypanosomen. A.K.O.A., xxii., p. 351. (498) — (1909). Kritische Bemerkungen zum Trypanosomenproblem. A.S.T.H., »ii., p. 301. (499) ROBERTSON, M. (1906). Certain Blood-inhabiting Protozoa. Proc. R. Phys. 8oc. Edinburgh., xvi.. p. 232. (500) — (1907). A Trypanosome found in the Alimentary Canal of PontdbdeUa muricata. Ibid., xvii., p. 83. (501) — (1909). Life-Cycle of T. vittatce. Q.J.M.S., liii., p. 665. (602) — (1909). A Trypanosome found in the Alimentary Tract of Pontobdella muricata. Q.J.M.8., Hv., p. 119. (503) — (1911). Transmission of Flagellates living in the Blood of Fishes. Phil Trans. (B.), ccii., p. 29. (504) RODENWALDT, E. (1909). T. lewisi in Hcematopinus spinulosus. C.B.B.P.K. (I Abth. Orig.), Iii., p. 30. 492 THE PROTOZOA (505) ROSENBUSCH, F. (1909). Trypanosomen-studien. A.P.K., xv., p. 263. (506) ROUBAUD, E. (1909). Les Trypanosomes pathogfenes et la Glossina palpalis. Rapport de la Mission d'Etudes de la Maladie du Sommeil au Congo Fran- cois (Paris, Masson et Cie.), p. 511. (507) — (1910). Phenomenes morphologiques du DeVetoppement des Trypano- somes chez les Glossines. G.R.A.S., cli., p. 1156. (508) STASSANO, H. (1901). La Fonction et Relation du petit Noyau des Trypano- somes. C.B.8.B., liii., p. 468. (509) STEPHENS, J. W W., and FANTHAM, H. B. (1911). Peculiar Morphology of a Trypanosome from a Case of Sleeping Sickness (T. rhodesiense). P.R.S. (B.), Ixxxiii., p. 28. (510) STOCKMAN, S. (1910). A Trypanosome of British Cattle. Journ. Comp. Pathol. Therapeut xxiii., p. 189. (51 1 ) STRICKLAND, C. ( 191 1 ). Mechanism of Transmission of T. lewisi by the Rat- Flea. Brit. Med. Journ., 1911, p. 1049. (512) — and SWELLENGREBEL, N. H. (1910). On T. lewisi and its Relation to Certain Arthropoda. Py., Hi., p. 436. (513) STUHLMANN, F. (1907). Die Tsetsefliegen (Glossinafusca und 01. tachinoides). A.K.O.A., xxvi., p. 301. (514) SWELLENGREBEL, N. H. (1909). Bau und Zellteilung von T. gambiense und T. equinum. Tijdschr. Ned. Dierk. Ver. (2), xi., p. 80. (515) — (1910). Fixation and Staining of T. lewisi. Py., iii., p. 226. (516) — (1910). Normal and Abnormal Morphology of T. lewisi. Ibid, p. 459. (517) — and STRICKLAND, C. (1910). The Development of T, lewisi outside the Vertebrate Host. Ibid., p. 360. (518) (1911). Remarks on Dr. Swingle's Paper, "The Transmission of T. lewisi by Rat-Fleas," etc. Ibid., iv., p. 105. (519) SWINGLE, L. D. (1907). On T. lewisi. Trans. Amer. Micr. Soc., xxvii., p. 111. (520) — (1611). Transmission of T. lewisi by Rat-Fleas. Three New Herpeto- monads. Journ. Infect. Diseases, viii., p. 125. (521) TAUTE, M. (1911). Die Beziehungen der Qlossina. morsitans zur Schlaf- krankheit. Z.H., htix., p. 553. (522) THIROUX, A. (1905). T. paddce. A.I.P., xix., p. 65. (523) — (1905). T. duttoni. Ibid., p. 564. (524) THOMSON, J. D. (1906). Blood-Parasites of the Mole. J.H., vi., p. 574. (525) — (1908). Cultivation of the Trypanosome found in the Blood of the Gold- fish. Ibid., viii., p. 75. (516) WERBITZKI, F. W. (1910). Blepharoplastlose Trypanosomen. C.B.B.L'.K. (I Abth. Orig.), liii., p. 303. (See also Bulletin of the Sleeping Sickness Bureau, vol. iii., pp. 221, 313, and 458, for further references.) (527) WOODCOCK, H. M. (1910). On Certain Parasites of the Chaffinch (Fringilla ccdebs) and the Redpoll (Linota rufescens). Q.J.M.S., Iv., p. 641. (528) YAXIMOST, W. L., KOHL-YAKIMOFF, N., and KORSSAK, D. W. (1910). T. korssaki of Mus agrarius, Piroplasmoses of M us agrarius, Reindeer, Yak, and Bears. C.B.B.P.K. (I Abth. Orig.), Iv., p. 370. (529) ZUFITZA, M. (1909). Die Vogel- und Fischtrypanosomen Kameruna. A.8.T.H., xiii., Beiheft 3, p. 101. (c) Crithidia, Leptomonas, Herpetomonas, etc. See also No. 84. (530) BOTTET, G., and ROUBAUD, E. (1911). LA Presence au Dahomey et Trans- mission du Leptomonas davidi. C.B.8.B., Ixx., p. 55. (531) CHATTON, E. (1909). Un Trypanosomide nouveau d'une Nycteribie, et les Relations des Formes Trypanosoma, Herpetomonas, Leptomonas et Crithidia. C.R.8.B., Ixvii., p. 42. (532) — and ALXLAIRJS, E. (1908). Coexistence d'un Leptomonas et d'un Trypano- soma chez un Muscide non vulnerant, Drosophila confusa. C.R.8.B., Ixiv., p. 1004. (533) — and LEGER, A. (1911). Eutrypanospmes, Leptomonas et Leptotrypano- somes chez Drosophila confusa (Muscide). C.R.8.B., Ixx., p. 34. (534) (1911). Quelques Leptomonas de Muscides et leurs Leptotrypano- somes. Ibid., p. 120. (535) DUNKERLY, J. S. (1911). Life-History of Lept. muscce-domesticce. Q.J.M.8., Ivi., p. 645. BIBLIOGRAPHY 493 (536) FLU, P. C. (1911). Die im Darm der Stnbenniege vorkommenden proto- zoaren Gebilde. C.B.B.P.K. (I Abth. Orig.), Ivii., p. 522. (537) FRANCA, C. (1911). L'Existence en Portugal de Lept. davidi dans le Latex de Euphorbia peplus et E. segetalia. B.S.P.E., iv., p. 532. (538) — (1911). Notes BUT Lept. davidi. Ibid., p. 669. (539) GEORGEWITCH, J. (1909). Le developpement de Crithidia aimulice. C.R.8.B., Ixvii., p. 517. (540) LAFONT, A. (1910). La Presence d'un Leptomonos . . . dans le Latex de Trois Euphorbiacees. A.I.P., xxiv., p. 205. (541) — (1911). La Transmission du Lept. davidi des Euphorbea par un Hemip- tere. C.R.S.B., Ixx., p. 58. (542) LEGER, L. (1902). La Structure et Multiplication des Flagelles du Genre Herpetomonas Kent. O.B.A.S., cxxxiv., p. 781. (543) — (1902). Un Magelle Parasite de I' Anopheles moculipennis. C.R.8.B., liv., p. 354. (544) — (1904). Un nouveau Flagelte, Parasite des Tabanides. C.R.S.B., Ivii., p. 613. (545) — (1904). Les Affinitea de Y Herpetomonas subulate et la Phylogenie des Trypanosomes. G.R.8.B., Ivii., p. 616. (546) — and DUBOSCQ, 0. (1909). Parasites de 1'Intestin d'une Larve de Ptychop- tera. Butt. A cad. Bdgique, No. 8, p. 885. (547) MACKINNON, D. L. (1910). New Parasites from Trichoptera. Py., iii., p. 245. (548) — (1910). Herpetomonads from Dung- Flies. Ibid., p. 255. (549) — (1911). More Protozoan Parasites from Trichoptera. Ibid., iv., p. 28. (550) PATTON, W. S. (1908). Life-Cycle of a Species of Crithidia parasitic in Oerris fossarum. A.P.K., xii., p. 131. (561) — (1908). Herp. lygcei. A.P.K., xiii., p. 1. (552) — (1909). Life-Cycle of a Species of Crithidia parasitic in Tabanus hilarius and Tabanut sp. A.P.K., xv., p. 333. (553) — (1910). Infection of the Madras Bazaar FJy with Herp. miiecce-domesticce. B.U.P.E., iii., p. 264. (554) POSTER, A. (1910). Crithidia melophagia. Q.J.M.S., Iv., p. 189. (555) — (1909). Crithidia gerridis. Py., ii., p. 348. (556) — (1909). Life-Cycle of Herp. jaculum. Ibid., p. 367. (557) PBOWAZKK, S. v. (1904). Die Entwicklung von Herpetomonas. A.K.O.A., xx., p. 440. (657'5) ROUBAUD, E. (1911). Cystolrypanosoma inteslinalis. C.S.8.B., Ixxi., p. 306. (558) STRICKLAND, C. (1911). A Herpetomonas parasitic in the common Green- bottle Fly, Lucilia sp. Py., iv., p. 222. (559) SWELLENOREBEL, N. H. (1911). Morphology of Herpetomonaa and Crithidia, etc. Ibid., p. 108. (560) WERNER, H. (1908). Eine eingeisselige Flagellatenform im Darm der Stubenfliege. A.P.K., xiii., p. 19. (d) Leishmania, etc. See also No. 84. For references to literature and critical summaries and reviews, see Kola Azar Bulletin (Royal Society, London). (561) BASILS, C. (1910). Leishmaniosi del Cane e 1'Ospite intermedio del Kala- Azar infantile. Rend. Ace. Lincei (5), xix. (2), p. 623. (562) — (1911). Trasmissione delle Leishmaniosi. Ibid. (5), xx. (1), p. 50. (563) — (1911). Leishmaniosi e suo Modo di Trasmissione. Ibid. (5), xx. (2), p. 72. (564) — LACAVA, F., and VISENTINI, A. (1911). L' Identita delle Leishmaniosi. Ibid., p. 150. (565) DARLING, S. T. (1909). Histoplasma tapsulatum and the Lesions of Uisto- plasmosis. J.E.M., xi., p. 615. (566) DONOVAN, C. (1909). Kala-Azar in Madras. Bombay Medical Congress, February 24, 1909. (567) LEISHMAN, W. B., and STATHAM, J. C. B. (1905). Development of the Leishman Body in Cultivation. Journ. S. A. Med. Corps, iv., p. 321. (568) MARSHALL, W. E. (1911). Pathological Report, Kala-Azar Commission. Rep. Wellcome Lab., iv., p. 167. (569) MARZINOWSKY, E. J. (1909). Cultures de Leishmania tropica. B.8.P.E.. ii., p. 691. 494 THE PROTOZOA (570) NIOOLI.B, C. (1909). Le Kala-Azar infantile. A.I.P., xxiii., p. 361. (571) — and COMTE, 0. (1908). Origine canine du Kala-Azar. C.B.A.8^ oxlvi., p. 739. (572) Now, F. G. (1909). Leishmania infantum. B.8.P.E., ii., p. 385. (573) PATTOH, W. S. (1908). The Leishman-Donovan Parasite in Cimex rotun- datus. S.M.I., xxxi. (574) — (1908). Inoculation of Dogs with the Parasite of Kala-Azar (Herpeto- tnonas [Leishmania] donovani). Py., i., p. 311. (575) — (1909). The Parasite of Kala-Azar and Allied Organisms. Trans. Soc. Trap. Med. Hygiene, ii., p. 113. (576) ROGERS, L. (1904). Trypanosomes from the Spleen Protozoic Parasites of Cachexial Fevers and Kala-Azar. Q.J.M.8., xlviii., p. 367. (577) — (1907). The Milroy Lectures on Kala-Azar. Brit. Med. Jov.rn., February 23, March 2 and 9. (578) Bow, R. (1909). Development of the Parasite of Oriental Sore in Cultures. Q.J.M.S., liii., p. 747. (579) THIROUX, A., and TEPPAZ, L. (1909). La Lymphangite epizootique des Equides au S6negal. A.I.P., xxiii., p. 420. (680) VISBNTINI, A. ( 1910). La Morfologia ed il CSolo di Sviluppo della Leishmania. Istituto d. Clin. Med. d. R. Univ. Roma. (581) WEIGHT, J. H. (1903). Protozoa in Tropical Ulcer (" Delhi Sore "). Journ. Med. Research, x. (n.s. v.), p. 472. (e) Prowazekia. (582) ALEXEIEFF, A. (1911). La Morphologic et la Division de Bodo caudatus. C.R.8.B., Ixx., p. 130. (582-5) DUNKEKLY, J.S. (191-)- Thelohania and Prowazekia in Ahthomyid Flies. C.B.B.P.K. (I Abth. Orig.), Ixii., p. 136. (583) HARTMANN, M. (1911). Die Flagellatenordnung Binudeata und die Gattung Prowazekia. A.P.K., xxii., p. 141. (584) MARTINI, E. (1910). Pr. cruzi und ihre Beziehungen zur Atiologie von ansteckenden Darmkrankheiten zu Tsingtau. Z.H., Ixvii., p. 275. (585) NAEGLEB, K. (1910). Pr. parva. A.P.K., xxi., p. 111. (586) WALKER. E. L. (1910). Trypanoplaama rawx. Journ. Med. Research, xxiii., (n.s. XVIII.), p. 391. (587) WHITMOBK, E. R. (1911). Pr. asiatica. A.P.K., xxii., p. 370. CHAPTER XIV SPOROZOA— TELOSPORIDIA (a) General Works. *(588) HAGENMULLEH (1899). Bibliotheca Sporozoologica. Ann. Mua. Nat. Hist. Marseille (2), i. *(589) MIKCHIN, E. A. (1903). The Sporozoa. A Treatise on Zoology (Lankester) (London, A. and C. Black), p. 150. (590) WOODCOCK, H. M. (1910). Sporozoa. Encydop. Brit., eleventh edition, xxv., p. 734. Coccidia. Ibid., vi., p. 615. Gregarines. Ibid., xii., p. 555. Haemosporidia. Ibid., xii., p. 806. Endospora. Ibid., ix. p. 383. (b) Qregarines. See also Nos. 72, 84, and 123. (591) AWBRINZBW, S. (1909). Die Vorgange der Schizogonie bci Gregarinen aus dem Darm von Amphiporus sp. A.P.K., xvi., p. 71. (592) BEATJCHAMP, P. de (1910). Une Gr&rarine nouvelle du Genre Porospora. G.R.A.8., cli., p. 997. (593) BKBNDT, A. (1902). Die im Darme der Larve von Tenebrio moHtor lebenden Gregarinen. A.P.K., i., p. 375. (594) BRASIL, L. (1905). La Reproduction des Gregarines monocystidees. A.Z.E. (4), iii., p. 17. BIBLIOGRAPHY 495 (595) BRASIL, L. (1905). La Reproduction des Gregarines monocystidees. A.Z.E. (4), iv., p. 69. (596) — (1907). La Schizogonie et la Croissance des Gametooytes chez Selenidium cautteryi. A.P.K , viii., p. 370. (597) — (1909). Documents BUT quelques Sporozoaires d'Annelides. A.P.K., xvi., p. 107. (598) COGNETTI DE MARTiis, L. (1911). Le Monocistidee e loro Fenomeni ripro- duttivi. A.P.K. , xxiii., p. 205. (599) COMES, 8. (1907). Der Chromidialapparat der Gregarinea. A.P.K., x., p. 416. (600) CKAWLEY, H. (1905). Movements of Gregarines. Proc. Acad. Philadelphia, Ivii., p. 89. (601) CUNNINGHAM, J. T. (1907). Kalpidorhynchut arenicolce. A.P.K., x., p. 199. (602) DOGIBL, V. (1906). Cystobia chiridotce. A. P.K., vii., p. 106. (603) — (1907). Schizocyetis sipunculi. A.P.K., viii, p. 203. (604) — (1909). Die Sporocysten der Colom-Monocystideae. A.P.K., rvi., p. 194. (605) — (1910). Callynthrochlamys phronimoe. A.P.K., xx., p. 60. (606) — (1910). Einige neue Catenata. Z.w.Z., xciv., p. 400.. (607) DBZEWECKI, W. (1903, 1907). Vegetative Vorgange im Kern und Plasma der Gregarinen des Regenwurmhodens. A.P.K., iii., p. 107. II. Stomato- phora coronata. Ibid., x., p. 216. (6Q8) DUKE, H. L. (1910). Metamera schubergi. Q.J.M.S., lv., p. 261. (609) FANTHAM, H. B. (1908). The Schizogregarines. Py., i., p. 369. (610) HALL, M. C. (1907). A Study of some Gregarines, with especial Bererence to Hirmocystis rigida. Stud. Zod. Lab. Univ. Nebraska, vii., p. 149. (611) HESSE, E. (1909). Les Monocystidees des Oligochetes. A.Z.E. (5), iii., p. 27. (611-5) HOFFMANN, R. (1908). Fortpflanzungserscheinungen von Monocystideen des Lumbricv* agriccla. A.P.K., xiii., p. 139. (612) HUXLEY, J. 8. (1910). Oanymedes anaaptdu, Q.J.M.S., lv., p. 155. (613) KUSCHAKEWITSOH, S. (1907). Vorgange bei den Gregarinen dea Mehlwurm- darms. A.P.K., SuppL L, p. 202. (614) LEQEB, L. (1904). LA Reproduction sexuee chez les Styloihynchus. A.P.K., iii., p. 303. (615) — (1904). Sporozoaires Parasites de YEmbia Solieri. Ibid., p. 358. (616) — (1906). Tceniocystis mira. A.P.K., vii., p. 307. (617) — (1907, 1909). Les Schizogregarines des Tracheates : L Ophryocystu. A.P.K., viii., p. 159. II. Schizocystis. Ibid., xviii., p. 83. (618) — and DUBOSCQ, 0. (1902). Les Gregarines et 1'Epitheliuai intestinal chez les Tracheates. Arch. Parasitd., vi., p. 377. (619) (1903). Le DeVeloppement des Gregarines Stylorhynchides et Steno- phorides. A.Z.E. (4), i., Notes et Revue, p. Ixxxix. (620) (1904). Les Gregarines et 1'Epithelium intestinal des Traoheates. A.P.K., iv., p. 335. (621) (1909). La SexualitS chez les Gregarines. A.P.K., xvii., p. 19. (622) (1911). Deux nouvelles Especes de Gregarines appartenant au Genre Porospora. Ann. Univ. Orcnoble, xxiii.,p. 401. *(623) LUHE, M. (1904). Die Sporozoiten, die Wachstumsperiode und die ausge- bildeten Gregarinen. A.P.K., iv., p. 88. (624) NUSBAUM, J. (1903). FortpSanzung einer Gregarine — SchaudineUa henlece. Z.w.Z., lxxv..p. 281. (625) PAEHLEB, F. (1904). Die Morphologic, Fortpflanzung und Entwicklung von Oregarina ovata. A.P.K., iv., p. 64. (626) PFEFFEB, E. (1910). Die Gregarinen im Dann der Larve von Tenebno molitor. A.P.K., xix., p. 107. (627) POBTER, A. (1909). Merogregarina amaroucii. A.P.K., xv., p. 228. (628) ROBINSON, M. (1910). On the Reproduction of Kalpidorhynchua arenicolce, Q.J.M.S., liy., p. 565. (629) SCHELLACK, C. (1907). Die Entwicklung und Fortpflanzung von Echinomera hispida. A.P.K., ix., p. 297. (630) — (1908). Die solitare Encystierung bei Gregarinen. Z.A., xxxii., p. 597. (631) SCHNITZLEB, H. (1905). Die Fortpflanzung von Clepaidrina ovata. A.P.K., vi., p. 309. (632) WOODCOCK, H. M. (1906). Life-Cycle of " Cystobia " irregulars. Q.J.M.S. 1., p. 1. 496 THE PROTOZOA (o) Coccidia. See also Nos. 47, 83, 94, 99, and 147. *(633) BLANCHAKD, R. (1900). Les Coccidies et leor Bole pathogene. Cauteries Set. 8oc. Zod. France, p. 133. (634) CHAGAS, C. (1910). Addea hartmanni. M.I.O.C., ii., p. 168. (635) DAKIK, W. J. (1911). Merocystis kathas. A.P.K., mi., p. 145. (635'5) DEBAISIEUX, P. (1911). Recherches sur les Coccidies. La Cellule, xxvii., pp. 89 and 257. (636) DOBELL, C. C. (1907). Life-History of Addea ovata. P.R.8. (B.), Ixxix.. p. 155. (637) ELMASSIAN, M. (1909). Coccidium rouxi, Zoomyxa legeri. A.Z.E. (5), ii. p. 229. (638) FANTHAM, H. B. (1910). Eimeria (Coccidium) avium. P.Z.S., 1910. p. 672. (639) — (1910). Avian Coccidiosia. Ibid., p. 708. (640) HADLEY, P. B. (1911). Eimeria avium. A.P.K., xxii., p. 7. (641) JOLLOS, V. (1909). Multiple Teilung und Reduktion bei Addea ovata. A.P.K., xv., p. 249. (642) KUNZE, W. (1907). Orcheobius herpobddlas. A.P.K., ix., p. 382. (643) LAVERAK, A., and PETTIT, A. (1910). Une Cocoidie de Agama colonorum. (Cocc. agamce). C.B.8.B., Ixviii., p. 161. (644) LEGER, L. (1911). Caryospora simplex, et la Classification des Coccidies. A.P.K., xxii., p. 71. (645) — and DUBOSCQ, 0. (1908). L'Evplution scbizogonique de YAggregata (Euc-occidium) eberthi. A.P.K., xii., p. 44. (646) (1910). Sdenococcidium intermedium. A.Z.E. (5), v., p. 187. (647) METZNEB, R. (1903). Coccidium cuniculi. A.P.K., ii., p. 13. (648) MOROFF, T. (1906). Addea zonula. A.P.K., viii., p. 17. (649) — and FIEBIGER, J. (1905). Eimeria subepithdialia. A.P.K., vi., p. 166. (650) PEREZ, C. (1903). Le Cycle evolutif de I' Addea mesnili. A.P.K., u., p. 1. (651) SCHELLACK, C., and REICFENOW, E. (1910). Lithobius-Coccidien. Z.A., xxxvi., p. 380. (652) SIEDLECKI, M. (1898). La Coccidie de la Seiche. A.I.P., xii., p. 799. (653) — (1907). Caryotropha mesnUii. B.A.8.C., 1907, p. 453. (654) STEVBNSON, A. C. (1911). Cpocidiosis of the Intestine of the Goat. Rep. Wellcome Lab. Khartoum, iv., p. 355. (655) TYZZER, E. E. (1910). Cryptosporidium muris of the Common Mouse. Journ. Med. Research, xxiii (n.s. XVIII.), p. 487. (656) WOODCOCK, H. M. (1904). On Klosnidla muris. Q.J.M.8., xlviii., p. 153. CHAPTER XV ' H^EMOSPORIDIA (a) General Works. *(657) LAVERAK, A. (1905). Hsemocytozoa. B.I.P., in., p. 809. *(658) SCHAUDINN, F. (1899). Der Generationswechsel der Coccidien und Haemo- sporidien. Zod. Centralbl., vi., p. 765. (659) WASIELEWSKI (1908). Studien und Mikrophotogramme zur Kenntnisse der pathogenen Protozoen. II. Untersuchungen fiber Blutschmarotzer. Leipzig : Barth. (b) Haemamcebae. See also Nos. 130 and 686. (660) ARAOAO, H. DE B., and NEIVA, A. (1909). Intraglobular Parasites of Lizards. PI. diploglossi and PI. tropiduri. M.I.O.C., i., p. 44. (661) BERENBERQ-GOSSLER, H. v. (1909). Naturgeschiohte der Malariaplas- modien. A.P.K., xvi., p. 245. (662) BERTRAND, D. M. (1911). Les Parasites endoglobulaires pigmentes des Vertebres. Paris : Jouve et Cie. (663) BILLET, A. (1905). Une Forme particuliere de 1'Hematozoaire du Palu- disme decrite par MM. Ed. et Et. Sergent. G.R.8.B., Iviii., p. 720. (664) — (1906). La Forme hemogregariniennc du Parasite de la Fievre quarte. C.R.8.B., lz., p. 891. BIBLIOGRAPHY 497 (666) BILLET, A. (1906). Diagnose diflferentielle des Formes annulaires des Hematozoaires du Paludisme. C.R.8.B., Ixi., p. 754. (666) — (1910). Evolution chez le meme Sujet du Paludisme tierce primaire en Paludisme tierce secondaire. B.S.P.E., iii., p. 187. (667) CABDAHATIS, J. P. (1909). Le Paludisme des Oiseaux en Gr&ce. Etude du Parasite de Danilewsky. C.B.B.P.K. (I Abth. Orig.). lii., p. 351. (668) CASTELLANI, A., and WILLEY, A. (1904). Haematozoa of Vertebrates in Ceylon. Spolia Zeylanica, ii., p. 78. (669) DARLING, S. T. (1910). Transmission and Prevention of Malaria in the Panama Canal Zone. A.T.M.P., iv., p. 179. (670) DOBELL. C. C. (1910). Life-History of Hcemocystidium simondi. Hertivig's Festschrift, i., p. 123. (671) FLTT, P. C. (1908). Affenmalaria. A.P.K., xii., p. 323. (672) GILBUTH, J. J., SWEET, G., and DODD, S. (1910). Proteosqma biziurce and Hcemogregarina megalocystis. Proc. Boy. Soc. Victoria (n.s.), xxiii., p. 321. (673) GBASSI, B. (1901). Die Malaria, Studien eines Zoologen. Jena : Gustav Fischer. (674) HALBERSTAEDTER, L., and PBOWAZEE, 8. v. (1907). Die Malariaparasiten der Affen. A.K.Q.A., xxvi., p. 37. (675) HABTMANN, M. (1907). Das System der Protozoen. Zugleioh vorlaufige Mitteilung iiber Proteosoma. AtP.K., x., p. 139. (676) MAYEB, M. (1908). Malariaparasiten bei Affen. A.P.K., xii., p. 314. (677) NEUMANN, R. O. (1908). Die Ubertragung von Plasmodium prcecox auf Kanarienvogel duichStegomyiafasciata. A.P.K., xiii., p. 23. (678) Ross, R. (1910). The Prevention of Malaria. London : John Murray. (679) SEEOENT, ET., and SEBOENT, ED. (1910). L'Immunit6 dans le Paludisme des Oiseaux, etc. G.R.A.8., cli., p. 407. (680) THIBOITX, A. (1906). Des Relations de la Fievre tropicale avec la Quarte et la Tierce. A.I.P., xx., pp. 766 and 869. (681) VASSAL, J. J. (1907). L'H&natozoaire de 1'Ecureil (Hcemamceba vassali). A.I.P., xxi., p. 851. (c) Halteridia. See also No. 132. (682) ANSCHTTTZ, G. (1910). Uebertragungsversuche von HcBmoproteus oryzivoras «nd Trypanosoma paddas. C.B.B.P.K. (I Abth. Orig.), liv., p. 328. (683) ABAOAO, H. DE B. (1908). Der Entwicklungsgang und die Ubertragung von Hcenuyproteus columbce. A.P.K., xii., p. 154. (684) MAYEB, M. (1910). Die Entwicklung von Halteridium. A.8.T.H., xiv., p. 197. (685) — (1911). Ein Halteridium und Leucocytozoon des Waldkauzes. A.P.K., xxi., p. 232. (685-5) MINCHIN, E. A. (1910). Report on Blood- Parasites collected by the Commission. Sep. Sleeping Sickness Comm., x., p. 73. (686) SEBOENT, ED., and SEROENT, ET. (1907). Les Hematozoaires d'Oiseaux. A.I.P., xxi., p. 251. (687) WOODCOCK, H. M. (1911). An Unusual Condition in Halteridium. Z.A., xxxviii., p. 465. (d) Leucocytozoa (Vera). See also Nos. 132, 473, and 686. (688) BEBESTNEFF, N. (1904). Das Leucocytozoon Danilewskyi. A.P.K., Hi., p. 376. (689) FANTHAM, H. B. (1910). Parasitic Protozoa of the Red Grouse. P.Z.8., 1910, p. 692. (690) WENYON, C. M. (1910). On the Genus Leucocytozoon. Py., iii., p. 63. (e) Hsemogregarines. See also Nos. 78, 84, and 89. (691) ADIE, J. R. (1906). "Leucocytozoon " ratti. Journ. Trop. Med.. ix., p. 325 (692) ABAOAO, H. DE B. (1911). Hamogregarinen von Vogeln. M.I.O.C., Hi. p. 54. (693) BALFOTTB, A. (1906). H. balfouri. Sep. Wellcome Lab. Khartoum, ii., p. 96. (694) — (1906). " Leucocytozoon " muris. Ibid., p. 110. 32 498 THE PROTOZOA (695) BERESTNEFF, N. (1903). Einc nouc Blutparasiten der indisohen Frosche. A.P.K., ii., p. 343. (696) BILLET, A. (1904). Ttypanoaoma inopinatum et Drepanidium. C.R.S.B,, Ivii., p. 161. (697) BOUET, 6. (1909). H6mogregaiines de 1'Afrique oocidentale fran^aiae. C.R.S.B., Ixvi., p. 741. (698) CABINI, A. (1910). " H. muris." Rev. Soc. 8ci. Sao Pavlo, v. (699) CHRISTOPHERS, S. R. (1905). H. gerbUli. S.M.I., 18. (709) _ (1906). Leucocytozoon cants. S.M.I., 26. (701) _ (1907). Leucocytozoon cams in the Tick. S.M.I., 28. (703) DANILEWSKY, B. (1886). Lea Hcmatozoaires des Lizards. Arch. Slav* Biol., i., p. 364. (704) — (1887). Les Hdmatozoaires des Tortues. Ibid., Hi., pp. 33 and 370. (705) — (1889). La Parasitologie comparer du Sang. I. Nouvelles Becherches BUT lea Hcmatozoaires du Sang dea Oiseaux. II. Reoherches BUT lea Hematozoaires dea Tortues. Kharkoff. (706) FANTHAM, H. B. (1905). Lankesteretta tritonis. Z.A., xxix., p. 267. (707) FLU, P. C. (1909). Hamogregarinen im Blute Surinamiseher Schlangen. A.P.K., xviii., p. 190. (708) FRANCA, C. (1908). Une H^mogregarine de 1'Anguille (H. bettencourli). A.LC.P., ii., p. 109. (709) — (1908). E. splendent (Labbe). Ibid., p. 123. (710) — (1909). Hemogregarines de Lacerta ocellata. Ibid., p. 339. (711) — (1910). Parasites endooellulairca du Psammodromus algirus. Ibid., iii., p. 1. (712) — (1910). Hemogregarines de Lacerta muralis. Ibid., p. 21. (713) HAHN, C. W. (1909). H. stepanowi in the Blood of Turtles. A.P.K., xvii., p. 307. (714) KOIDZUMI, M. (1910). H. gp. in Clemmysjaponicus. -A.P.K., xviii., p. 260. (715) LAVEBAN, A., and PBTTIT, A. (1909). Lea H6mogregarinea de quelquea Sauriens d'Afrique. B.8.P.E., ii., p. 506. (716) (1910). Les Formes de Multiplication endogene de //. sebai. C.R.A.S., cli., p. 182. (717) (1910). H. agamas. C.R.S.B., Ixviii., p. 744. (718) (1910). Le Role d'Hyalomma AZgyptium L. dans la Propagation de H. mauritanica. C.-R. Assoc. France (Lille), p. 723. (719) MILLER, W. W. (1909). Hepatozoon perniciosum and its Sexual Cycle in the Intermediate Host, a Mite (Lelaps echidninus). Hygienic Laboratory Bulletin, No. 46 (June, 1908). (720) NEBESHEIMEB, £. (1909). Das Einclringen von Lankesteretta spec, in die Frosohblutkorperohen. A.P.K., xvi., p. 187. (721) PATTON, W. S. (1906). On a Parasite found in the Blood of Palm Squirrels. S.M.I., 24. (722) — (1908). The Haemogregarines of Mammals and Reptiles. Py., i., p. 319. (723) PORTER, A. (1908). Leucocytozoon musculi. P.Z.8., 1908, p. 703- (724) PROWAZEK, S. v. (1907). Ueber Hamogregarinen. A.K.Q.A., xxvi., p. 32. (725) ROBERTSON, M. (1910). Life-Cycle of H. nicorice. Q.J.M.8., lv., p. 741. (726) SAMBON, L. W.,and SELIQMANN, C. G. (1907). Hsemogregarines of Snakes. Trans. Pathol. Soc. London, Iviii., p. 310. (727) SETTZ (1910). Die Hartmannsohe Binukleaten. C.B.B.P.K. (I. Abth. Grig.), Ivi., p. 308. (f) Piroplasms. See also No. 528. (728) BETTENCOUBT, A., FBANCA, C., and BORGES, I. (1907). Piroplasmose bacilliforme chez le Daim. A.I.C.P., i., p. 341. (729) BOWHILL, T. (1905). Equine Piroplasmosis, or "Biliary Fever " J.H., v., p. 7. (730) BREINI,, A., and HINDLE, E. (1908). Morphology, etc., of Piroplasma canis. A.T.M.P., ii., p. 233. (731) BBTJCE, D., HAMEBTOK, A. E., BATEMAN, H. R., and MACKIE, F. P. (1910). Amakebe : a Disease of Calves in Uganda. P.R.S. (B.), Ixxxii., p. 256. *(732) CHBISTOPHERS, S. R. (1907). P. canis and its Life-Cycle in the Tick. S.M.I., 29. (733) DSCHUNKOWSKY, E., and LTTHS, J. (1909). Protozoenkrankheiten des Blutes des Haustiere in Transkaukasien. Ber. IX. Int. Tierarztl. Kongr. Haag. BIBLIOGRAPHY 499 (734) DSCHXTNKOWSKY, E., and LUHS, J. (1909). Entwickelungsformen von Piroplasmen in Zecken. Ibid. (735) FANTHAM, H. B. (1907). The Chromatin-Masses of P. bigeminum (Babesia bovis. Q.J.M.S., li., p. 297. (736) FRANOA, C. (1910). La Classification des Piroplasmes et Description de deux Formes. A.I.C.P., iii., p. 11. (737) GONDEB, R. (1906). Achromaticus vesperuginis. A.K.O.A., xxiv., p. 220. (738) — (1910). Die Entwicklung von Theileria parva. A.P.K., xxi., p. 143. (739) — (1911). Th. parva und Babesia mutatis Kiistenficberparasit and Pseudo- kiistenfieberparasit. Ibid., p. 222. (740) _ (1911). Die Entwicklung von Th. parva. II. A.P.K., xxii., p. 170. (741) KTNOSHITA, K. (1907). Babesia cants. A.P.K., viii., p. 294. (742) KLEENE, F. K. (1906). Kultivierungsvereuch der Hundepiroplasmen. Z.H., liv., p. 10. (743) KOCH, R. (1906). Entwicklungsgeachichte der Piroplasmen. Ibid., p. 1. (744) MAYER, M. (1910). DasostafrikanischeKustenfieberderRinder. A.8.T.H. xiv., Beiheft 7, p. 307. (745) NsuitANrN, R. 0. (1910). Die Blutparasiten von Vesperugo. A.P.K., xviii., p. 1. (746) NICOLLB, G. (1907). Une Piroplasmose nouvelle d'un Rongeur. C.S.S.B., Ixiii., p. 213. (747) NUTTALL, G. H. F., and FANTHAM, H.B. (1910). Theileria parva. Py., iii., p. 117. *(748) — and GBAHAM-SMITH, G. S. (1906, 1907). Canine Piroplasmosis V. and VI. J.H., vi., p. 585 ; vii., p. 232. (749) (1908). Multiplication of Piroplasma bovis, P. pithed in the circu- lating Blood compared with that of P. cants. Py., i., p. 134. (750) (1908). Development of P. cants in Cultures. Ibid., p. 243. (751) SMITH, T., and KILBOBNB, F. L. (1893). Southern Cattle Fever. U.S. Dept. of Agriculture, Eighth and Ninth Reports Bureau Animal Industry, 1891, 1892, p. 77. (752) THEILEB, A. (1910). Texasfieber, Rotwasser und Gallenkrankheit der Kinder. Zeitschr. /. Infektionskrankheiten der Haustiere, viii., p. 39. (753) YAKIMOFF, W. L., SxounKOFy, W. J., and KOHL-YAKMOFF, N. (1911). L. Achromaticus vesperuginus. A.P.K., xxiv., p. 60. (g) Incerte Sedis. (754) NICOLLE, C., and MAKCEAUX, L. (1909). Un Protozoaire nouveau du Gondi. C.S.A.8., cxlviii., p. 369. (755) PATELLA, V. (1910). Corps de Kurloff-Demel dans quelques Mononucl^aires du Sang des Cobayes. La Genese Endotheliale des Leucocytes Mono- nudeaires du Sang (Siena, Imprimerie St. Bernardin), p. 211. (756) SEIDELIN, H. (1911). Protozoon-like Bodies in Yellow-Fever Patients. Journ. Pathol. Bacterial., xv., p. 282. (757) _ (1911). Etiology of Yellow Fever. Tettow Fever Bureau Bulletin, i., p. 229. CHAPTER XVI SPOROZOA— NEOSPORIDIA A. CNIDOSPOEIDIA (a) General Works. *(758) ATIEBBACH, M. (1910). Die Cnidosporidien. Leipzig : Werner Klinkhardt. (b) Hyxosporidia. (759) AWEBLNZEW, S. (1909). 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(768) — (1910). Die Anlage der Sporooyste (Pansporoblasten) bei Sphoeromyxa sdbrazcsi. A.P.K., xix., p. 1. (0) Actinomyxidia. (769) CAULLBBY, M., and MBSNIL, P. (1905). Sphceractinomyxon stdci. A.P.K., vi., p. 272. (d) Microsporidia. (770) AWERINZEW, S., and FBBMOB, K. (1911). Die Sporenbildung bei Olugea anomala. A.P.K., xxiii., p. 1. (771) CHATTOH, E., and KBEMPP, A. (1911). Les Protistes du gKare-Octoeporea. B.S.Z.F., xxxvi., p. 178. (772) HESSE, E. (1904). Le Developpement de Thdohania legeri. C.R.S.B., Ivii., p. 571. (773) _ (1905). Myxocystis mrazeki. C.B.S.B., Iviii., p. 12. (774) LBQER, L., and DUBOSCQ, 0. (1909). Perezia lankesterice. A.Z.E. (5), i., Notes et Revue, p. Ixxix. (775) — and HESSE, E. (1910). Cnidosporidies des Larves d'Eph^meres. C.R.A.S., cl., p. 411. (776) MEROIER, L. (1908). Neoplasie du Tissu Adipeux chez les Blattes Parasitees par une Microsporidie. A.P.K., xi., p. 372. (777) MRAZEK, A. (1910). Auffassung der Myxooystiden. A.P.K., xviii., p. 245. (778) PEREZ, C. (1904). Une Microsporidie parasite du Carcinus mcenas. C.R.8.B., Ivii., p. 214. (779) — (1905). Microsporidies Parasites des Crabes d'Arcachon. Bull. Stat. Bid. Arcachon, viii. (780) — (1908). Duboscqia legeri. C.R.S.B., Ixv., p. 631. (781) SCHRODER, O. (1909). Thdohania chcstogastris. A.P.K., xiv., p. 119. (782) SCHUBERG, A. (1910). Microsporidien aus dem Hoden der Barbe. A.K.O.A., xxxiii., p. 401. (783) SHIWAGO, P. (1909). Vermehrung bei Pleistophora periplanetce. Z.A., xxxiv., p. 647. (784) STBMPELL, W. (1904). Nosema anomalum. A.P.K., iv., p. 1. (785) — (1909). Nosema bombycis. A.P.K., xvi., p. 281. (786) — (1910). Morphologie der Miorosporidien. Z.A., xxxv., p. 801. (787) WEISSENBEHO, R. (1911). Einige Mikrosporidien aus Fisohen (Nosema lophii, Olugea anomala, 01. Hertwigii). 8.B.O.B., p. 344. (787-5) WOODCOCK, H.M. (1904). On Myxosporidia in Flatfish. Trans. Liverpool Bid. floe., xviii., p. 126. (e) Sarcosporidia. See also NOB. 18, 25, and 26. (788) BETEGH, L. v. (190*). Entwicklungsgange der Sarcosporidien. C.B.B.P.K. (I Abth. Orig.), lii., p. 566. (788'5) CRAWLEY, H. (1911). Sarcocystis rtieyi, Proc. Acad. Philadelphia, 1911, p. 457. (789) DARLING, S. T. (1910). Experimental Sarcosporidiosis in the Guinea-Pig. J.E.M., »i., p. 19. (790) ERDMANN, R. (1910). Kern und metaobtomatische Korper bei Sarko- sporidien. A.P.K., xx., p. 239. (791) — (1910). Sarcocystia muris in der MuskuJatur. S.B.O.B., p. 377. (792) FIBBIGEB, J. (1910). Sarkosporidien. Yi-*h. Zod.-Bot. Oes. Wien, lx., P- (73). BIBLIOGRAPHY 501 (793) LAVERAN, A., aud MESNIL, F. (1899). La Morphologic des Sarcosporidies. G.R.8.B., li., p. 245. (794) NEGRE. L. (1910). Le Stade intestinal de la Sarcosporidie de la Souris. G.R.S.B., Ixviii., p. 997. (795) NEGRI, A. '1908, 1910). Ueber Sarkosporidien, I. and II. C.B.B.P.K. (I Abth. Orig.), xlvii., pp. 56 and 612 ; III., Ibid., lv., p. 373. (797) TEICHMANN, E. (1911). Die Teilungen der Keime in der Cyste Von Sarco- cyatis tenetta. A.P.K., xxii., p. 239. (798) VxttiiEMiN, P. (1902). Sarcocyatia tenetta. C.R.A.S., cxxxiv., p. 1152. (799) WATSON, E. A. (1909). Sarcosporidiosis : Its Association with Loco- Disease, etc. Journ. Comp. Pathd. Therapeut., xxii., p. 1. B. HAPLOSPORIDIA. (800) BEATTIE, J. M. (1906). Rhinosporidium kinealyi. Journ. Pathol. Bacterial., xi., p. 270. (801) CAULLERY, M., and CHAPPELLIER, A. (1906). Anurosporidium pelseneeri. C.R.S.B., lx., p. 325. (802) — and MESNIL. F. (1905). Les Haplosporidies. A.Z.E. (4), iv., p. 101. (803) CHATTON, E. (1907). Gautterya mesnili. C.R.S.B., Ixii., p. 529. (804) — (1908). Blaetulidium pcedophthorum. G.R.S.B., Ixiv., p. 34. (805) CRAWLEY, H. (1905). Codosportdium blatettce. Proc. Acad. PMaddphia, Ivii., p. 158. (806) KING, H. D. (1907). Fertramia bufonia. Ibid., lix., p. 273. (807) LAVERAN, A., and PETTIT, A. (1910). Une Epizootie des Truites. C.R.A.S., cli., p. 421. (808) MINCHIN, E. A., and FANTHAM, H. b. (1905). Rhinosporidium kinealyi. Q.J.M.8., xlix., p. 521. (809) RIDEWOOD, W. G., and FANTHAM, H. B. (1907). Neuroaporidium cephalo- disci. Q.J.M.S., li., p. 81. (810) ROBERTSON, M.'(1908). A Haplosporidian of the Genus Ichthyosporidium. Proc. R. Phya. Soc. Edinburgh, xvii., p. 175. (811) — (1909). An Ichthyosporidiau causing Disease in Sea-Trout. P.Z.S., 1909, p. 399. (812) STEMPELL, W. (1903). Die Gattung Pdycaryum. A.P.K., ii., p. 349. (813) WARREN, E. (1906). Bertramia kirkmanni. Ann. Natal. Govt. Mus., i., p. 7, (814) WRIGHT, J. (1907). Rhinosporidium kinealyi. New York Med. Journ., December 21. C. INCERTJE SEDIS. (815) AWERINZEW, S. (1908). Lymphocyslis johixtonei. A.P.K., xiy., p. 335. (816) — (1911). Die Entwicklungsgesohichte von Lymphocystia johnstonei. A.P.K., xxii., p. 179. (817) CHATTON, E. (1906). La Biologie, etc., des Amo&idium. A.Z.E. (4), v., Notes et Revue, p. xvii. (818) — (1907). Pansporetta perplexa. G.R.S.B., Ixii.. p. 42. (819) — (1910). Gaatrocystis gilruthi. A.Z.E. (6), v., Notes et Revue, p. cxiv. (820) GILRTTTH, J. A. (1910). Gastrocystis gilruthi. Proc. Roy. Soc. Victoria (n.s.), xxiii., p. 19. (821) GRANATA, L. (1908). CapUlus intestinalis. Biologica, ii., p. 1. (822) KRASSILSTSCHIK, J. M. (1909). Neue Sporozoen bei Insekten. A.P.K., xiv., p. 1. (823) LEGER, L., and DUBOSCQ. 0 (1909). Les Chytridiopsis. A.Z.E. (5), i. Notes et Revue, p. ix. (824) WOODCOCK, H. M. (1904). Lymphocystis johnatonei. Trans. Biol. Soc. Liverpool, xvii) p. 143. 602 THE PROTOZOA CHAPTER XVII INFUSORIA (a) General Works. (825) HABTOO, M. (1910). Infusoria. Encydop. Brit., eleventh edition, xiv., p. 557. *(826) HICKSON, 8. (1903). The Infusoria. A Treatise on Zoology (Lankcster) (London : A. and C. Black), p. 361. (b) Ciliata. See also, Nos. 16, 32, 33, 38-5, 44, 50, 53, 73, 93, 96, 102, 104, 106-109, 111-113. 115, 121, 122, 134-126, 136-143, 148, 149, 155, 162, 165-167, 170-173, 177, 181-183, 197-199, 201, 205, 206, 208, 209, 211, 214-220, and 346. (827) BKATTCHAMP, P. DB, and COLLIK, B. (1910). Sur Hastatella radians. A.Z.E. (5), v., Notes et Revue, p. xxviii. (828) BUSCHKIBL, A. L. (1911). Ichthyophthirius multifiliis. A.P.K., xxi., p. 61. (829) CAULLBKY, M., and MESNIL, F. (1903). LA Structure nucleaire d'un Infu- aoire Parasite des Actinies (Fcettingeria actiniarum). C.B.S.B., lv., p. 806. (830) (1907). L'Appareil nucleaire d'un Infusoire (Rhizocaryum concavum). C.R.AM. Franc. Reims. (831) CEPEDB, C. (1910). Les Infuspires astomes. A.Z.E. (5), iii., p. 341. (831'6) CHATTON, E. (1911). Perikaryon cesticola and Conchophrys davidofli. A.Z.E. (5), viii., Notes et Revue, p. viii. (832) COLLIN, B. (1909). Deux Formes nouvelles d'Infusoircs Discotriches. A.Z.E. (5), ii. Notes et Revue, p. xxi. (833) DOBELL, C. C. (1909). Infusoria parasitic in Cephalopoda. Q.J.M.S., liii., p. 183. (834) FATJBE"-FBEMIET, E. (1905). L'Appareil fixateur chez les VorticeUidce. A.P.K., vi., p. 207. (835) — (1907). Mitochondries et Spheroplastes chez les Infusoires cilies. C.R.S.B., Ixii., p. 523. (836) — (1908). Tintinnidium inquilinum. A.P.K., xi., p. 225. (837) — (1908). L' Ancystropodium maupasi. A.P.K., xiii., p. 121. (838) — (1909). Le Macronucleus des Infusoires cilies. B.S.Z.F., xxxiv., p. 55. (839) — (1910). Le Mycterothrix tuamotuensis. A.P.K., xx., p. 223. (840) GONDEB, R. (1905). Kernverhaltnisse bei den in Cephalopoden schmarot- zenden Infusorien. A.P.K., v., p. 240. (841) HAMBTTRGEK, C. (1903). Trachelius ovum. A.P.K., ii., p. 445. (842) — (1904). Die Konjugation von Paramcecium burtaria. A.P.K.,iv.,ip.l99. (843) — and BUDDENBBOCK, v. (1911). Nordische Ciliata mit Ausschluss der Tintinnoidea. Brandt and Apstein, Nordisches Plankton. (844) JOSEPH (1907). Kernverhaltnisse von Loxodes rostrum. A.P.K., viii., p. 344. (845) KASANZEFF, W. (1910). Loxodes rostrum. A.P.K., xx., p. 79. (846) KIEBNIK, E. (1909). 'Chilodon hexastichus. B.A.S.C., p. 75. (847) KOFOID, C. A. (1903). Protophrya oincola. Mark Anniversary Volume, p. 111. (848) LEGER, L., and DUBOSCQ, 0. (1904). Les Astomata representent-ils un Groupe naturel ? A.Z.E. (4), ii., Notes et Revue, p. xcviii. (849) (1904). Les Infusoires endoparasites. A.Z.E. (4), ii., p. 337. (850) MABTINI (1910). Uber einen bei amobeuruhrahnlichen Dysenterien vor- kommenden Ciliatcn. Z.H., Ixvii., p. 387. (851) MAST, S. O. (1909). The Reactions of Didinium iiasutum. B.B., xvi., p. 91. (851-5) MAUPAS, E. (1888). La Multiplication des Infusoires oilies. A.Z.E. (2), vi., p. 165. (862) METCALF, M. M. (1907). Excretory Organs of Opalina. A.P.K., x., pp. 183. 365. (853) — (1909). Opalina : Its Anatomy, etc. A.P.K., xiii., p. 195. (854) MEUKIER, A. (1910). Microplankton des Mers de Barents et de Kara. Duo d'Orleans, Gampagne Antique de 1907. Brussels. BIBLIOGRAPHY 603 (855) MmsorHANOW, P. (1905). La Structure, etc., des Trichooystes des Para- mecies. A.P.K., v., p. 78. (856) NERESHBIMEB, E. R. (1003). Die Hdhe histologischer Differenzierung bei heterotrichen Ciliaten. A.P.K., ii., p. 305. (857) NERBSHEIMER, E. (1907). Die Fortpflanzung der Opalinen. A.P.K., Suppl. i., p. 1. (858) — (1908). Fortpflanzung eines parasitischen Infusore (Ichthyophthirius), 8.B.G.M.P., xxiii. (859) PROWAZEK, S. v. (1904). Der Encystierungvorgang bei Dileptua. A.P.K., iii., p. 64. (860) — (1909). Conjugation von Lionotua. Z.A., xxxiv., p. 626. (861) — (1909). Formdimorphismus bei Ciliaten Infusorien. M.I.O.C., i., p. 105. (862) Roux, J. (1899). Quelquea Infusoires ciliea des Environs de Geneve. Rev. Suiaae Zool., vi., p. 557. (863) SCHEWIAKOFF, W. (1893). Die geographische Verbreitung der Susswasser- Protozoen. Mem. Acod. Imp. St.-Peterebourg (vii.), xli. (864) SCHRODER, 0; (1906)v Campanula umbettaria. A.P.K., vii., p. 75. (865) — (1906). Epietylis plicatilis. Ibid., p. 173. (866) — (1906). Vorticetta monilata. Ibid., p. 395. (867) *— (1906). Stentor coeruleus und St. roisdii. A.P.K., viii., p. 1. (868) SCHUBOTZ, H. (1908). Pycnothrix monocystoidee. Dcnkschr. Gee. Jena, xiii., p. 1. (869) SCHWEYER, A. (1909). Tintinnodeenweichkorper, etc. A.P.K., xviii., p. 134. (870) SIEDLECKI, M. (1902). UHerpetophrya astoma. B.A.S.C., p. 356. (871) STEIN, F. v. (1859, 1867). Der Organismus der Infusionthiere : I. Hypo- tricha ; II. Heterotricha. Leipzig : W. Engelmann. (872) STEVENS, N. M. (1904). On Licnophora and Boveria. A.P.K., iii., p. 1. (873) THON, K. (1905). Bau von Didinium nasutum. A.P.K., v., p. 281. (874) WALKER, E. L. (1909). Sporulation in the Parasitic Ciliata. A.P.K., xvii., p. 297. (c) Acinetaria. (875) AWERINZEW, S. (1904). Astrophrya arenaria. Z.A., xxvii., p. 425. (876) CHATTON, E., and COLLIN, B. (1910). Un Acinetien commensal d'un Copepode, Rhabdophrya trimorpha. A.Z.E. (5), v., Notes et Revue, p. cxxxviii. (877) COLLIN, B. (1907). Sur quelques Acinetiens. A.Z.E. (4), vii., Notes et Revue, p. xciii. (878) — (1908). Sur Tokophrya cy'dopum. A.Z.E. (4), viii., Notes et Revue. p. xxxiii. (879) — (1909). La Conjugaison, gemmiforme chez les Acinetiens. C.R.A.S., cxlviii., p. 1416. (880) — (1909). Les Formes hypertrophiques et la Croissance degenerative cbez queiques Acinetiens. C.R.A.S., cxlix., p. 742. (881) — (1909). Sur deux Acinetiens. Ibid., p. 1407. (882) — (1909). La Symetrie, etc., des Embryons d'Acinetiens. A.Z.E. (5), ii., Notes et Revue, p. xxxiv. (883) FtLirjEv, J. (1910). Tocophrya quadripartite. A.P.K., xxi., p. 117. (884) HARTOO, M. (1902). Notes on Suctoria. A.P.K., i., p. 372. (885) HICKSON, S. J., and WADSWORTH, J. T. (1902). Dendrocometee paradoxus. Q.J.M.S., xlv., p. 325. (886) (1909). Dendrosoma radians. Q.J.M.S., liv., p. 141. (887) ISHIKAWA, C. (1897). Eine in Misaki vorkommende Art von Ephdota. Journ. Coll. Sci. Tokyo, x., p. 119. (888) MARTIN, C. H. (1909). On Acinetaria. Parts I. and II. Q.J.M.S., liii., p. 351. Part III. Ibid., p. 629. (889) PEREZ, C. (1903). Lernceophrya capitata. C.R.8.B., lv., p. 98. 504 THE PROTOZOA CHAPTER XVIII (a) Classification. (890) AWERINZEW, S. (1910). Die Stellung im System und die Klassifizierung der Protozoen. B.C., xxx., p. 465. (891) DOFLEIN, F. (1902). Das System der Protozoen. A.P.K., i., p. 169. (892) HARTMANN, M. (1911). Das System der Protozoen. Vide Prowazek (14),. p. 41 ; and No. 675. (b) Spirochsetes. *(893) BOSANQTJET, W. C. (1911). Spirochsetes. Philadelphia and London : W. B. Saunders Company. (894) — (1911). Sp. anodontce Keysselitz. Q.J.M.S., Ivi., p. 387. (895) DOBELL, C. C. (1911). On Cristispira veneris and the Classification of Spiro- chsetes Q.J.M.S., Ivi., p. 507. (896) FANTHAM. H. B. (1911). Life-Cycle of Spirochsetes. A.T.M.P., v., p. 479. (897) GROSS, J. (1910). Cristispira nov. gen. Mitt. zoot. Stnt. Neapd, xx., p. 41. (898) — (1911) Freilebende Spironemaceen. Ibid., p. 188. (899) — (1911). Nomenclatur der Sp. pallida. A.P.K., xxiv., p. 109. (900) HINDLE, E. (1912). Life-Cycle of Sp. gattinanim. Py., iv., p. 463. (901) KKZYSZTALOWICZ, F., and 'SIEDLECKI, M. (1905). La Structure, etc., de Sp. pallida. B.A.S.C., p. 713. (902) LEISHMAN, W. B. (1910). Mechanism of Infection in Tick Fever and Heredi- tary Transmission of Sp. duttoni in the Tick. Trans. Soc. Trap. Med. Hyg., iii., p. 77. (903) SCHAOTJINN, F. (1905). Sp. pallida. Deutsch. Med. Wochenschr., xxxi., p. 1665. (904) ZUELZER, M. (1911). Sp. plicatilis. A.P.K., xxiv., p. 1. (c) Chlamydozoa. (906) ACTON, H. W., and HARVEY, W. F. (1911). Negri Bodies. Py., iv., p. 255. (907) AWERINZEW, S. (1910). Die Krebsgeschwulste. C.B.B.P.K., Ivi. (I Abth. Orig.), p. 506. (908) CALKINS, G. N. (1904). Cytorydes variolas, Guarnieri. Journ. Med. Research (Special Variola Number), xi., p. 136. (909) HARTMANN, M. (1910). Chlamydozoen. C.B.B.P.K. (I Abth. Ref.), xlvii., Beiheft, p. 94. (910) NEGRI, A. (1909). Die Morphologie und der Entwicklungszyklus dcs Para- siten der Tollwut. Z.IL, etc., Ixiii., p. 421. (911) PROWAZEK, S. v. (1907). Chlamydozoa. A.P.K., x., p. 336. (912) — and ARAGAO, H. DE B. (1909). Variola-Untersuchungen. M.I.O.C., i., p. 147. (913) — LiPSCHttTZ, B., and Others (1911). Chlamydozoa, etc. Vide Erowazek (14). (914) SIEGEL, J. (1905). Die Atiologie der Pocken und der Maul- und Klauen- seuche : des Scharlachs : der Syphilis. Abhandl. k. preusa Akad. Wise. (Anhang. ) The numerals printed in hoaviec black type refer to pages on which the meaning of the word or the systematic position of a genus, family, or order are fully explained. ACANTHAIUA, 251, 256 Acanthin, 37, 253 Acanthocystis, 37, 48, 91, 245, 243 — aciOeata, 117, 118 (Fig. 64), 123 (Fig. 68) — chcetophora, 37 (Fig. 18) Acanthometra, 256 — elastita, 250 (Fig. 105) — pellucida, 255 Acanth&metridm, 37 Acephalina, 339 Achromaticus, 364, 382 — vesperuginis, 382 Aohromatin, 65 Acineta, 461 — grandis, 11 (Fig. 10) — papillifera, 16 Acinetaria, 430, 455 Acinetidco, 461 Acrasise, 243 Acrasis, 243 Actinobolus radians, 441 ActinocephalidcB, 339 Actinocephalusoligacanlhus, 327 (Fig. 142) Actinomma aster acanthion, 254 (Fig. 107) Aotinomyxidia, 409 Actinophrys, 117, 215, 245, 248 — sol, 90 (Fife. 46), 132 (Fig. 71), 151 Aotinopoda, 218 ActinospluBrium, 43, 50 (Fig. 22), 68, 74, 77, 78, 80, 91, 138, 144, 150, 193, 198, 207, 209, 214, 216, 245, 248 — eichhorni, 7 (Fig. 3), 81 (Fig. 37), 115 (Fig. 62), 116 (Fig. 63) Adaptive polymorphism, 164 Adelea, 175, 176, 348, 352, 393 — hartmanni, 344, 347, 348 — ovata, 344, 345 (Fig. 153), 346, 347 (Fig. 154), 352 AdeleidcB, 352, 354, 355 Adeleidea, 352, 394 Adinida, 278 Adoral spiral, 442 Adult, 212 .T.thalium, 242 Aflagellar, 287 Agametes, 180,181 Agamogony, 181 Agamont, 181 Agglomeration, 128, 209, 305 Agglutination, 128 Agglutinin, 128 Aygregata, 23, 168, 325, 348, 353 — jacyuemeti, 121 (Fife. 67) Aggregatida, 353 Alcohol, effects of, 204 Alloffromia, 230 — ovoidea, 235 Alternation of generations, 181 Alveolar layer, 436 Alveoli, 42 Amicronucleato, 211 Amitosis, 105 Amoeba, 219 — albida, 221 (Fig. 87) — Mnudcata, 78, 95, 214, 223 — diploidea, 222 (Fig. 88) — diplomifotica, 108, 109 (Fig. 56) — flava, 221 — Umax, 46, 47 (Fig. 20), 206, 217, 219 — minuia, 221, 223 — mticicola, 220 — proteus, 6 (Fig. 2), 47, 191, 205, 209, 215, 216, 217, 219, 220, 222, 230 — radiosa, 217, 219 — terricola, 48, 190 (Fig. 82), 214, 220 — verrucosa, 32, 45, 48, 50, 51 (Fig. 23), 198, 214, 219 — vespertilio, 217 Amoeba, form-changes, 216 (Fig. 85) Amcebeea, 217 Amcebidium, 428 Amoebodiastaso, 193 Amoeboflagellata, 463 Amcebogenise, 325, 466 Amosboid, 30 Amogbula, 169 Amphikaryon, 96 Amphileptus, 439 Amphimixis, 150, 164 Amphinuoleus, 96 Amylum, 188 Anaerobic, 196 Anaplasma maryinale, 383 Ancyslropodium, 441 Angeiocystis audouinice, 349 Anisogamy, 126, 132, 175 Anisonema, 274 — yrande, 53 (Fig. 25) AnisonemincB, 274 Anisospore, 215, 254 Annulus, 276 Anopholime, 358 Anoplophrya, 171, 439, 443, 449, 452 — branchiarum (reduction), 145 (Fig. 74) Anoplophryinat, 197, 452 Anurosporidium, 424 Aphrothoraca, 247 Apiosoma, 379 Apodinium, 278 505 506 THE PROTOZOA Aposporogony, 368 Arcella, 64, 65, 72, 78, 128, 148, 173, 199, 201, 215, 216,229 — tndgari9, 67 (Fig. 32), 110 (Fig. 57), 177, 178 (Fig. 80) Archeeocytes, 133 Arohoplasm, 79, 103 Arenaceous, 34, 231 Asporooystea. 388 Aspirigera, 439 Arrhenoplasm, 129 Artificial classification, 463 Assimilation, 187 Association, 127, 330 Astasia, 274 — tenax, 33 (Fig. 15) Astasiidce, 274 Astomata, 438, 439, 451 Astrodisculus, 248 Astrophrya, 461 — arenaria, 456 Athene noctua, 390 Attraction-sphere, 103 Attraction-spindle, 104 Aulacantha, 256 Autooyst, 417 Autogamy, 138, 306 Automixis, 140 Autophya, 34 Avoiding reaction, 202 Axopodium, 48, 53, 60, 87, 199, 465 Axostyle, 36, 259, 289, 311 Azoosporidae, 218 Babesia, 357, 379, 394 — bovis (bigemina), 379, 384 — mutans, 380, 382 Babesioses, 378 Bacteria, 5, 98 Jiadhamia, 242 — utrieularis, 240 (Fig. 99), 241 (Fig. 100) BalantWum, 439, 440 — coli, 440 — minutum, 440 Banana-tree, 136 Barroussia, 352 — alpina, 344, 345 (Fig. 153) — caudala, 348 — ornata, 346, 352 — spircUis, 344, 348 Baro taxis, 202, 207 Basal granule, 82, 92, 200, 443 — rim, 443 Benedenia, 853 Bertramia, 424 — asperospora, 424 — bujonis, 424 — - capitellce, 424 — kirkmanni, 424 Bertramiidce, 424 Bilateral symmetry, 31, 250 BUoculina depressa shells, 233 (Fig. 94) Binary fission, 100 Binuclearity, 96 Binucleata, 85, 280, 388 Bioblast, 40, 41 Bionomics, 15 Black spores, 364 Blastoooalo, 133 Blastodinidce, 278 Blaslodinium, 278 Blastogenea, 418 Blastomere, 133 Blastulidium pmdophthorum, 424 Blepharoplast, 52, 59, 82, 262, 286, 288, 289 Bodo, 270, 281, 319 — edax, 319 — gracilis, 271 (Fig. 115) — lacertm, 270 — aattans, 271 (Fig. 115), 319 Bodonidce, 268, 270 Body-form, 29 Bud, 122 Buetschlia, 439 Bursaria, 439 Bursaridce, 439 Callyntrochlamys phronimce, 327 Calonympha, 276 Calymma, 251, 252 Calyx, 89 Campanella, 440, 446, 447 — umbellaria, 434 (Fig. 183) Camptonema, 51, 248 — mOans, 91 (Fig. 47) Cancer, 473 Capillitium, 241 Capillus intestinalia, 428 Capsulogenous cell, 399. 403 Carcheaium, 145, 192, 194, 440, 441, 449 Caryoryctes, 473 Caryospora, 349, 352 — simplex, 352 Caryotropha, 195, 344, 348, 352 — mesnilii, 349, 352 CaryotrophidiB, 352 CauUerya, 424 — mesntti, 424 Cell, 1, 98, 464 Cell-anus. 433 Cell-division, 121 Cell-membrane, 45 Cell-mouth, 63 Cell-theory, 133 Central capsulo, 250 — grain, 91 — spindle, 103 Centriole, 73, 80, 97, 262 Centrodesmose, 36, 58, 59, 82, 103 Cenlropyxis, 14&, 173, 229 — aculeata, 36, 23.0 Controsomo, 58, 59, 73, 79, 262, 288 Centrosphere, 80 Cephalina, 339 Cephaloidophora, 337 Cephalont, 181,326 Ceratwmyxa, 242 Ceratium, 278 Ceratocorys, 278 — horrida, 277 (Fig, 121) Ceratomyxa, 408 — drepanopsettcB, 402 (Fig. 166), 403 — spharulosa, 409 Ceratomyxidw, 408 CeratophyUus fasciatua, 291 Cerpomonodido), 268, 270 Cercmnonos, 270, 271 — crossicauda, 271 (Fig. 114) Chagasia hartmanni, 344, 347 Chalaiothoraca, 248 Chemotaxis, 202 Chiliferida), 439 Chilodon, 145, 439, 448 INDEX 507 ChUodon cucullulus, 435 (Fig. 184) — dentatus, 440 Chilomonas, 208, 274 Chlamydodoniidce, 439 Chlamydomonadidai, 275 Chlamydomonas, 275 CMamydomyxa, 214, 243, 244 Ohlamydophora, 248 Chlamydophrya, 237 — schmidinni, 237 — stercorea, 17, 237 Chlamydozoa, 470 Chloromyxidae, 407, 409 Chloromyxum, 409 — lei/dipt, 400 (Fig. 164), 409 Chlorophyll, 13, 63, 188, 261 Choanoflagellata, 261, 271 Choanoflagellidcf, 271 Chondriosomo, 41 Chromatin, 65, 69 Chromatoid grains, 67, 289, 311 Chromatophoro, 13, 63, 188, 261 Chromidia, 6, 65, 97, 150, 215, 328 Chrornidial fragmentation, 101 Chromidina, 452 Chroinidiogamy, 126, 416 Chromidiosome, 65, 103 Chromomonadina, 274 Chrornophyll, 188 Chromoplast, 13, 63 Chromosome, 103 Chromulina, 274 — flavicans, 15 Chrysamceba, 274 Chryso monad ina, 14, 274 Chytridiopsis, 428 — socius, 428 Ciliary apparatus, 442, 444 (Fig. 186) Ciliata, 430, 432 (Fig. 181) Cilioflagollata, 277 Ciliophora, 462 Ciliophrys, 248 Ciliospore, 169 Cilium, 12, 53, 92, 199, 200, 442, 454 Circumfluonoo, 189 Circumvallation, 189 Cirrus, 55, 445 Cladomonas, 270 Cladothrix pelomyxcs, 227 Classification, 462 Clathrulina, 39, 245, 248 — elegans, 38 (Fig. 19) Clepsydrina, 335, 339 Cnidosporidia, 399 Coccidia, 341, 389 CoccidiidcB, 352 Coccidioides immitis, 17 Coooidiomorpha, 388, 395 Cocoidiosis, 343 Coccidium, 101, 166, 173, 174, 346, 352 — cuniculi, 341, 351 — mitrarium, 344 — oviforme, 341 — rouxi, 349 — schuberai, 102 (Fig. 50), 106 (Figs. 51, 52), 127 (Fig. 69), 146 (Fig. 75), 204, 342 (Fig. 152), 353, 354 — stiedce, 341 Coocoid bodies, 468 Coocolith, 274 Coccolithophoridce, 274 Coccomyxa, 409 Cdccomyxa morovi, 409 Cochle&ria faurei, 442 Cochliopodium, 229 Codonosiga botrytis, 260 (Fig. 110. Ccelosporidiidce, 399, 424 Caelosporidium, 424 — blateUm, 424 Ccelozoic, 324 Coleps, 439, 441 Collar, 57, 89, 261 Collecting-pusule, 277 Collodagia, 255 Collozoum, 256 Colpidium, 208 Colpoda, 439 Conchophrys, 439 Conjugant, 126, 448 Conjugation, 126, 448 Conorhinus megistus, 291, 302 Contact-stimulus, 207 Contractile vaouole, 60, 196, 197, 262, 437, 447 — system, 445, 446 Contractility, 200, 201 Copromonas, 171, 274 — major, 268 — mbtilin, 264 (Fig. Ill), 268 Copromyxa, 243 Copularium, 355 Copulation, 126 Corps en barillet, 344 Cortex, 45 Cortical layer, 32 Corticate, 45 Costia, 258, 272 — necatrix, 16, 272 Cothurnia, 440 Craspedomonads, 261, 271 Craspedotella, 279 Cristispira, 466, 469 — anodontte, 468 — balbianii, 467 — peclinis, 469 (Fig. 194) Crithidia, 281, 282.-2S7, 308, 312, 320, 321 — campanulaia, 313 — gerridis, 313 — melophagia, 290 - minuta, 312 (Fig. 135) Cryptocystes, 412 Cryptodijfflugia, 229, 230 Cryptomonadina, 15, 274 Cryptomonas, 274 — schaudinni, 15 Cryptosporidium, 349, 352 — muria, 344, 352 Crystal-spores, 254 Cuirass, 33, 45, 276 Culicinae, 358 Culture d'attente, 304 Cuticle, 45 Cyclasterium, 470 Cyclical transmission, 290 CyclochcBta, 440, 441 Cycloposthium, 439 Cyclosis, 192, 194, 437 Cyclospora, 352 — caryolitica, 176, 198, 344, 348, 349, 352 Cyst, 154 Cystal residuum, 349 Cysiobia chiridotce, 341 508 THE PROTOZOA Cyatobia holothuricB, 128 (Fig. 70) — irregularis, 331 — minchinii, 336 Cystoflagollata, 257, 278 Cystotrypanosoma, 304 Cytooyst, 344 Cytomere, 344 Cytomiorosome, 41 Cytopharynx, 63, 261, 433, 442 Cytoplasm, 6, 7, 99 Cytopyge, 433 Cytorhyctea, 471 — aphtharum, 471 — luis, 471 — scarlatina), 471 — vaccines, 471 Cytoryctes, 470, 471 Cytostome, 63, 190, 191, 261, 433, 452 Cytozoio, 324 . Dactylosoma splendens, 378 Defalcation, 233 Degeneration, 208 Dendrocometes, 457, 460, 461 Dendrocometidco, 461 Dendrosoma, 456, 458, 461 — radians, 78, 460 (Fig. 193) Dendrosomidw, 461 Dendrosomides paguri, 455 Depression, 131, 135, 197, 208 Derbesia, 90 Dosmothoraca, 248 Deutoblast, 426 Doutomeritc, 327 Deutoplasmic, 41 Devescovina, 276 Dexiotricha, 440 Dictyostelium, 243 Didinium, 145, 439, 442, 449 Didymophycs, 330 Diffluffia, 34, 35, 50, 65, 66, 78, 126, 140, 149, 199, 215, 216,229 — spiralis, 34 (Fig. 16) - urceolata, 214, 229, 230 Diffuse infiltration, 400 Digestion, 192 JHlcptus, 439 Dimastioamoeba, 268 Dimarpha, 249 — nuians, 249 (Fig. 104) JUnc-nymphn, 276 J)i;. ; era, 278 LHndbryon, 274 Dinoflagellata, 257, 276 DinophysidcB, 278 Dinophysis, 278 Diphtheria, 470 ZHplocysiis minor, 128 (Fig. 70) Diplodina, 174 Diplosome, 79 Diplozoa, 273 Direct division, 101 Direct transmission, 290 Discophrya, 439 Discorbina, 232 (Fig. 93, vii) Disporea, 408 Dizoic, 349 DoliocyslidcB, 339 Dourine, 26, 285, 289 Drehkrankhoit, 400 Drepanidia, 395 Drepanidium, 372 Duboscyia, 418 — legeri, 418 Earth-amoebae, 220 Echinomera, 333 Echinopyxis, 101 Botoplasm. 43, 45, 435 Eotosaro, 43 Eotoeohiza, 339 Eotosporea, 325 Kimeria, 346, 352 — falciformis, 3.46 — nepcB, 346 EimeridcB, 352 Eimeridea, 352, 394 Electrical stimuli, effects of, 208 Elementary corpuscles, 472 Klleipsisoma, 387 — thamsoni, 387 Enchelidof, 439 Enchyloma, 41, 72 Encystment, 164 Endogenous budding, 124 — cycle, 184 Endoparasita, 462 Endophrys rotatorium, 249 Endoplasm, 43, 62, 437 Endoral membrane, 445 Endosarc, 43 Endoschiza, 339 Endosomo, 73 Endospore, 335 Endosporese, 242, 825 Endotrypanum, 307 — schaudinni, 307 (Fig. 133) End-piece, 443 Kiiorgid, 121 Entamceba, 220 — africana, 226 — blaUa, 47, 220, 223 — buccalis, 220 — coli, 18, 138 (Fig. 73), 139, 223, 224 (Fig. 89), 225 — histolytica, 18, 46, 223. 224, 225 (Fig. 90) — minuta, 226 — muris, 220 — ranarum, 220 — tetragcna, 226 — vnlliamsi, 225 Entodinium, 439, 441 Entozoio, 16 Enzymes. See Ferments Ephelota, 457, 461 — buefschliana, 457 — gemmipara, 460 Epicyte, 45, 327 Epimerite, 45,326 Bpispore, 335 Epistylis, 440, 441 — plicalilis, 444 (Fig. 186, K), 446 — umbellaria, 447 Epithelioma oontagiosum, 470 Epizoic, 16 Equating division, 104 Equatorial plate, 103 Ergastoplasm, 41 Erythropsis, 277 Etheogenesis, 138, 315 Eucocoidia, 352 Eucoccidium, 353 Eucyrtidium, 256 INDEX 509 Eucyritidium craniaides, 258 (Fig. 109) Euflagollata, 257 Euglena, 14, 33, 52 (Fig. 24), 107, 202, 274 — oradlis, 188 — spiroffyra, 8 (Fig. 4) — viridis, 188, 205 (Fig. 84) Euglenidce, 274 Euglenoid movement, 33 Euglenoidina, 273 Euglypha, 34, 35, 214, 287 — alveolata, 111, 112 (Fig. 59), 113 (Fig. 60) Eugregarinoe, 328, 339 Euplasmodida, 242 Euplotea, 194, 440, 448 — harpa, 433 (Fig. 182) — patella, 433 (Fig. 182) Euplotidoe, 440 Eutrypanosome, 292 Ex-conjugant, 153 Excretion, 197 Excretory canals, 447 Exflagellation, 357, 362, 364, 365, 390 Exogenous cycle, 184 Eye-spot, 205 Falciform body, 324 Fat, 194 Fatty degeneration, 210 Feeding canals, 437 Female sex, 159 Ferments, 193, 194 Fertilization-spindle, 127, 348 Filoplasmodida, 243 Filose, 48 Fission, 100 Fixation, 441 Flagellar, 287 Flagellata, 82, 257 Flagellispore, 169 Flagellosis, 313 Flagellula, 169 Flagellum, 6, 51, 199, 200, 289, 454, 465 Fcettingeria, 439 Fcettingeriida), 439 Food-vaouole, 50, 62, 191, 194, 437 Foot-and-mouth disease, 470 Foraminifera, 217, 231 Form-production, 31 Framboesia, 467 FrondimUaria, 232 (Fig. 93, iv.) Fronionia, 439, 442 — leucas, 206, 447 (Fig. 187) Fulcra, 441 Fuligo, 242 — septica, 239 (Fig. 97) Oalvanotaxis, 202, 208 Gamete, 125, 448 Gametid, 334 Garnet ooyte, 126 Gamogony, 181 Gamont, 126, 181 Ganymedes, 330 Gas-vaouole, 64 Gastrocystis gUruthi, 427, 428 (Fig. 179) Gemmation, 122 Gemmula.^459 Gemmule, 471 Generative ohromatin, 71 Geotaxis, 207 Germ, 165 Germ-cells, 130 Gormen, 130 Germinative infection, 24 Glaucoma, 439 — colpidium, 197, 206 — sciniillans, 445 Glenodinium, 278 — cinctum, 277 (Fig. 120) Globidium, 387 — multifidum, 387 Globigerina, 231, 232 (Fig. 93, vi. Glossina morsitans, 291 — palpalis, 291, 303, 304 Glugea, 412, 417,418 — anomala, 411, 415, 417 (Fig. 174), 418 — stephani, 412 Gonium, 276 — perforate, 275 (Fig. 119) Granellse, 238 Granellarium, 238 Gregarina, 174, 335, 339 — blattarum, 339 — munieri, 58 (Fig. 29) — ovata, 332, 333 (Fig. 146), 335, 339 — polymorpha, 9 (Fig. 7), 339 Grogarines, sporogony, 331 (Fig. 144) Gregariniform phases, 315 Gregarinoidoa, 326 Gregarinula, 169, 324 Gromia, 231 — oviformis, 49 (Fig. 21) Guarnieri's bodies, 470 Gurleya, 418 Gymnamcebee, 219 GymnoainidcB, 278 Gymnodinium, 278 Gymnospore, 165 Gymnostomata, 439, 442 Gymnozoum, 439, 442 — vimparum, 439 Hiemamcebre, 357, 389 Hsematochrome, 188 Hcsmatococcus, 188, 275, 379 — pluvialis, 111 (Fig. 58) Hcematomonas, 308 Hwmatopinus spinulosus, 291, 301 Haimocystidium, 358, 364 — diploglossi, 358, 365 — metschnikovi, 358 — simondi, 358, 365 — tropiduri, 365 Hoemoflagollatos, 258, 280 Hcemogregarina, 372 — agamoe, 373 — balfouri, 376 — bicapsulata, 372 — earns, 377 — funambuli, 377 — gerbitti, 376, 377, 390 — jaculi, 876 — muris, 23, 376, 390 — musculi, 352, 377 — nicoricB, 373, 375 — peramelis, 376 — sebai, 377 — stepanowi, 107 (Fig. 53), 372, 373, 374 (Fig. 159), 375, 390 Heemogregarines, 357, 371, 390 HcBmogregarinidcB, 378 Uaimoprotcus, 365, 391 — columbce, 365, 366 (Fig. 157), 390, 391 510 THE PROTOZOA Hcemoprotcus danilewskyi, 365 — noctuw, 365, 390 — oryzivorcB, 368 Haemosporidia, 356 Hatiphysema, 35, 231 — tuman&wiczii, 35 (Fig. 17) Halteria, 439 HaUeridce, 439 Halteridia, 389, 391 Halteridium, 357, 365, 391 Haplosporidia, 399, 423 HaplosporidiidcB, 423, 424 Haplosporidium, 424 Hastatella radians, 441 Helcosoma tropicum, 393, 412 Heliozoa, 90, 218, 244 Hemiclepsis marginata, 291, 298, 303 Hemispeira asterice, 441 Henneguya, 409, 426 Hepatozoon, 372 — muris, 376 — perniciosum, 376 Hereditary transmission, 24, 290 Herpetamonas, 281, 282, 292, 313, 319, 320 — muscae-domestica). 137, 138, 282 (Fig. 124), 315 Herpetophrya, 452 Heterokaryote, 449, 453 Heteromastigoto, 259 HeteronemincB, 274 Heterophrys, 248 — fockei, 248 (Fig. 103) Heterotricha, 433 Hexactinomyxon, 409 Hexamitus, 258, 272 Histocytes, 130, 133 Histoplasma, 319 — capsulatum, 319 Histozoic, 324 Holomastigina, 270 Holomastigote, 259 Holophrya, 439 Holopbytic, 13, 187, 188, 261 Holotrioha, 439 HolotriohouB larvae, 459 Holozoio, 8, 13, 187, 261 Homaxon, 39, 250 Hoplitophrya, 452 House, 33, 45 Hyalosphcera gregarinicola, 341 Hyalosphenia, 34 — cuneata, 34 (Fig. 16) Hydrophobia, 470 Hymenostomata, 439, 442 Hyper*hromasy, 71 Hypnocyst, 166 Hypocoma, 460 — acinetarum, 460 Hypocomidce, 460 Hypothallus, 240 Hypotricba, 433, 440 Hypotrichoue larvae, 459, 460 IchthyophthiriasiB, 450 Ichthyophthirius, 448, 453 — muUiflliis, 16, 21, 450, 451 Ichthyosporidium, 424 Idiochromatin, 71 Idiochromidia, 150 Immanoplasma, 388 — scvllii, 388 Imperforate, 231 Import, 189 Incubation-period, 292, 361 Incurvation, 468 Indirect division, 101 — transmission, 290 Infusoria, 2, 12, 152 (Fig. 77), 153, 430 Ingestion, 204 Initial body, 472 Intestinal flagellates, 258 Invagination, 189 Involution stages, 296 Isogamy, 126, 175 Isomastigote, 259 Isospore, 215, 254 Isotricha, 439 Isotrirhido), 439 J&nia, 276 Kala-azar, 316 Kalpidorhynchus, 332 Karyogamy, 126 Karyokinesis, 101, 119 Karyolysus, 372 — lacertarum, 372, 378. Karyosome, 76, 288 Kataphoric action, 208 KerUrochona, 440 Kentrochonopsis, 440 Kinetonucleus, 78, 85 200,286,288,289 392 Klossia, 348, 352 — heUcina, 352 Klossiella, 352 — muris, 352 Kurlofl-Demel bodies, 388 Ldbyrinthula, 243, 244 Labyrinthulidea, 243 Lagena, 232 (Fig. 93, ii.> LagenophryincB, 440 Lagenophrys, 440 Lamblia, 272 — intestinalis, 31, 272, 273 (Fig. 117) — sanguinis, til LankestereUa, 189, 372, 378 — ranarum, 372, 378 Lankesteria ascidice, 327, 329 (Fig. 143) — culicis, 327 Latent bodies, 296 Laverania, 358 Legendrea loyezce, 441 Lcgerella, 348, 349, 352, 355, 388 — nova, 352 LegereUidce, 352 Legeria, 353 Leyerina, 353 Leishmania, 258, 281, 316,. 320, 321» 393, 394 — donovani, 316 (Fig. 138), 317 (Fig. 139), 473 — infantum, 316, 317 — tropica, 87, 316, 317, 318 (Fig. 140), 412, 473 Lentospora cerebralis, 400 Leptodiscus, 279 Leptomonaa, 52, 281, 282, 882, 308, 313, 319, 320, 321 — butschlii, 282 — joculum, 314 (Fig. 136), 315 Leptotheca, 408 INDEX 511 Ltptotheca agilis, 201, 401 (Fig 165) ranarum, 408 Leptotrypanosome, 292, 314 Lernceophrya, 461 Lethal, 19 Lcwcocytogregarina, 372 Leucocytozoa, 372 Leiitocytnzoon, 357, 360, 390, 392 — tovaii, 370 — muris, 376 — piroplasmoides, 319 — ratti, 376 — sabruzesi, 371 — ziemanni, 369 (Fig. 158), 370, 371 Leucophrys, 439 — patula, 440 Leucoplasts, 188 Leydenia gemmipara, 237 Licnophora, 440, 441, 446, 449 Licnophoridat, 440 Life-cycle, 129, 130 Light-perception, 201 Light-production, 201 Linellee, 238 Linin, 72 Lionntus, 439 Lithocircus, 256 — productus, 252 (Fig. 106) Lithocysti* schneideri, 331 Lobopodia, 47, 199 Lobosa, 217, 219 — testacoa, 229 Lobose, 47 Lophomonadidai, 276 Lophomonat, 36, 88, 261, 276 — blattarum, 17, 18, 89 (Fig. 45), 263, 276 — striata, 276 Lophophora, 276 Lorica, 33, 45, 276,441 L'jxoctes, 439, 448 Luminosity, 201 Lymphorystis, 426 — johnntonei, 426, 427 (Fig. 178) Lymphocytozoon, 388 — cobayco, 388 Lynchia, 365 Macramceba, 148 Macroconjugant, 153, 449 Macrogamete, 126 Macrogamy, 131, 161, 172 Maorogonidiae, 267 Maoromerozoite, 373 Macront, 426 MaoronucleuB, 78, 107, 430, 437, 448, 458 MaoroBohizogony, 344 Maoroscbizont, 344, 373 • Macrospores, 254, 255, 416 Macrostoma, 272 — 'inesnili, 272 Mai de caderas, 285 Malaria, 358, 359 Male sex, 159 Mai lory's bodies, 470 Malpighiella refringent, 229 Mantle-fibres, 103 Mastigceinceba, 213, 261, 268 Mastigella, 77, 268 — vitrea, 83 (Fig. 40), 265,266 (Fig. 112) Mastiyina, 265, 267, 268 — setosa, 82 (Fifts. 38, 39) Mastigophora, 12, 257 Mastigotrioha, 455 Maturation, 142 Maupasia, 454 — paradoxa, 454 (Fig. 189, B) Measles, 470 Mechanical stimuli, effects of, 207 Mechanical transmission, 290 Megaloephaerie, 184, 233 Megastuma, 272 — entericum, 272, 2-73 (Fig. 117) Melanin, 64, 198, 357 Membrane (nuclear), 76 Membranellse, 55, 443, 445 Membranulee, 445 Merocystis, 352 — kathcB, 352 Merogregarina amaroutii, 336 Merogregarinida, 341 Meront, 398, 413 Merozoite, 169, 325 Merozoon, 210 Mesomitosis, 111 Metabolic, 33 Metaohromatinio grains, 67, 420. 421 M etacineta, 460 Metacinetidm, 460 Metagenesis, 266 Metamera, 332, 339 Metamitosis, 111 Metaplaatio, 40, 63 Metazoa, 2 Micramoeba, 148 Microconjugant, 153, 172, 448 Microgamete, 126, 448 Microgamy, 132, 172 Miorogonidia, 267 Microklosiiia. 426 Micromarozoite, 373 Mioront, 426 Micronucleus, 78, 113, 114 (Pig. 61), 288. 332, 333, 4SO, 437. 448 Microechizont, 344, 873 Miorosome, 40 Micrbspheric, 184, 233 Miorospore, 254, 255, 416 Miorosporidla, 411 Microthoracidcv, 489 Microthorax, 439 Miesoher'e tubes, 419, 422 Minchiiiia, 352 — caudata, 348 — chitonis, 349, 352 Mitochondria, 41, 448 Mitosis, 101 Mixotrophic, 188 Molluscum contagiosum, 17, 470 Monad, 466 Monadidce, 270 Monas, 270 Monaxon, 39, 250 Monera, 78 Moniliform, 77 Monocercomonas, 272 Monocystis, 23, 174 (Fig. 79), 328, 336, 339 — coronata, 328 — pareudrili, 331 — rostrata, 332, 333, 335 Monokaryon, 121, 255 Monomast igoto, 259 Monomastix, 455 612 THE PROTOZOA Monomasiix cilialus, 454 (Fig. 189, A), 455 Monomorphio fepecios, 163 Monopylaria, 251, 256 Monospora, 339 Monosporea, 409 Monothalamous, 36, 232 Monozoa, 273 Monozoic, 349 Mother-cyst, 138 Movement, 199 Movements of gregarines, 327 Mullicilia, 249, 261, 270, 454 — lacustris, 269 (Fig. 113), 270 — palustris, 269 (Fig. 113), 270 Multiple fission, 100, 120 — gemmation, 122 — promitosis, 120 Multiplicative phase, 20, 166 Multipolar mitosis, 120 Murrina, 285 Mycotosporidium, 243 Mycetozoa, 218, 239, 268 Mycterothrix, 446 Myocyte, 327 Myonemes, 57, 201, 253, 259, 286, 445 Myophiisks, 253 Myxamoeba, 239 Myxidiidce, 409 Myxidium, 409 — bergense, 407 — lieberkuhni, 400, 401, 409 — sp., 406 Myxobolidatt 22, 23, 409 Myxobolus, 409 — cerebralis, 400 — neurobius, 400 — pfeifferi, 405, 406 (Fig. 168) Myxocystis, 417, 418 Myxoflagellate, 239 Myxogaetres, 242 Myxomycetes, 239, 242 Myxopodia, 253 Myxosporidia, 399 Myxothcca, 231 Nagana, 19 Narcotics, effects of, 204 Nassollaria, 256 Nassula, 439 Natural classification, 463 Nebenkern, 95 NebenkSrper, 278 Negri's bodies, 470 Nomatocyst, 447 Neogamous, 127, 330 Neosporidia, 325, 398, 466 Nephroselmis, 275 Nervous system, 446 Nenronemes, 446 NeurorycUs, 470 — hydrophobia, 471 Neurosporidium, 424 — crphalodisci, 424 Nicollia, 380 — guadrigemina, 380, 381 Nina. See Ptcrocephalw Noctiluca, 201, 213, 279 — mUiaris, 119 (Fig. 65), 279 Nodosaria, 232 (Fig. 93, 3) Nosema, 418 — apis, 412 Nosema bombycts, 24, 411, 413, 414 (Fig. 172) Nuclear membrane, 76 — sap, 72 Nuclearia, 248 Nuclearia -stage, 177 Nucleo-cytoplasmio ratio, 70 Nuoleolo-oentrosome, 95 Nuoleolus, 76, 103 Nucleophaga, 473 Nucleus, 6, 7, 65, 96 — seoundus, 95 Nuda, 217, 219 Nummulites, 232 (Fig. 93, 11) Nutation, 51 Nutrition, 187 NuUallia, 380 — equi, 380 — herpestidis, 380 Nyctolherus, 439, 440, 447 — cordiformis, 10 (Fig. 9), 444 (Fig. 186, F) — faba, 440 X Octomitidce, 272 Octoniitus, 36, 258, 272 — dujardini, 272 (Fig. 116) Octosporca, 418 — tnvtccB domesticcB, 138 Ootozoic, 349 (Ecomonas, 270 (Esophagus, 261, 433 Oikcmonas, 270 Oligosporea, 418 Oligosporulea, 424 Oligotrioha, 439 Odoyst, 348 Odcyte, 143 Ofikinoto, 305, 362 Opalina, 196, 198, .208, 209, 439, 440, 447, 448, 452, 454 — cavdala, 452 — inUstinalis, 452 — ranarum, 447, 452, 453 OpalinincB, 452 Opalinopsis, 452 Opercularia, 145, 440 — faurei, 442 Operoulum, 441 Ophrydivm, 438, 440 Ophryocyetidm, 341 Ophryocystis, 337, 339 Orcheobius, 352 OphryodendridcB, 461 Ophryodendron, 455, 461 OphryoscolecidcB, 439 Ophryoscolex, 439, 441 Orcheobius herpobdellce, 346, 348, 349, 352 Organella, 1 Oriental sore, 316 OsmotaxiB, 203 Ovum, 125 Oxyrrhis, 52, 278 — marina, 278 (Fig. 123) Oxytricha, 202, 440 Oxytrichidai, 440 PansporeUa perplexa, 427 Pansporoblast, 405, 417, 423 Pantastomina, 268 Parabasal apparatus, 89 Paracoccidium prevoti, 349 INDEX 513 Paraglycogen, 41, 63, 195, 327 Paramastigoto, 259 Paramecidce, 439 Paramecium, 61, 114 (Fig. 61), 171, 191, 192, 194, 196, 197, 198, 203 (Fig. 83), 205, 206, 208, 210, 437, 489, 442, 443 — bursaria, 449 — caudatum, 107 (Fig. 53), 436 (Fig. 185), 444 (Fig. 186, D, E), 447 (Fig. 187) Paramceba, 228 — eilhardi, 94 (Fig. 49), 95, 228 — hominis, 228 Paramylum, 63, 188, 195 Paramyxa, 243, 409 — paradoxa, 409 Paramyxidia, 409 Paraplasma flavigenum, 379 Parasite, 8, 14 Parietal cell, 403 Parthenogenesis, 137 Parthenogonidia, 267 Partial karyogamy, 128, 153, 453 Pathogenic, 19 — amcebse, 226 Paulinella, 214 Pearl -stage, 334 Pebrine, 24, 411 Pectinellifi, 442 Peduncle, 31 Pellicle, 32, 45, 435 PetomyZka, 78. 144, 150, 205, 214, 227 — palustris, 227 (Fig. 91) PeMomyces, 243 Peneroplis, 15, 235 Peranema, 274 — trichophorum, 273 PeranemidcB, 274 Perezia, 418 Perforate, 231 Peridiniales, 276 Peridinidce, 278 Peridinium, 278 — diver gens, 278 (Fig. 122) Peridium, 241 Perikaryon, 439 Periplast, 45, 259 Peripylaria, 251, 255 Peristome, 433, 442 Peritricha, 433, 438, 440, 441, 442, 448 Peritrichous larvee, 459 PeritromidcB, 440 PerUromus, 440 Pernicious malaria, 358 Peroral membrane, 445 Phacus, 274 - triqueter, 274 (Fig. 118). Phsenocystes, 412 Phrcodaria, 256 Phseodium, 252 Phosphorescence, 201, 278 Phototaxis, 202, 205 Phylogeny, 463 Physurum didermoides, 242 Physodes, 244 Phytoflagellata, 274 Phytomonadina, 274 Phytomy xinas, 243 Piroplasma, 24, 357, 379, 393, 394 — bigeminum (bovis), 379 (Fig. 160), 384, 385 (Fig. 162) — cabatti, 379 Piroplasma canis, 382, 383 (Fig. 161), 384, 385 (Fig. 162), 387 — donovani, 393 — hominis,~319 Piroplasmoees, 378 Piroplasms, 378, 390 PlagiotomidcB, 439 Planont, 398, 408, 413, 423 Planorbvlina, 232 (Fig. 93, 9) Plasmodiophora, 243 — brassicte, 149 (Fig. 76), 243 Plasmodium, 100, 128, 240, 398, 423 Plasmodium, 357 — brasilianum, 364 — cynomolgi, 364 — diploglossi, 358 — falciparum, 358, 359, 3CO (Fig. 156) — inui, 364 — kochi, 364 — malance, 358, 359 — pithed, 359, 364 — prcBcoij 358 — relictum, 358 — vivax, 137 (Fig. 72), 358, 359, 3GO (Fig. 156) — vassali, 364 Plasmodioma, 462 Plasmogamy, 128 Plasmotomy, 100 Plastin, 73, 103 Plastinoid granules, 41, 195, 346 Plastogamy, 128, 209 Plegopoda, 462 Pleistophora, 418 — longifUia, 413 (Fig. 171), 415, 416 — periplanetiB, 416 — species, 413 Pleodorina californica, 267 Pleuronema, 55, 439, 442 — chrysalis, 56 (Fig. 27) PleuronemidcB, 439 Podophrya, 461 — fixa, 456 (Fig. 190, C), 458 — gemmipara, 108 (Fig. 55) — mollis, 456 (Fig. 190, A) Podophryidee, 461 Polar bodies, 143 — capsule, 399 (Fig. 163) — cones, 117 — filament, 399 — masses, 110 — platos, 117 Polycaryum, 424 Polychromophiliis, 364 Polycystid, 326 Polycyttaria, 256 Polyonorgid nuclei, 121, 151, 255 Polykaryon, 121, 255 Poly mast igidcu, 272 Polymastigina, 271 Polymastigote, 259 Polymastix, 272 Polymoiphism, 162, 163, 297, 311 Polyspora, 339 Polysporoa, 409, 418 Polysporulea, 424 PolystomfUa, 210 — crispa, 139, 234 (Fig. 95), 235, 236 (Fig. 96) Polythalamous, 36, 232 Polytomella agilis, 86 (Fig. 43) Polvtrema, 231 33 514 THE PROTOZOA Polytricha, 439 Polyzoic, 349 Poneramceba, 224 Ponlobdella muricata, 291, 303 Pontomyxa flava, 218 Parospara, 337, 340 — gioantea, 74 (Fig. 35), 336, 339 (Fig. 150) — legeri, 336 PorosporidcB, 341 Pouchetia, 62 — cornuta, 61 (Fig. 31) Prehensile tentacle, 457 Proboscidiform individuals, 456 Proboscidium, 442 Prococcidia, 352 Proflagellata, 469 Promitosis, 109 Pronucleus, 127 Propagativo cell, 405 — phase, 21, 166 Propulsive psoudopodium, 401 Prorocentraccffi, 276 Prorocenlrum, 278 Prorodon, 439 — teres, 32 (Fig. 14), 444 (Fig. 186, B, C), 446 Proteomyxa, 217, 268 Proteosoma, 358, 364, 365, 393 Protista, 4, 5 Protoblast, 426 Frotococcaceee, 15 Protoentospora ptychoderce, 229 Protokaryon, 75> 87, 108 Protomerito, 327 Protomonadina, 270 ProtopJirya, 452 — ovicola, 452 Protophyta, 8 Protoplasm, 29, 40 Protozoa, 2, 10, 464 Prowazok's bodies, 470 Prowazekia, 260, 271, 281, 319, 321, 322 — asialica, 319 — cruzi, 319 — parva, 319, 320 (Fig. 141) — weinbergi, 319, 320 (Fig. 141) Psevdochlamys-st&go, 170, 177 Pseudoplasmodida, 243 Pseudoplasmodium, 242 Pseudopodiospore, 169 Pseudopodium, 30, 46, 90, 199, 214, 400, 465 Pseudospora, 213, 218, 249 Psorosperm, 165, 323 Pterocephalus, 173, 327, 329, 330, 339 — ffracilis, 174 (Fig. 79), 332 (Fig. 145), 334 (Fig. 147) — nobilis, 339 Pulsellum, 52, 259 Pusule, 277 Pycnothrix, 452 — monocystoides, 443, 446, 447, 452 Pyramimonas, 275 Pyrenoid, 63, 188, 261 Pyrodinium, 201, 278 Pyrosoma, 379 Pyxinia, 329, 330 Quartan malaria, 358, 359 Radiolariii, 218, 249 Radium-rays, effects of, 205 Rainoy's corpuscles, 419 Reactions of protozoa, 201 Recapitulative forms, 170 Reducing division, 104 Reduction, 142, 145, 335 Reduction-nuclei, 144 Redwater, 378 Regeneration, 208, 210 Rejuvenescence, 155 Relapse (malarial), 363 Reserve-materials, 195, 196 Rescrvoir-vacuole, 262 Respiration, 195 Koticulosa, 217, 218 Roticulofio, 48 Retioulum (nuclear), 75, 103 — (protoplasmic), 41 Uhabdogonise, 325, 466 Jlhabdophrya, 461 — trimorpJitt, 455 Rhaphidiophrys, 245 Rhootaxis, 207 lihinosporidium , 424 - kinealyi, 424, 425 (Fig. 177) Rhizomastigina, 265, 268, 465 Rhizoplast, 82 Rhizopoda, 213, 217 Rhyncheta, 457, 460 Rhynchoflagellata, 278 Right hosts, 291 Rod-apparatus, 433, 439 Rbntgen-rays, effects of, 206 Rostrum, 326 Saccamina, 232 (Fig. 93, 1) Sack-pusulo, 277 Sapropelic, 14 Saprophytic, 8, 14, 187, 194, 262 Saprospira, 467, 469 — glandis, 468 Saprozoic, 14 Sarcocystine, 20, 420, 421 Sarcocystis, 20, 419 — bertrami, 420 — muris, 419, 420, 421, 422 (Fig. 176) — rileyi, 420 — tenella, 419, 420, 421 (Fig. 175) Sarcocyte, 327 Sarcode, 40 Sarcodina, 11, 213 Sarcosporidia, 20, 419 Scaiotricha, 440 Scarlet fever, 470 Schaudinne'.la, 355 Schewiakovella schmeili, 425 Schizocystidce, 339, 341 Schizocystis, 339 — gregarinoides, 336, 338 (Fig. 149) Sohizogonea, 418 Schizogony, 166, 324, 392 Schizogrogarinffi, 328, 339 Schizokineto, 373 Schizont, 166, 181, 324 Schlzontocyte, 344 Schizotrypanum, 285, 307, 392 — cruzi, 28, 295 (Fig. 128), 296, 302 (Fig. 132), 307 Schizozoite, 344, 428 Sclerotium, 166, 240 Scopula, 441, 456, 459 Scyphidia, 440, 441 Secondary nuclei, 66 INDEX 515 Secretion, 197, 198 Selenidiidco, 341 Selenidium, 339 — caiUleryi, 336, 337 (Fig. 148) Selenococcidium, 352, 354 — intermedium, 344, 350 (Fig. 155), 351 Senility, 131, 135, 155 Sensory organs, 201, 446 Separation -spindle, 104 Septate, 326 Serumsporidia, 425 Sex, 154 Sexual differentiation, 160, 170, 176 — phases of try panosomes, 305 Shel], 33, 45, 232 (Fig. 93) Siedleckia, 339, 352 Silicoflagellata, 274 Sleeping sickness, 26 Smithia, 380 — microti, 380 Soma, 130 Somatic number, 143 Sorophora, 243 SorosptKsra, 243 Soru8,242 Souma, 304 Spasmoneme, 446 Species, 141, 162 Spermatocyte, 143 Spermatozoon, 125 Sphmractinomyxon, 409 — stolci, 409, 410 (Fig. 170) Spheerellaria, 255 Sphceromyxa sabrazesi, 404 (Fig. 167), 405 Sptuerophrya, 461 Spheerozoa, 256 Sphere, 95 Spheroplast, 41, 448 Spicule, 36 Spindle (nuclear), 103 Spirigera, 442 Spirillacea, 467 Spirillar forms, 319 Spirillum, 467 Spirochceta, 466 — plifatUis, 466 — ziemanni, 371, 468 Spirochaetes, 466 Spirochona, 440 SpirochonidcB, 440 Spiroloculina, 232 (Fig. 93, 5) Spironema, 467, 469 Spironemacea, 469 Spiroschaudinnia, 467 — (inserina, 467 — dvttoni, 467, 468 — gattinarum, 467 — obermeieri, 467 — recurrentis, 467 Spirostamum, 196, 197, 208, 438, 439, 445 — ambiguum, 431 (Fig. 180) Sponyomonas, 270 — splendida, 84 (Fig. 41) - uvella, 85 (Fig. 42) Sporal residuum, 349 Sporangium, 240, 241 Spore, 165, 166, 323 Spore-formation, 166 Sporetia, 150 Sporoblast. 325 Sporocyst. 165 Sporocyst-mothor-eell, 403 Sporoduct, 335 Sporogony, 181, 325 Sporomyx ', 243 Sporont, 166, 181, 325, 326 Sporophoro, 242 Sporoplasm, 405 Sporozoa, 12, 323, 462, 466 Sporozoito, 169, 324 Sporulation, 122, 165, 168 Spumellaria, 255 Stannomidco, 238 Starvation, 195, 210 Stemonitis flaccida, 82 — fusca, 240 (Fig. 98) Stemm-pseudopodium, 401 Stempellia, 418 — mutabttis, 418 Stenophora, 329 Stentor, 61, 202, 211, 437, 438, 439, 441, 445, 446' — casndeus, 444 (Fig. 186, A, I) — niger 444 (Fig. 186, O) — roeselii, 10 (Fig. 8) Stentoridce, 439 Stephanosphasra, 267, 276 Steroomarium, 238 Stercome, 194, 233 Stigma, 81, 205, 262 Stomatophora coronata, 328 Streaming movements, 199 Strongyloplasmata, 470 Stylonvchia, 438, 440 — histrio, 444 (Fig. 186, H) — mytUus, 211, 459 (Fig. 192) Stylorhynchus, 173 329, 330, 339 — longicollis, 174 (Fig. 79), 327 (Fig. 142), 339 Suctoria, 455 Suctorial tentacle, 190, 458 SulcuB, 276 Surface-tension, 200 Surra, 26 Swarm-spore, 169, 396 Symbiosis, 15 Symbiotic algse, 197 Synactinomyxon, 409 Syngamy, 128, 438 Synkaryon, 127 Syphilis, 467 Syzygy, 330 Tachyblaston, 460 Tactic, 202 Tactile bristles, 443 — organs, 201 Tceniocystis, 327, 339 Taxis, 202 Technitella thompsoni, 34 Tcloblast. 426 Telomyxa, 418 — glugeiformis, 418 Telosporidia, 325, 395, 466 Temperature, effects of, 206 Tentaculifera, 455 Tertian malaria, 358, 359 Tost, 33 Testacea, 217,219 Tetramyxa, 243 Telratrichomonas, 272 Totrazoic, 349 Thalamophora, 219 516 THE PROTOZOA Thalassicolla, 255 — pelagica, 30 (Fig. 13) Thalassophysa, 255 Thocamoebae, 219, 229 Theileria, 379 — parva, 380, 382, 386 Thelohania, 418 — Chmtogastris, 416 (Fig. 173) — contejeani, 412 — mcenadis, 416 Thelyplasm, 129 Thormotaxis, 202, 208 Thigmotaxis, 207 Thyroid extract, efloctc ot, 204 Tinctin-body, 458 Tinlinnidce, 439, 441, 443, 447 Tocophrya, 461 — cyclopum, 461 — limbata, 460 — quadripartita, 210, 456 (Fig. 1£0- B) 460 Toddia, 387 — bufonis, 387 Tolerant, 21 Tonicity, effects of, 207 Total karyogamy, 126, 453 Toxocystis homari, 426 Toxoplasma, 319, 387 — canis, 387 — cuniculi, 387 — gondii, 387 — talpce, 387 TrachelidcB, 439 Trachelius, 439 — otrum, 441, 448 Trachelocerca, 439, 448, 453 — phcenicopterus, 120 (Fig. 6ft), 449, 450 (Fig. 188) Trachoma, 470 Tractellum, 52, 259 Trailing flagellum, 53, 260 Transmission of trypanosomcs, 289 Transmutation of energy, 199 Treponema, 467 — pallidum, 467, 468 — pertenue, 467 Triactinomyxon, 409 TricMavaria, 241 (Fig. 101) Triohites, 442 Triohocyst, 46, 435, 447 (Fig. 187) Trichodina, 440, 441 Trichomastix, 260, 271 Triehomonas, 17, 36, 56, 258, 260, 271 — eberthi, 8 (Fig. 5), 36 — hominis, 272 — vaginalis, 272 Trichonympha, 276 — hertwigi, 276 Trichonymphida, 463 Trichonymphidw, 89, 276, 454 Trichophrya, 461 TrichophryidcB, 461 Trichorhynchus, 446 Trichosphcorium, 51, 216, 229 — sieboldi, 73 (Fig. 34), 182 (Fig. 81) Trimastigamceba, 268 Tripylaria, 251, 256 Tritoblast, 426 Trizoic, 349 Trophic phase, 324 Trophochromatin, 71 Trophocbromidia, 150 Trophonucleus, 78 85, 286, 288 Trophozoito, 324 Tropical malaria, 358 Trypanomonad, 282, 298, 299 Trypanomorphp, 308 Trypanophia grobbem, 309 Trypanoplasma, 56, 78, 87, 260, 271, 281, 287, 308,309,321,322 — abramidis, 310 (Fig. 134) — borreli, 312 r— congeri, 312 — dendrocceli, 309 — gryllotalpcB, 309, 310 — gurneyorum, 78 (Fig. 36) — helicis, 309, 311, 312 — intestinalis, 312 — keysselitzi, 310 (Fig. 134) — ranee, 319 — vaginalis, 309 Trypanosoma, 270, 280, 283, 308, 320, 321 — baUnanii, 467 — blanchardi, 25 — brucii, 19, 25, 26, 27 (Fig. 12), 285, 291, 296, 305, 306, 308 — caznlboui, 304 — cruzi, 285, 295, 296 — cuniculi, 25, 26 (Fig. 11) — dimorphon, 304 — drosophilcB, 315 (Fig. 137) — dvttoni, 25, 26 (Fig. 1.1) — elyomis, 25, 26 (Fig. 11) — equinum, 285, 305 — equiperdum, 22, 26, 285 — evaniri, 26, 27 (Fig. 12), 285 — gambiense, 19, 26, 27 (Fig. 12), 285, 291, 296, 297, 303, 304, 305, 306 — granulosum, 288, 297, 298 (Fig. 129) — grayi, 304 — hippicum, 285 — inopinalum, 290 — letoisi, 19, 25, 26 (Fig. 11), 28, 263, — 286, 291. 292, 293 (Fig. 127), 297, 299, 300 (Fig. 130), 301 (Fig. 131). 305, 306, 308 — longocaudense, 294 — mega, 284 (Fig 125) — microti, 25, 26 (Fig. 11) — nanum, 27 (Fig. 12) — noctuce, 59 (Fig. 30), 137, 144, 158, 283, 297, 305, 306, 308, 391 — pecaudi, 304 — percce (myonomos), 58 (Fig. 28) — rabinmcitschi, 25 — raw§, 291, 303 — remaki, 9 (Fig. 6) — rhodesiense, 26, 286 — rotatorium, 59 (Fig. 30), 283, 297, 307 — sanguinis, 283 — vittatcB, 303 — vivax, 27 (Fig. 12), 287, 291, 304 Trypanosomos, syngamy, 136 Trypanosomidce, 268, 270 Trypanotoxin, 20 Trypanozoon, 308 Ultramicrosoopic stages, 3C6 Unciform individuals, 455 Undulating membrane, 55, 260. 286, 287, 443 Undulina ranarum, 283 Unicellular, 1, 3 INDEX 517 Urceolarince, 440 Urhsemoflagellat, 468 Urnula, 457, 480 — epislylidis, 457 (FJ8 191), 460 UrnulidcB, 460 Urospora lagidis gametes, 174 (Fig. 79) Urosporidium, 424 Urostyla, 440 Vaccinia, 470 Vacuole, 43 Vaffinicola, 440 Vampyrella, 218 — lateritia, 219 (Fig. 86) Variola, 470 Vegetative chromatin, 71 Vermiform individuals, 455 Vestibule, 433 Volutin, 68, 195, 289 Volvocidce, 267, 275 Volvox, 3, 131, 206, 267, 276 Vorticella, 440, 441, 445, 446 Vortice.Ua microstoma, 172 (Fig. 78) — inonilata, 446 VorticeUidcB, 440 Vorlicellinai, 440 Wagnerella, 48, 51, 92, 120, 245, 246, 248 - borealis, 93 (Fig. 48), 247 (Fig. 102) Wrong hosts 291 Xenophya, 34, 238 Xenophyophora, 218, 237 Yaws, 467 Yellow cells, 252 Zoochlorollw, 15, 252 Zoomyxa, 243 Zoospore, 169, 262 ZoosporidcB, 218 Zoothamnium, 440 Zooxanthcllcc, 15, 252 Zygote, 125 THE END Mnnul ImproMion. 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