AN INTRODUCTION TO THE STUDY OF THE PROTOZOA AN INTEODIJCTION TO THE STUDY OF THE PROTOZOA WITH SPECIAL EEFERENCE TO THE PARASITIC FORMS BY E. A. MINCHIN, M.A., Ph.D., F.KS. PBOFESSOR PBOTOZOOLOGY IN THE UNIVEBSITY OF LONDON LONDON EDWAED AKNOLD 1912 [Ml rights reserved] PREFACE This book, cas its name implies, is intended to serve as an mtro- 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 m form, structure, and modes of hfe. In recent years the recognition of the importance of the Protozoa to mankind in various ways, and especiaUy 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 iriore extensive than that of common language ; and with this aim in view, care has been taken to define or explain f uUy aU 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 their 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 mterests 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 acquamted 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 receivuag continually new additions, and in which most of the data are based upon an elaborate and dehcate 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 mdicating more or less decisively what is, in his opmion, the most probable solution of the problem under discussion. Such judgments, how- ever, are not mtended to be put forward in a dogmatic or polemical spirit, suace the author recogriizes fully that any conclusion now reached may be upset entirely by fresh evidence to the contrary. The vast hterature of the Protozoa would, if cited in fuU, easily fiU by itself a volume of the size of the present one. It has been necessary, therefore, to restrict the Hmits of the bibliography as much as possible, both by selecting carefully the memou's 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 m which fuU references to older works will be found ; secondly, c assica memoirs on particular subjects, also contaming, as a rule full bibhographies ; and, thirdly, such miemoirs 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 pubhshed recently by Mathis and Leger (473) covers the ground of the earlier memoirs pubhshed by these authors, which are therefore not cited; similarly, the memoir upon amoebae by Nagler (95) covers the earher work of Hartmann and Nagler upon Amoeba difloidea. 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 acquiruig complete information with regard to modern knowledge concerning aU 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 pubhcations 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 pubhshed. The present edition does not, however, profess to deal with works pubhshed later than 1911. In order to further the object of making this book a guide to the technicahties of the subject, the plan has been adopted of prmting in heavier black type in the index the numbers of those pages on which the term cited is fully 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 significaiace of a technical term, or, though necessarily to a more hmited 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 m current use, or has made additions to it. The adjective in general use relating to chromatin is " chromatic." with its various deriva- viii PREFACE tives (" achromatic," etc.) ; since, however, these adjectives have a totally different meanmg 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 Binnenkdrper, 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 bibUography 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. BagshaAve, 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. LiSTKR Institute of Preventive Medicine, Chelsea, S.W., July 1, 1912. CONTENTS CHAPTER PAGES I. Inteoductoby — The Distinctive Chakacters of the Peotozoa AND OF THEIE 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. III. The Organization of the Protozoa — External Form and Skeletal Strtjctures .... 29 — 39 IV. The Organization of the Protozoa {continued) — The Proto- plasmic Body 40 — i4 V. The Organization of the Protozoa (continued) — Diffeebntiations of 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 Ntjcleab, Apparatus — Chromatin, Nucleus, Chromidia, Centeo- somes, and Blepharoplasts .... 65 — 99 VII. The Repeoduotion of the Protozoa- - . . lOO 124 Types of Fission, 100 ; Division of the Nucleus, 101 ; Division of the Cell-Body, 122. VIII, Syngamy and Sex in the Protozoa - - - . 125 I6I 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. ix X CONTENTS CHAPTER PAGES IX. Polymorphism and Life-Cycles of the Protozoa - • 162 — 185 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. Amoebaea, 218 ; II. Foraminifera, 231 ; III. Xenophyophora, 237 ; IV. Mycetozoa, 239. B. Actinopoda— V. Heliozoa, 244 ; VI. Radiolaria, 249. XII. Systematic Review of the Protozoa : The Mastioophora 257 — ^279 I. Flagellata, 257 ; II. Dinoflagellata seu Peridiniales, 276 ; III. Cysto flagellata seu Rhyncho flagellata, 278. XIII. The H^moflagellates 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 Gregarines 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^mosporidia - - - 356—397 (1) Hajmamoebae, 357; (2) Halteridia, 365; (3) Leucocytozoa, 369 ; (4) Hsemogregarines, 371 ; (5) Piroplasms, 378 ; Affimties of the Hsemosporidia, 388 ; of the Telosporidia, 395. XVI. The Sporozoa: III. The Neosporidia - - - * ^ I. Myxosporidia, 399; II. Actinomyxidia, 409; III. Micro- sporidia, 411 ; IV. Sarcosporidia, 419 ; V. Haplospondia, 423. Incertce Sedis, 425. - 430--461 XVII. The Infusoria L Ciliata, 430 ; II. Acinetaria, 455. CONTENTS xi PAGES CHAPTER XVIII. Affinities and Classification of the Main Subdivisions — DoxTBTFUL Groups .... - 462 — 474 General Phylogeny of the Protozoa, 463. Spirochsetes, 466 ; Chlamydozoa, 470. BiBLioaRAPHY ------- 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 (chromatin) 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 (" organella "), 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 mterest 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 I 2 THE PROTOZOA cultivated zealously even by many who are not professed biologists, Avith 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 keej) 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 Avith 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 it 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 {Infusionsthierchen), used to denote the swarnas 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 stm 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 (tt/jStos) in the ascending scale of animal life or in the course of organic evolution ; the Metazoa, m which the' individual is an organism composed of many cells, come after (/xera) the simpler forms of life in rank and time. * For the detaUed history of the growth of scientific knowledge of the Protozoa, see Butschli (2), pp. i-xviii. 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 ^\•hich 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 aU cases of this kind, however, the constituent cells are morphologically equivalent, and are to be regarded as complete individuals more or less inde- pendent, sho-\ving 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, the 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 Metazoa have been evolved from simple unicellular ancestors, similar to the 4 THE PROTOZOA Protozoa, then there must heave 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 appear- 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 aU 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 " Idngdom " under the title Protista {Protistenreich, Haeckel), irrespective of their habit of life and metabolism, whether animal or vegetable. The kmgdom 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 5 understood. The term Protista thus unites under a single systematic category the vast assemblage of simple and primitive livint^ beings from which the animal and. vegetable kuigdoms have taken origin, and have developed, by a continuous process of natural evolution, in difierent dkections in adaptation to divergent modes of life. The conception of a Protistan kingdom separate from the animal and vef^etable 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 animal 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 Pro. 1 difficult problem — that, namely, of givmg a natural classification of the most primitive forms of life. Graphic representa- tion of the relation of the animal and vegetable king- doms to the kingdom of the Protista (Protisienreich). The Protozoa are represented by the portion of the triangle representing the animal ]?ingdom 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 inn atiire, 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 aiid specialization. These organisms do not conform to the type of structm-e 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 cliromatin is distributed through the proto- plasmic body in the condition of scattered lumps or granules (" clu'omidia "), and in many cases it constitutes, apparently, the whole or a very large proportion of the substance of the body. A B j'jQ. 2. — Amwha 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 miilberry- like apj)earance ; c.v., contractile vacuole ; /., faecal 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 its prey (Pi), an Infusorian {Urocentrum) ; two pseudopodia have flowed round it, as shown by the arrows, and met at the point c, enclosing the prey ; another Infusorian (P^) is seen in a food vacuole in the body ; N., nucleus ; other letters as in ^. 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 natm-e 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 oecur amongst them. Although thcur metabolism is in general distinctly of a vegetative or saprophytic ty^e, 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 diffei-entia- tion of the living substance into two quite distinct pait -the cytoplasm, or general body-protoplasm, in which is lodged at least ono 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, cliromidial condition .N. \ ■-. \\ \ \ \ u I ////// ■./ /Av X \ \ I U I / / //// / / V //7//^//ll w Fig. 3. — Actinosphwrimn eichhornii. ecL, Ectoplasm; end., endoplasm ; c.v.'^, a contractile vacuole at its full size ; c.v.^, a contractile vacuole which has just burst if.v., f.v., food vacuoles ; D., a large diatom engulfed in the protoplasm ; ps., 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 firm 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 caj)ture 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 " Fermentation " in Thorpe's " Dictionary of Applied Chemistry " (Longmans). 8 THE PROTOZOA do«cribod in more detail in the next chapter-namely, animal-like holozo.c), plant-hke (holophytie), fungus-like (saprophytic), or a 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 alga?, and fungi) and Protozoa respectively. It has been pointed out abeady, however, that this e.r- -N ?0i ^0^ If Fig. 4. — Ewjhna spi- rorjyra. oes., (Esopha- gus; si., stigma; c.r., reservoir of the con- tractile vacuole; P,P, ]5aramylum - bodies ; N., nucleus. After Stein. Fig. 5. — Trichomonas ehertJii, from the intestine of the common fowl, fll., Anterior flagella, throe in number ; p.fl., posterior flagellum, forming the edge of the undidating membrane ; chr.L, " chromatinic lino," forming the base of the undulating membrane; chr.b., "chromatinic blocks " ; U., blopharoplast f rom which all four flagella arise ; mouth-opening ; N., nucleus ax., axostylc. After Martin and Robertson. DISTINCTIVE CHARACTERS OF THE PROTOZOA 9 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 Ave are obliged to include in it some forms which are plants in every sense of the word. The systematist who deskes 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 impaii- the universal applicability of the definition given. Such difficulties arise in every attempt to apply rigid verbal definitions to natural groups of livmg 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. ii N B Fici. Ci.—Trypanosoma remaki of the pike. A, Slender form ("var. parva"). B, Stout form (" magna "). After Minchin, x 2,000. var. wm wmMmmL Fig. 7. — Gregarina polymorpha, parasite of the digestive tract of the mealworm ; ." syzygy " of two individuals attached to one another. In each individual, N., nucleus; pr., proto- morite, 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, 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 entu'e ceU- aggregate by the formation of e.v. C.V- FiG. S.—Slentor roeseZw, fully expanded. (Es., CEsophagus; N, band-like macro- nucleus ; C.V., contractile vacuole, con- nected with a long feeding-canal (/.c.) stretching down the body ; H, gelat- inous house into which the animal can retract itself completely ;/., fibres attaching the extremity to the stalk to the house. After Stein. an. Fig. 9. — Ntjclotherus cordijormis . parasite of the rectum of the frog. N, Macronucleus ; n, micronuclcus ; gr., mass of granules in front of the macronucleus; ces., oesophagus; c.v., contractile vacuole ; an., auus (cytopygc). After Stein. tissues. The body protoplasm is naked or clothed with a fii-m envelope, which is usually not of the natm^e of cellulose. Proto- plasmic organs are usuaUy present for purposes of locomotion and for the capture and ingestion of food. Cldorophyll is usually absent as a cell-constituent, and the metabolism is usually of the animal type. To these characters it may be added, though not as special peculiarities of Protozoa, that reproduction takes place DISTINCTIVE CHARACTERS OF THE PROTOZOA 11 always by some form of fission— that is to say, division of the body into smaller parts ; that the phenomena known as " syngamy " and " sex " occur, perhaps universally, throughout the group ; and that it is very characteristic of Protozoa, as compared with other Protista, to exhibit in their life-history a develop- mental cycle, more or less complicated, in. the course of which the organism may appear under very different forms at different stages in its develop- ment. The Protozoa, as thus under- stood, are commonly divided into four main subdivisions, termed "classes." Other methods of classifying the Protozoa have been suggested, which will be considered later ; for the present the old- established subdivisions are sufficient for our purpose. Class I., Sabcodina.* — Protozoa in which the proto- plasmic body is naked or non- corticate — that is to say, without a hmiting envelope in the form of a cuticle, membrane, or stiff cortical layer ; consequently the body tends to be either more or less spherical in floating forms, or to have an irregular, con- tinually changing shape in c reeping forms . 0 rgans serving for locomotion and capture of food are furnished by tem- porary extensions of the Uving protoplasm, termed pseudo- podia. A skeleton or shell may be present. Examples Fig. 10. — Acineta grandis. sL, Stalk ; lli., theca ; s., suctorial tentacles. After Saville Kent. The name is derived from sarcode, the tor m coined by Dujardin to denote m now 12 THE PROTOZOA are Amoeba (Fig. 2), Difflufjia (Fig. 16), AclinosphcBrium (Fig. 3), etc. Class II., Mastigophora.* — Protozoa in which the organs of locomotion and food-capture in the adult are flagella, slender fila- ments which are capable of performing active whip-Uke, lashing movements. The body-protoplasm may be naked or corticate. Examples are Euglena (Fig. 4), Trichomonas (Fig. 5), Trypanosoma (Fig. 6), etc. Class III., Sporozoa. — Protozoa occurring always as parasites of other organisms, and without definite organs for locomotion or ingestion of food in the adult condition. The reproduction takes place, typically, by formation of resistant seed-like bodies, termed spores, containing one or more minute germs, termed sporozoites. Examples are Gregarina (Fig. 7), Coccidium (Fig. 152), the malarial parasites (Fig. 156), etc. Class IV., Infusoria. — Protozoa in which the organs of loco- motion and food-capture- are cilia, small vibratile filaments dis- tinguished from flagella by their smaller size, by differences in their mode of movement, and by being present usually, in primitive forms at least, in great numbers like a fine fur over the whole or a part of the surface of the body. The cilia may be present tlxrough- out life (subclass Ciliata), or only in the early stages of the life- history (subclass Acinetaria). The body -protoplasm is always cor- ticate. Examples are Stentor (Fig. 8), Nyctotherus (Fig. 9), Acinela (Fig. 10), etc. Bibliography. — For a list of general works on Protozoa, see p. 476. • * Derived from the Greek juao-ri^, a whip, equivalent to the Latin flagellum. CHAPTER II THE MODES OF LIFE OF THE PROTOZOA Protozoa, as simple protoplasmic organisms, can only exist in an active state in a fluid medium. Hence the free-living, non-parasitic forms are aquatic, either marine or fresh-water in habitat. A certain number of species, however, are semi-terrestrial in their mode of life, creeping on damp surfaces or living in a minimum of moisture. Examples of such forms are the Amoebae, etc., found in the soil, or Mycetozoa, which in the plasmodial phase (p. 239) creep on tree-trunks, logs, and so forth. None of these forms,, however, can remain active in perfectly dry surroundings, but pass into a resting state when desiccated. It has been stated already that the methods by which Protozoa gain their livelihood vary greatly in different cases. Considered generally, these methods may be classified under four types : I. The majority of Protozoa nourish themselves after the manner of animals — that is to say, they are entirely dependent for food and sustenance on other organisms which they capture, devour, and digest. Such forms are said technically to be holozoic, a word sig- nifying " completely animal- like "; they are unable to utilize simpler chemical substances in order to build up the protein constituents of the living body, but require proteins ready-made for their sustenance. II. A certain number of Protozoa— all, with rare exceptions, belonging to the class Mastigophora — possess in their body-sub- stance peculiar colour-bearmg corpuscles, so-called chromatopJiores or chromoplasts, containing chlorophyll or a pigment of allied nature, by means of which they are able to decompose carbon dioxide in the sunlight, hberating the oxygen and making use of the carbon m order to build up the protein and other constituents of the hving body. Such organisms are entirely similar in their metabohsm to the ordinary green plants, and are hence termed holophytic or ' completely plant-Uke." nJ^r>/'?J,°^^^^'^''°"'^'J'??'^'? '^^'''^ chlorophyll-bodies form an integral lu jL ? body, and are to be regarded simply as proto- plasmic organs, must be distinguished carefully from a state of things often 13 14 THE PROTOZOA found in holozoic Protozoa of all classes— namely, the presence in the body substance of symbiotic independent organisms of vegetable nature, as described below. III. A certain number of Protozoa that have no chlorophyll or similar pigment in their bodies are, nevertheless, free from the necessity of preying upon other organisms in order to obtain their sustenance, since they are able to live upon organic substances in solution, the products of the metabolism or decay of other living organisms. Such forms are termed saprophytic (or saprozoic). since their mode of Uf e is similar to that of a saprophyte, such as a fungus. It is not necessary that they should be supplied Avith ready-made proteins in their food, since they are able to build up their protein constituents from substances of simpler chemical nature. Many examples of saprophytic forms are found amongst the free-hving Flagellata. Lauterborn (17) has coined the useful term sapropelic (from the Greek Trr]X6s, mud) to denote a mode of life which must bo regarded as a special type of the saprophytic method, partly also of the holozoic — namely, the mode of life of those fresh- water organisms that live in a mud or ooze composed almost entirely of the decaying remains of dead plants and other debris of a similar nature. A very characteristic fauna occurs under these conditions. IV. Finally, many Protozoa of all classes live as parasites — that is to say, at the expense of some other living being, which is termed the host. These four modes of life can be used only to a very limited extent for classificatory purposes ; it is only possible to do so in those cases where a particular habit of life, long' continued, has resulted in definite structural characteristics, and more especially in the loss of organs requisite for other modes of life — as, for example, in the case of the subdivision PhytoflageUata, of the order FlageUata, where the holophytic habit has become so ingrained that only structural features proper to vegetable life are retained. In other cases it is clear that a given habit of life in different organisms does not necessarily indicate close affinity between them. In the first place, we find closely allied forms living in different ways. Examples of all the four methods of metabolism described above are to be found in the single order FlageUata, and through- out the Protozoa there are commonly to be found parasitic forms closely allied to free-living forms. In the second place, different types of metabolism may be found as transitory phases in the life of one and the same individual or species. Thus the common Euglena (Fig. 4), a flagellate possessing chromatophorcs and living normally in a holophytic manner, is able to maintain itself as a saprophyte if deprived of the sunlight necessary for a holophytic mode of life (p. 188) Striking examples of variability m the mode of nutrition are seen also in the section Clirysomonadma of THE MODES OF LIFE OF THE PROTOZOA 15 the Flagellata, where a given species may be either holozoic or holophytic,* according to circumstances. The bionomics of Protozoa — that is to say, their relations to their environment and to other organisms— constitute a very important branch of knowledge, both practical and theoretical, especially in the case of parasitic forms. Considering the subject from the point of view of the four modes of life already described, it is clear, in the first place, that the holophytic forms are entirely independent of all other living organisms, and require for their continued existence only sunlight and a suitable environment, containing the necessary inorganic substances, at a temperature which permits the continuance of vital processes and activities. Saprophytic organisms, however, in so far as they require for their sustenance materials produced by living bodies, are dependent directly or indirectly upon other organisms for their existence. Purely holozoic forms, also, cannot exist without other forms of life upon which, or upon the products of which, they can feed. But neither holozoic nor saprophytic organisms are dependent, as a rule, upon any other particular form of life, but only upon living things generally ; though in some cases such forms may be specialized in their nutrition to such an extent as to be unable to exist without some particular food. A parasitic form, on the other hand, is entirely dependent, as a rule, for its existence on some particular organism or limited group of organisms which constitute its host or hosts. It must, however, be understood clearly that an organism living in or upon the body of another organism is not necessarily a parasite by any means. In the first place, a distinction must be drawn between parasitism and symbiosis, by which is meant an association of two organisms for mutual benefit. j Good examples of symbiosis are seen in some of the Sarcodina, Radiolaria, and Foraminifera, the proto- plasm of which contains constantly intrusive organisms, known as zoocMorellce or zooxanthellce, according as they contain a green or a yellow pigment. Zoochlorellse are green algae of the order Proto- coccacece; zooxanthellse are holophytic flagellates of the suborder Cryptomonadma— e.(/., Cryptomonas schaudinni, symbiotic in the foraminifer Peneroplis (Winter, 28). These organisms penetrate * For example, the species GAromzi/ma ^amcaws. See BiitscWi (2), vol. ii. p 865 T liie term symbiosis " is often much misused, especially by medical writers, by wliom it is commonly applied to any association of two distinct organisms • Xro r"''' '^^''^ "^'""^'^ 'i''^*."'^' " ^i*^ so^ie species of bacillus "svmwll'^.U^'' '"I'/rP^^ ^"-^ spoken of as ciation Tf'=l, rv?^ ^^/'^'}t^g« 13 clearly only on one side in such an asso- o iZ^nidt? , ,^indorstood that the term " symbiosis " is a technical term n fcanofnf^ ^^^fl' ^"^.V' ''^"'^ '"^''''1^ etymological sig- toScthor fl ther," but in tlie special and restricted sense of "living together for the mutual benefit of the two organisms concerned." 16 THE PROTOZOA into the body of their host, lose their flagella, and nourish them- selves by means of their pigment, which has the nature and proper- ties of plant-chlorophyll ; that is to say, it decomposes carbon dioxide in the sunlight and liberates oxygen. The carbon dioxide is obtained from the respiratory processes of the host, which in its turn utilizes the oxygen produced by the symbionts (p. 197), and thus each organism supplies the needs of the other. When the host enters upon its reproductive processes and breaks up into a vast number of swarm-spores, the symbionts develop flagella and swim o£E, doubtless to seek for lodging elsewhere. It is a matter of convenience to distinguish as epizoic those organisms which live upon, or are attached to, and as entozoic those which live within, the body or substance of the particular form of life with which they are associated. Epizoic forms may be entirely harmless to the creature upon which they occur ; they may simply utilize its body as a coign of vantage where they readily obtain their food, which may consist in some cases of nutritious substances dropped or reje6ted by the animal that carries them ; or they may obtain the benefits of shelter or transport, especially when the epizoic form in question is itself of sedentary habit. Every naturalist is acquainted with the sea-anemones that live habitually upon hermit-crabs, probably to the advantage of both animals — at all events, to the detriment of neither. There are many similar cases among Protozoa. The appendages of Crustacea, especially of the Cladocera and Copepoda, are often thickly beset with sessile Vorticellids and Acinetaria, which obtain a convenient lodging, but provide their own board. Other forms occur similarly on the stems of hydroids, as, for example, Acineta papilUfera on Gordylophora lacustris. Amoebae are found creeping on the exterior of Calcareous Sponges, nourishuig themselves on diatoms and other organisms. Similar instances could be multiplied indefinitely. On the other hand, epizoic forms may be dangerous parasites, nourishing themselves at the expense of the animal they infest, and sometimes inflicting much damage upon it. It can be easily understood that an epizoic form which at first lived harmlessly upon some animal, drawing its supplies of food from the surrounding medium might acquke the habit ultimately of obtaining its nourish- ment from the living substratum upon which it has planted itself. Examples of epizoic parasites are the flagellate Gostia necatnx (V 272) and the ciliate IcUlnjopUhirius muUifihis (p. 450), both ot which are epizoic parasites of fishes, attachmg themselves to the skin and destroying the epidermis ; as a result, the way is left open for fungi and bacteria to penetrate the skin, and so produce ulcera- tion and suppuration, which may be fatal. -.^^^^^ AH certain instances of Protozoa acting as external parasites are THE MODES OF LIFE OF THE PROTOZOA 17 found amongst aquatic animals, and it can bo readily understood that a delicate protoplasmic organism could only pass from one host to another in a fluid medium, or by the help of special mechan- isms adapted to aerial transport or transmission by contact. It should be mentioned, however, that some human contagious skin- diseases arc suspected to be due to the agency of parasites of the nature of Protozoa.* Like the epizoic forms, there are many entozoic Protozoa which inhabit the bodies, and especially the intestines, of other animals, but Avhich are in no way to be regarded as parasites ; they feed merely on various substances to be found there, such as waste particles of food, excreted or faecal matter, or on other organisms, such as bacteria, yeasts, and the like — in short, on substances which from the point of view of the host are superfluous, or even harmful. Many examples of such organisms could be cited ; a good one is the common Ghlamydophrys stercorea, found in the faeces and digestive tract of man and many animals. The common intestinal flagellates belonging to the genus Trichomonas (Fig. 5) and other genera are, similarly, not to be regarded as true x^arasites in any sense of the word. The common Lophomonas blattarum (Fig. 45) from the intestuae of the cockroach feeds chiefly upon bacteria and yeasts. Many of these intestinal Protozoa are perhaps useful, rather than harmful, to their hosts. On the other hand, the vast majority of organisms. Protozoa or otherwise, that live in the interior of other living creatures are there for no good or useful purpose ; their habitat is alone sufficient to render them suspect. Two modes of parasitism may be distin- guished from a general point of view. On the one hand, the para site may merely intercept the food of the host and rob it of its sustenance. On the other hand, the parasite may nourish itself upon the living substance or vital fluids of its host. Organisms which rob the host of its food may do so in one of two ways. They may appropriate the raw food-material, which they then ingest and devour after the strictly holozoic method of feedint^ • examples of this mode of life are possibly to be found in the extensive nofusorian fauna to be found in the stomachs of ruminants Or they may absorb the fluid products of the digestion of the host by diffusion through the sm-face of the body of the parasite ; examples ot this mode of parasitism are to bo seen, probably, in the case of the Gregarmes so common in the guts of insects. Parasites of tho * For example the so-called Coccidioides immilis, a name given to bodies found in certain &outh Amencan skin diseases ; see Blanchard (033f p 1(S Molh so^ m con agu^sum has also been attributed to parasites referred by some to the Sozol "om e5"°thrrnrS^f^ ""-^^^ the^Karasi^e bodi st foi- toThe S amytJSl a contagiosum should probably be referred 18 THE PROTOZOA Ij'pe that may bo denoted as food-robbers are in general very harmless to their hosts. Those parasites, however, that liourish themselves on the sub- stance of the host may produce the most dangerous effects on its health and weU-being. As in the case of the food-robbers, parasites of this kind may absorb then- food in one of two ways. They may devour solid j)ortions of the host's body in a holozoic manner ; an example of this is seen in Entamoeba histolytica (Fig. 90), the parasite of amoebic dysentery, which devours portions of the host's tissue, such as epithelial cells, or blood-corpuscles. But more usually the parasites absorb then nourishment in a fluid form tlnrough the surface of their body, doubtless by the help of enzymes secreted by them. Hence it is typical of true parasites to have lost all trace of special organs for the capture, ingestion, or digestion, of solid food. Just as in the epizoic mode of life a harmless or even beneficial commensalism may degenerate by insensible gradations into dangerous parasitism, so the same is true of the entozoic habit. An organism which begins by being a scavenger readUy becomes a food-robber. Lophonionas, for instance, may be seen to contain starch-grams and other substances which probably belong to the food of its host. A fm-ther but easy gradation leads to the entozoic organism devoming portions of its host. A good example of this is seen in two of the entozoic amoibaj of the human intestine : the common Entamoeba coli (Fig. 89) appears to be chiefly a scavenger, harmless to its host, and not deserving the reproach of parasitism ; on the other hand, E. Ustolylica is a dangerous parasite. So also an entozoic organism, Avhich begins by merely absorbing the pro- ducts of digestion, may end by absorbing the substance of its host. It is higlily probable that in many entozoic organisms the mode of feeding may vary according to circumstances, and that an organism which may be a harmless commensal under some conditions may become a more or less dangerous parasite under others. The entozoic Protozoa which are truly parasitic may inhabit a variety of situations in the bodies of their hosts. In some cases the host is another species of Protozoon, into the body of which the intruder penetrates, living either in the cytoplasm or the nucleus. Amosbaj are very subject to the attacks of intranuclear parasites, and the young stages of many Acinetans are parasitic upon other Infusoria When the host is one of the Metazoa, the invading organism may be in like manner mtracellular or intranuclear in habitat ; or it may penetrate into the tissues, living amongs and between the constituent cells ; or it may inhabit, finally, one of the internal cavities of the 'body, such as the digestive tract genera iody cavity, spaces containing blood or lymph, cavities of the renal THE MODES OF LIFE OF THE PROTOZOA 19 or ui-iuary organs, etc., cither living free iu the cavity it inhabits, or attached to the linmg epithelium. As diverse as the modes of iDarasitism among Protozoa are the elTects they produce on then' hosts. Some parasites produce no perceptible disturbance in the well-bemg of then- host ; oven when they destroy cells and portions of tissues, the damage may be slight, and is quickly made good without appreciable permanent uajury being done. From this condition of more or less perfect harmless- ness there is a continuous gradation in the ascending capacity for harmfulness, culminatuag m species which bring about the death of then- hosts with greater or less rapidity. Hence parasitic Protozoa are commonly distmguished as pathogenic or non-pathogenic ; but smce there is no precise limit to the degree of sickness or indis- position which justifies the application of the term " pathogenic," it is perhaps more convenient to distinguish them as lethal or non- lethal. It is not possible, however, to lay down hard-and-fast distmctions in these matters, since a parasite which is not lethal under some ckcumstances may become so under others ; for instance an animal living a free and natural life may be quite well able to resist the attacks of parasites to which it succumbs in captivity Moreover, it must be borne in mind that such terms as "lethal " or " pathogenic " can only be applied to a parasite in its relation to a particular host, smce, as wUl be shown below, a parasite which is harmful to one host may be harmless to another. It is far fi-om clear in what way the pathogenic effects of parasitic Protozoa are produced. If the action and reaction of host and parasite were relations dependent simply on the number or relative total bulk of parasites present in a given host, the problems of parasitism would be comparatively simple ; but in many cases this IS obviously very far from being the case. The effect produced bv a given species of parasite upon a given species of host is a specific «sthf ''^""-'^'y one of the two .rat ZlZe t^o^f^^ 1 " "^^ommon to find insects with their digestive ract or body-cavity crammed with parasitic Gregarines of relatively large size, but apparently none the worse for T On the other hand, large mammals may succumb to the Jln^. f minute pai-asites in relatively scanty Lmbers-in^l^^se i^^^^^ or hi a run;;:ii:^^;,\^ tz^::^:::' r r - -c., the trypaLii -:;z ^ti;!^ 20 THE PROTOZOA difficult to detect.* These facts suggest strongly that the parasites produce specific toxins ; but the " sarcocystine " produced by para- sites of the genus Sarcocystis (Sarcosporidia)| is almost the only case up to the present, in which a toxin has been isolated from a Pro- tozoan parasite. Laveran and Pettit (19), however, claim to have obtained " trypanotoxins " from trypanosomes. Considering the facts of parasitism generally, as a problem of natural history, two guiding principles must be borne m mind clearly : the first is that any organism, parasitic or otherwise, tends to be adapted in the best possible manner to the circumstances of its natural environment ; the second is that, so long as a parasite is entirely dependent on its host for its existence, it is to its utmost disadvantage to bring about the death of its host. When, therefore, a given parasite is constantly lethal to a particular host or hosts, one of two explanations must be sought for : either the case is one of a disharmony — that is to say, of imperfectly-adjusted relations between the host and parasite ; or the parasite must obtain from the death of the host advantages in the matter of the continuance of the species sufficient to compensate for the temporary loss through destruction of mdividuals. The conditions to which a parasite requires to be adapted are different in many ways from those that influence the life of a free- living organism. When once a parasite has obtained a footing in its proper host, the problem of food-supply is solved for it, smce it finds itself lodged in the midst of abundant nutriment so long as its host lives. On the other hand, if the species is to be main- tained, it is essential that the parasite should be able to infect new hosts, a difficult undertakuig, and one iia which the chances are all against the parasite in most cases. To insure dissemination of the species a large number of offspring must be produced, and special mechanisms and adaptations may be necessary to insure thek reaching then- destmation. Hence, the more parasites become specialized and adapted to thek peculiar mode of life, the more the organs and functions of nutrition tend to become simplified, and the greater the tendency to elaboration and extreme fertility of the reproductive function. Considered generaUy, a parasitic Protozoon reproduces itselt within a given host with one of two results : in the first place, with that of overrunning the host and establishing itself there ; in the second place, with that of producing forms destined to infect new hosts Forms produced in the fii'st manner may be termed the " multiplicative phases " ; theii- function is to produce a stock of the parasite. From the stock are given off what may be termed * Coraware Laveran and McsnU (391), pp. Ifli-l^O. t Lavemu and Mesnil (18) ; Teichmann (25) ; Teichmann and Braun (26). THE MODES OF LIFE OF THE PROTOZOA 21 tho " propagativo phases," which as a rule do not multiply further in tho host in which they are produced, but await their chance of being transferred to a new host ; and if such a chance be not given to them, they die off and are replaced by fresh propagative forms from the stock (see further beloAV, Chapter IX., p. 166). So long as the nutritive or multiplicative function is tho most important one in the life of a parasite, and until it has matured its propagative phases, the death of the host is the greatest disaster that can befall it. The ideal host, from the point of view of a para- site, is one that is " tolerant " to it — that is to say, one that can support the presence of the parasite and keep it supplied with the nutriment it requires, without suffering in health or vigour to any ■"marked extent. When once, however, the parasite has made the necessary provision for propagating the species, the life or death of the host may become a matter of indifference to the parasite, or may even in some cases be necessary for the dissemination of the offspring. This will be apparent from a consideration of the methods by which parasitic Protozoa infect new hosts. The passage of a parasite from one host to another includes two manoeuvres : the passing out from the first host, and the passing into the second. Primitively it may be supposed that tliis migra- tion was effected simply by the unaided efforts of the parasite itself — that is to say, that the active motile parasite would force its way out of one host, move freely in the surrounding medium, and sooner or later attack and penetrate a fresh host. This primitive method of transference doubtless occurs in many cases, especially amongst epizoic forms {e.g., IchthyophtMrius, p. 450). In the case of entozoic parasites its occurrence is less common, but it is found in a certain number of cases. The young stages of many Acinetaria, parasitic in Ciliata, probably seek out their hosts and penetrate uato them ; after a period of juvenile parasitism they leave the host's body and become free-living, non-parasitic organisms. Active migration of this kind, however, is very rare amongst entozoic parasites. In the first place, the conditions of life within a living body, in the midst of organic fiuids, are so different from those in the open water, whether salt or fresh, that it is hardly to be expected that a delicate unicellular organism adapted to the one mode of life could stand the sudden change to tho other. In the second place, it is clear that active migration of parasitic Protozoa could only be effected Avhen the host js an aquatic animal, and not when it leads a terrestrial life. The only instances of active migration known with certainty to occur m the case of Protozoa parasitic on terrestrial animals are those in which tho parasite can penetrate a mucous membrane and is thus able to pass from one host to another when two such surfaces are m contact. In this way the trypanosome of dourine in horses 22 THE PROTOZOA {T. equiperdum) passes from one host to another during coitus, and the transmission of the parasite of syphilis is another instance. Speaking generally, and excluding for the moment those cases in which the transmission is brought about by means of an inter- mediary host, the propagative phases of the parasitic Protozoa take the form of inactive, resting stages in which the body of the parasite is protected against adverse external conditions by tough protective membranes. In the form of resistant cysts or spores, the parasites in a dormant state offer a passive and inert resistance to the elements ; they are disseminated like seeds, and they ger- minate when they reach a suitable environment, but not till then. Many, perhaps the majority of parasitic Protozoa, occupy posi- tions in the body of the host whence the propagative phases can pass without difficulty to the exterior. This is the case when the para- site is lodged in organs which have ducts or passages leading directly or indirectly to the exterior — such as, for instance, the digestive tract and its dependencies, or the urinary organs and ducts. In all such cases the propagative stages of the parasite pass harmlessly to the exterior. The host may in this manner get rid entirely of its parasites, without, however, necessarily acquiring immunity to- fresh infections ; or, on the other hand, the parasite may keep up its numbers in the host by continual multiplication to produce a stock from which are sent forth incessant relays of the propagative phases destined to infect new hosts. In the majority of parasitic Pro- tozoa the relations to the host are of this type, and the parasites are neither lethal nor pathogenic to any great extent. On the other hand, there are many instances in which parasitic Protozoa occupy a position in the body of the host whence escape by anatomical channels is not possible. This is the case when the parasite inhabits some closed space in the body, such as the coelome or general body-cavity, or the blood-system ; or when it attacks deeply-situated cells or tissues of the body. In some cases where natural means of exit from the body occur, they may be unsuitable for the dissemination of the parasite, as in the case of those forms parasitic in the genital organs of one sex of the host. In cases of this kind there are at least six known methods whereby parasitic Protozoa are disseminated and transferred to fresh hosts. 1. The resistant stages of the parasite may be set free by the death and decay of its host. This appears to be the manner in which some of the tissue-infecting parasites of the order Myxo- sporidia, especially the family Myxobolidce, are disseminated ; they are for the most part parasites of fishes, and are often very deadly in their effects. 2. The parasite may cause tumours and ulcers, Avhich suppurate, and so set free the cysts or spores of the parasite. This, again, is THE MODES OF LIFE OF THE PROTOZOA 23 an effect often produced by tissue-parasites, such as the MyxoboUdce, or by species of Microsporidia. In such cases also the parasite is pathogenic to its liost, and frequently lethal. 3. The parasite remains in the host until the latter is eaten by some animal which preys upon it. The propagative phases of the parasite are able, however, to resist digestion by the animal that has devoured their former host, and pass unaltered tlirough its intestine, to be finally cast out with the dejecta. This is almost certamly the method by which the common Monocystis of the earth- worm infects its host. The parasite produces resistant spores in the worm ; the worm is eaten by a bu-d, mole, frog, or some other animal, through the digestive tract of which the spores pass un- altered ; they are scattered abroad with the fseces, and may then be swallowed by another earthworm, in which they germinate and produce an infection. 4. As in the last case, the host, together with its parasites, is devoured by some animal, in which, however, the parasite is not merely carried passively, but again becomes actively parasitic. Hence in this case there is an alternation of hosts, one of the two hosts becoming infected by devouring the other. This mode of infection, which is well known to occur commonly among parasitic worms, such as Cestodes, is probably also frequent among Pro- tozoa ; but at present only two cases of it are known with certainty. One is that of the species of the genus Aggregata {vide infra, p. 353), parasites of crabs and cephalopods, such as the cuttle-fish and the octopus. In the cephalopod the parasite forms resistant spores which pass out with the faeces, and may then be devoured by crabs. In the crab the spores germinate and give rise to a second form of the parasite, which lives and multiplies in its new host. If, as fre- quently happens, the crab is eaten by a cephalopod, the parasite completes its life-cycle by becoming once more a parasite of the cephalopod. Another case is that oi Hcemogregarina muris in the rat-mite (p. 376, infra). 5. The Protozoa parasitic in the blood of vertebrates are dis- seminated by blood-sucking invertebrates, such as leeches, ticks, or insects, which take up the parasites by sucking the blood of an infected animal. Later on the parasite may be inoculated into a second vertebrate host by the invertebrate when it sucks blood at a later feed. In some cases the transference of the blood-parasite may be effected in a purely direct and mechanical manner by the invertebrate, but in most cases the invertebrate plays the part of a true host, in which the parasite multiplies and goes tlirough a cycle of development. Hence in such cases also there is an alternation of hosts and a complicated life-cycle, of which the life-history of the malarial parasite is a good example {vide infra, p. 359). It 24 THE PROTOZOA need only be noted hero that in such cases resistant spores or cysts become unnecessary and superfluous for the propagation of the parasite, and tend to disappear from its developmental cycle. 6. In some cases the parasite may penetrate the ovary of its host, pass into the ova, and thus infect the embryo and the next genera- tion. Transmission of this kind is known in a certain number of cases ; it is never the sole method of transmission, but is always supplementary to other methods. For instance, in " pcbrine " of silkworms, caused by Nosema bombycis, the spores of the parasite arc liberated in the ordinary way from the caterpillar either with the faeces or by its death, and are then eaten accidentally on the leaves by other silkworms ; but a certain number of the parasites pene- trate into the ovary and form spores, which pass through the pupal and imaginal stages of the host into the next generation of silk- worms, which are born infected. In this way the parasite is able to tide over the winter season, when the ordinary method of infec- tion would be impossible. The blood-parasitea of the genus Piroplasma (p. 384, infra) afford another example of germinative infection in the ticks which transmit them. To turn now to the methods by which parasitic Protozoa pene- trate into new hosts ; there are four known methods, which, after what has been said, can be summarized very briefly. The com- monest is the method of casual or contaminative infection, where the host infects itself accidentally by taking up the propagative phases of the parasite from its surroundings — most usually by way of the mouth, with the food, but it may be by way of the respira- tory organs. Other modes of infection are the contagious, as in dourine, already mentioned ; the inoculative, as in malaria and other diseases caused by blood-parasites ; and the so-called " heredi- tary " or " germinative " method, as in Nosema homhijcis and other cases. From the foregoing summary of the methods by which parasitic Protozoa are propagated from one host to another, it is clear that there are very few cases m which it is of direct advantage to the parasite to cause the death of its host. Even where it is necessary, for the propagation of the parasite, that the host should be destroyed by some other animal, as in the case of the Monocystis of the earth- worm, the interests of the parasite are not furthered, and may, indeed, be damaged, if it cause disease or death to the host. In the case of blood-parasites, transmitted by the inoculative method, it may be necessary for the propagation of the parasite that the required phases should be sufficiently abundant in the blood of the vertebrate host to insure the invertebrate host becommg infected when it sucks the blood ; then large numbers of the parasite may be detrimental to the well-being of the host to a greater or less extent, THE MODES OF LIFE OF THE PROTOZOA 25 and one interest of the parasite may, so to speak, clash with another. But in all cases alike it is perfectly clear that the death of the host before the parasite has matured its propagative phases leads simply to the extirpation of the parasite, and is a suicidal policy on its part, a glaring disharmony in Nature. This conclusion is borne out by a general survey of the facts of parasitism in the Protozoa, since the vast majority of these parasites are quite harmless to their hosts, and lethal parasites, greatly in the minority when compared with harmless forms, must be considered as exceptional and aberrant types of parasites, from a general point of view. The parasitic Protozoa of lethal properties present a problem which can be best attacked by considering and comparing two cases of closely allied parasites, the one harmless, the other lethal, to their hosts. Very instructive cases of this kind are furnished by trypanosomes {vide infra, p. 285). The common parasite of the rat, Trypanosoma lewisi, is perfectly harmless as a rule to its host, and the infection runs a very definite course. When the parasite is introduced into the blood of a healthy and susceptible rat, it enters at once upon a period of rapid multiplication, which lasts about twelve days. At the end of that time the parasite swarms in the rat's blood, without perceptibly affecting its general health. After about twelve or thirteen days the multiplication of the parasite ceases entirely ; the swarming period lasts generally about a month, and after that the parasites begin steadily to diminish and dis- appear, until after a variable length of time, usually three to five months, the blood is quite free from them, and the rat, cured from the attack, is now quite immune to the parasite, and cannot be infected by it a second time. The behaviour and effects of a pathogenic trypanosome, such as T. brucii, when introduced into a rat's blood, contrast sharply with that just described. Not only do the trypanosomes begin to multiply at once, but they never cease to do so while the host remains alive. By the fifth or sixth day there are practically more trypanosomes in the blood than blood-corpuscles, and the death of the host soon follows when this stage has been reached. Trypanosoma lewisi is a type of a well-marked group of try- panosomes, which may be conveniently denoted the lewisi-gron-p (Fig. 11). Such are T. cuniculi of the rabbit ; T. duttoni of the mouse ; T. rdbinowitschi of the hamster ; T. blanchardi of the dor- mouse ; T. microti of Microtus arvalis ; and T. elyomis of the lerot {Eliomys quercinus). All these species of trypanosomes are ex- ceedingly similar in their appearance and structure ; each species, however, appears to be perfectly specific to its particular species of host. The trypanosome of the rat, for instance, will not flourish in any other host, not even in a mouse, under normal circumstances. 26 THE PROTOZOA Roudsky suggests thcat all this group of trypanosomes constitutes in reality a suigle species ; in any case, it is reasonable to regard them as forms recently evolved from a common ancestor, incipient species which have not advanced beyond the stage of phvsiolo<^ical differentiation. In like manner, T. brucii is a type of a group of trypanosomes which may be termed the 6mcu"-group (Fig. 12) ; other members of it are T. gamhiense, the parasite of human sleeping sickness ; T. evansi, causing surra in horses ; T. equiperdum, of dourine in horses ; and several other species. These forms also are exceedingly similar in appearance and structure, though easily distinguishable from members of the lewisi -grouip. They are all of them very lethal, as a rule, to their hosts ; and they differ further from the try- FiG. 11. — Trypanosomes of the leivisi-growp. A, T. lewisi (rat) ; B, T. duiloni (mouse) ; C, T. cuniculi (rabbit) ; D, T. microti {Microtus arvalis) ; E, T. elyomis (Eliomys quercinus) ; A and C, from preparations ; B, after Thiroux ; D, after Laveran and Pettit; E, after Franca. All figures magnified 2,000 diameters. panosomes of the lewisi-gron-p in the fact that a given member of the 6rMcn-group is not specific to a particular host, but can flourish and exert its lethal powers in a great variety of vertebrate hosts — a fact which, coupled with their very similar morphology, renders the exact determination of the species of this group very difficult, and often a matter of controversy. All these facts point to the irwm-group being also descended from a common ancestral form ; they may be regarded as incipient species in which the process of evolution has not yet the degi'ee of physiological speciaUzation reached in the lewisi-gron^. This view receives support from the fact that a new race or species of the &racn'-group has been made known this year (1911)— namely, T. rJiodesiense, a trypanosome pathogenic to human beings which appears to have come into existence as a species very recently. THE MODES OF LIFE OF THE PROTOZOA 27 A further point of great interest in this connection is that T. hrucii in Africa appears, from the observations of Bruce, to occur as a natural parasite of Avild game, and to be as harmless to these its natural hosts as T. lewisi is to rats. The physiological difference between these two species is that T. lewisi is perfectly specific to its natural host, whereas T. hrucii is capable of flounsh- ing in others, with most deadly effects. Hence the pathogenic properties of T. hrucii would appear to be exerted on hosts to which Fro. 12.— Trypanosomes of the Irucii-gvoxcp. A, B, C, T. hrucii of " nagana," three forms— slender, intermediate, and stumpy ; D, E, F, T. gamUense oi sleeping sickness, the three corresponding forms ; G, H, T. evansi of " surra," two forms ; I, T. vivax ;J,T. nanum. A to C, I, and J, after Bruce, Hamerton, Batoman, and Mackie (411); G and H, after Bruce (404); D to F. from preparations. All figures magnified 2,000 diameters. it is a new parasite, and not on those to which, like T. lewisi, it has established harmonic relations in the course of evolution. The pathogenic properties of T. hrucii, and doubtless of other similar forms, may from this point of view be characterized as a disharmony associated with the first steps in the origin of species. The problem of the origin of diseases caused by parasites is essentially a problem of the same nature as the origin of species. The first step in the formation of new species is a process of varia- tion in an established species. Similarly, in the process of forma- 28 THE PROTOZOA tion of now species of parasites, the first step would be the acquisi- tion by the parasite of the power of living in hosts other than that to which it is specific. How such a variation might arise in Nature is impossible to conjecture in the present state of knowledge ; but some experiments that have been carried out upon T. lewisi show that conditioiis can modif}^ thn apparent fixity of its characters. Roudsky (22, 23) found that after prolonged culture on artificial media, and subsequent rapid passages tlirough rats, it was possible to infect mice with T. leivisi. Wendelstadt and Fellmer (27) have shown that T. leivisi, if inoculated into cold-blooded vertebrates, can persist there for a time, and then becomes virulent to rats.* In both cases it is evident that the normal specific properties of the parasite have been induced to vary by changes in the conditions of life, with the result that they become similar to those characteristic of the pathogenic trypanosomes. If it be true that a parasite attacking a new host is at first patho- genic to it, but tends in the course of evolution to establish more harmonic relations with the host, the question arises as to how such relations are brought about. There are two organisms con- cerned, and the problem affects them both. In the case of the host the adaptation to the effects of the parasite may be both individual and racial, in the latter case to be perhaps largely ex- plained by the elimination of indi^'iduals less fitted by their con- stitution to resist the parasite. In the case of the parasite, also, the problem may be considered from both points of view ; deadly strains of the parasite contribute to their own destruction. Interesting observations bearing on the individual adaptability of strains of Schizotrypanum cruzi have been made by Chagas (425). This para- site, when inoculated into guinea-pigs, was found to kill them in about six days ; this is its initial virulence to this host. After repeated passages through guinea-pigs, it Avas found that the viru- lence diminished, until guinea-pigs inoculated with strains of attenu- ated virulence lived as much as six weeks before they succumbed to the effects of the parasite. If, when this result had been attained, the parasite was given a single passage tlirough a marmoset, it was then found to have regained its primary virulence to guinea-pigs. The study of the exact mechanism of the physiological relations between parasites and their hosts is the task of the investigations upon immunity and kindred problems which now engross so large a share of the attention of scientific workers, but which cannot be considered here in detail. BibliograpJiy. — For references, see p. 476. * See also Sleeping Sickness Bulletin, No. 22, p. 412, and No. 24, p. 81. CHAPTER III THE ORGANIZATION OF THE PROTOZOA— EXTERNAL FORM AND SKELETAL STRUCTURES A UNICELLULAR organism of any kind is a more or less minute mass or corpuscle of the living substance, protoplasm, containing usually other substances, fluid, solid, or even in some rare instances gaseous, in greater or less amount — substances which are either the product of its own vital activity or have been taken up into the body from without. As will be shown in more detail in the next chapter, proto^olasm is a substance or complex of substances which, considered in the aggregate, exhibits the physical proxoerties of a viscid fluid. Some samples of protoplasm may be less, others more fluid, but the essentially fluid nature of the whole mass of protoplasm composing the cell-body is very obvious, as a rule, in the case of Protozoa. A drop of a fluid substance, when suspended in another fluid with which it is not miscible, tends immediately, under the action of the physical laAvs of surface-tension, to assume the geometrical form in which the surface is least in proportion to the mass ; that is to say, it tends to become a perfect sphere, except in so far as this tendency may be altered or modifled by the contact or pressure of other bodies, or by the operation of other forces or conditions which oppose the action of surface-tension. The sphere may therefore be regarded as the primary form of the livmg cell — the form, that is to say, which the organism tends to assume under the influence of iDhysical forces when not checked or inhibited in their operation by other factors. A great many Protozoa exliibit the spherical form m a striking manner, especially those species which float more or less freely in the water, such as the Heliozoa (Fig. 3) and Radiolaria (Fig. 13). But the majority of Protozoa depart more or less widely from the primitive spherical form, for reasons which must be considered in detail. In the first place, departure from a spherical form may be merely temporary, the result of vital activity producing altered conditions of surface-tension. In order that a drop of fluid may assume a spherical form as the result of surface-tension, its surface must be 29 30 THE PROTOZOA homogeneous-that is to say, of simUar nature in all parts ; if However, its surface be heterogeneous, and differs in different parts, local niequahties of surface-tension may be the result, and then a perlectly spherical form cannot be maintained so long as the surface remains heterogeneous. Thus an organism, such as an amoeba, in which the protoplasm is quite naked and exposed at the surface of the body, tends always to have a spherical form in the resting state ; but when it enters upon a phase of vital activity, it may assume various forms which can be explained by supposing that the surface- tension is altered at one or more regions of the surface as the result Fig. 13. — Tlialassicdla {Thcdassophysa) pelagica, Haeckel, an example of a species of floating habit combined with radiate symmetry and spherical body-form. OK, Central capsule ; EP, extracapsular protoplasm ; al, vacuoles in the calymma (see p. 251) ; ps., psoudoiJodia. The small dots in the calymma represent " yellow cells " (p. 252). After Lankester, magnified 25 diameters. of local changes in chemical constitution, brought about by the vital activity of the protoplasm (Rhumbler, 34, and j). 200 infra). In consequence, the spherical form characteristic of the resting state undergoes modification in various ways when the organism becomes active. In floating forms the sphere throAvs out radiating processes, so-called " pseudopodia," in all directions (Figs. 3, 13). In creeping species the body assumes the indefinite and constantly changmg form, with pseudopodia extruded in every direction, which is characteristic of the amoeba (Fig. 2), and hence commonly termed " amoeboid." In all such cases, when the animal passes into a THE ORGiVNIZATION OF THE PROTOZOA 31 resting, inactive condition, or when the vital activity is temporarily hihibited by some shock or stimulus, such as an electric current suddenly turned on, lohysical forces reassert their sway, and under the influence of surface-tension the pseudopodia are retracted, and the body rounds itself off and returns to the spherical form. Apart, however, from temporary and variable departures from the primary and fundamental spherical form, many unicellular organisms exliibit a constant body-form which is often widely different from the sphere, and which is characteristic of particular species, or for the corresponding stages in the life-history of a given species, and varies only within the narrowest limits, if at all. The problem of form-production in Protozoa, like all other bio- logical problems, may be considered from two points of view. In the first place, there is the question why a particular species has such and such a form. The answer to this question must be sought in the habits and mode of life of the species and its relation to the environment. In general it may be said that each species pos- sesses, or tends to possess, the body-form best adapted to its par- ticular mode of life, though it is not always easy to trace the correlation of form and habit in special cases. A broad distinction may be drawn, however, between species which move freely in their environment and those which are fixed and sessile in habit. In freely-moving species, again, a further distinction can be drawn between those which float or swim in the medium, and those Avhich creep on a firm substratum. Pree-swimming species tend to the form of an ovoid, more or less elongated, with the longitudinal axis lying in the direction of forward movement (Mg. 14). Creeping forms tend to be more or less flattened, and spread, as it were, upon the substratum, leading in extreme cases to the differentiation of a ventral surface, in contact with the substratum, from a dorsal surface on the opposite side. Sedentary forms tend to be more or less vasiform, often with the point of attachment drawn out into a stalk or peduncle of greater or less length. A frequent peculiarity of the body-form in Protozoa, whether fixed or free, is the tendency to a more or less pronounced spiral twist. Bilateral symmetry, on the other hand, is a comparatively rare phenomenon in these organisms ; examjDles are found among the Flagellata — e.g., Lamhlia intestinalis (Fig. 117). The second question which arises is, Given a particular specific form, how is the form developed and maintauaed, on physiological or mechanical principles ? To this question the answer must be sought in the structure of the individual, and more especially in the formation and possession of special structural elements, more or less rigid in nature, which determine the form and support the soft body. Such structures may be external to the body, in the 32 THE PROTOZOA ttZuf^ r T '^^^^^'lopes of various kinds, or intcinal, lu the lorm of an axis or framework. Both these types of form-determming or skeletal elements, as they may be termed broadly, may be present together in a given organism 1. Gutzcular and Exoskdelal Slructures.~\n the Sarcodina gener- ally, and m a few examples of the Mastigophora and Sporozoa, the body.protoplasm is quite naked at the surface, as abeady stated and not covered by any cuticle or firm covering. With these exceptions, the bodies of Protozoa are clothed by a firm cortical 7Vr~ N- Fio. 14. — Prorodon teres. N, Macronucleus ; n, micronucleus ; o, mouth ; as., oesophagus surrouuded by rod-apparatus (p. 433) ; f.v., food vacuoles ; c.v., contractile vacuole surrounded by feeding- vacuoles ; id., alveolar layer ; st, meridional rows of cilia ; a., aual pore. After Schewiakoli, magnified 660 dia,meters. layer, which is produced either as a differentiation of, or secretion by, the most superficial layer of the protoplasmic body, and which receives various names in different cases. The very first beginnings of a cortical layer are seen in some species of amoebfe, such as Amoeba verrucosa — species in which the X3rotoplasni, extremely viscid and slow-flowing, forms a delicate investing XDellicle at the surface. In these cases the pellicle is so thin that it does not hinder the amoeboid movement appreciably (Fig. 23). A further advance is seen in some of the Flagellata, THE ORGANIZATION OF THE PROTOZOA 33 m where a thin cuticle is present Avliich permits changes in shape, caused by the contractility of the enclosed protoplasmic body. Such forms are not amoeboid, but exhibit rhythmical changes of form produced by contractions of the superficial body-layer in a manner somewhat recalling peristaltic movement, and are com- monly said to be metabolic (Fig. 15) ; and since such movements are characteristic of some species of the genus Euglena, they are sometimes called euglenoid. In most cases, however, in which a cuticle or firm cortex is present, a definite and characteristic body-form is main- tained, subject only to. such changes, as may result from curvatures of the body, or temjoorary shortening of its axis in a particular direction, brought about by the contractility of the living body. An envelope of this kind, which may vary in consistence from a thin, flexible cuticle to a rigid inflexible cuirass, or " lorica," inliibits completely the natural tendency of the fluid protoplasmic body to round itself oil — a tendency, however, which frequently reasserts itself during resting phases of the organism, when the cortex may be softened or absorbed. Hence it is very common to find that the resting phases of Protozoa revert to the primi- tive spherical form, whatever the shape characteristic of the organism in an active state. A close-fitting cortex or cuticle which is essentially a part of the body itself must be distinguished clearly from struc- tures buflt up by the organism externally Fig. IS.— AstasiarteJiax, two to the body to afford shelter or support. Such a structure is termed variously a "shell," "test," or "house." The formation of protective shells, into which the body can be completely retracted, and from which it can emerge to a greater or less extent, is of extremely common occurrence amongst the naked-bodied Sarcodina. The forms of these shells, their structure and mode of formation, exhibit an almost infinite variety, and can only be described here in a quite general manner. individuals showing the changes of form duoj^to metabolic movement, ocs., Oesophagus ; c.r., reservoir of the contractile vacuole ; N., nucleus. After Stein. 34 THE PROTOZOA As regards material, the shells may be composed of elements secreted by the organism (" autophya,',' Hacckel). as in Ilyalosphenia (Hig. 16, B), or of foreign particles taken up by the animal from its surroundings (" xenophya "), as in Difflurjia (Fig. 16, A). Skeletal elements secreted by the organism may be of organic or inorganic nature. In the former case they are probably chitinous in most cases, or composed of a substance allied to chitin ; in the latter they are either calcareous or siliceous. A good example of the formation of a shell is seen in Euglyjjha (Fig. 59), where the chitinous plates composing it are formed first of all in the interior of the proto- plasmic body, and pass to the surface to build up the shell. When the shell is built up of foreign particles, the material employed may vary greatly, and consists generally of particles of sand, grit, etc.. Fig. 16. — Examples of shells or houses formed by Protozoa. A, Difjlw/ia sjnralis, which forms a house built up of foreign bodies ; B, Ilyalosphenia cuneala, in which the house is buUt up of plates secreted by the animal itself (compare also Euglyyha, Fig. 59). Both these species belong to the order Amcebwa ; the i^seudopodia (ps.) arc seen streaming out of the mouth of the shell. After Leidy ; A magnified 250, B 500 diameters. taken up at hazard from the environment. Such shells are de- scribed technically as " arenaceous." In the case of Difflugia, Verworn (36) was able to cause it to build up its test of various materials, such as particles of coloured glass or other substances, when these were supplied to it exclusively. Many species of Foraminifera, however, form their tests exclusively of particular materials under natural conditions. Thus, in the genus Haliphy- sema (Fig. 17) the test is formed of sponge-spicules ; in Technitella thom'psoni the calcareous plates of echinoderms are selected ; and other instances could be cited in which the organism selects habitually for its shell certain materials from a varied environment in ■v\-hich the particular materials required may be far from common in occurrence relatively to other particles apparently equally suitable (see eopecially Heron-Allen and Earland). Verworn (36) found that THE ORGANIZATION OF THE PROTOZOA 35 in the case of Difflugia the foreign particles used are taken up by the pseudopodia during the process of being retracted ; the surface of the pseudopodium then becomes wrinkled, and particles of debris are caught in these \n-inkles, and so drawn into the interior of the protoplasmic body, in which they are stored up in the fundus of the shell, like the plates in Euglypha, and are utilized in the growth of the shell, or in repairing damages to it, or in building a new shell when the animal reproduces itself by division. -Fig. 17. — Haliphysema iumanowiczii, a foraminifer which buikls up its house out of sponge-spicules. A, part of the protopLasm stained to show the nuclei (n.) ; B, a living specimen with expanded pseudopodia (p. ). After Lankester (11). The simplest architectural type of shell or test is a simple spherical or oval capsule, usually with a largo aperture at one pole through which the protoplasm is able to creep out in order to capture food or perform the function of locomotion (Fig. 16). The wall of tlie test may be imperforate, or may have fine pores througJi which also the protoplasm can stream out. With continued growth of the organism, the original shell may become too small for Its requirements. Then the organism may reproduce itself by fission. 3^ THE PROTOZOA sTolltrnln,?hv7l "''^^^^^^^ ^ ""^ ^^^^l^- I" "^^ny cases the slioll toimod by the daughter is larger tlian that of tlie jjareut ; for instance nnh fT'^" '"^^^ ^^^^'^^^ "P^'^i^^' ^vhicli tl e young hid V ral^ Zn 'tho o^ld ''on"' ""ri'n^ for^liid is ar,^^^^ Sxi dinn ni ' H ^^^"'t individual is reached (bcliaudum, 131), alter vvhich point the new shell formed after the process 111 such o.^'offf ^'fT' T ^''^ r'^"^ ^"^ daughter-indivldual as moSlSmotf ' ' ^'"^ ''^^^^^^^^ In other cases, however, the organism does not multiply by fission when It has outgrown its first shell, but forms a new shell of larger size which is in continuity vvith its first shell ; the protoplasmic body now occupies both the chambers of the shell formed m this way. With further gro^vtli more chambers are formed givmg rise to a complex " polythalamous " shell composed of many chambers all occupied by the protoplasmic body (p. 232, infra). For a detailed study of the developmental mechanics of shell-formation, see Knumbler (35). 2. Internal Skeletal Structures. — In many cases in which the proto- plasmic body is naked at the surface, or bears only an extremely tliin cuticle, a definite body-form may be maintained by means of internal supporting fibrils or other similar structm^es (Koltzoff, 30, 31). In some cases such structures may be of temporary nature. A beautiful example of this is seen in the dehcate organic axes formed in the pseudopodia of Heliozoa (Fig. 22), in the form of slender needle-like rods secreted by the protoplasm to stiffen the pseudopodia, and absorbed agam when the pseudopodia are re- tracted. In other cases, supporting structures of orgaiuc natme may be permanent constituents of the protoplasmic body ; such are the axial rods, or " axostyles," found in many flagellates, such as Trichomonas (Fig. 6, ax.), Lophomonas (Fig. 45), etc., slender flexible rods of organic substance which form a supporting axis for the body. Previous to division the axostyle is absorbed, and new axostyles are formed in the daughter-mdividuals. The axostyles are stated to arise from a centrodesmose (p. 103, injra) formed in the process of division of the blepharoplast (Dobell, 236) or of the centriole of the nucleus (Hartmann and Chagas, 62) ; the centrodesmose per- sists after division is complete, and its two halves become the axostyles of the two daughter-individuals. In Trichomonas eberthi, however, Martin and Robertson (348) find that the axostyles arise after division quite independently of the centrodesmoses or other nuclear structures. In Octomitus (Fig. 116) two axostyles are present. From supporting structures of organic nature, such as the axostyles or the organic axes of the pseudopodia mentioned above, it is not difficult to derive the more rigid and permanent elements Imown as " spicules," in which the organic basis becomes uidurated by deposits of inorganic mineral substance. In some cases spicules may perhaps consist entirely of muieral substance deposited directly within the living substance without any organic basis. In THE ORGANIZATION OF THE PROTOZOA 37 either case the spicules grow by accretion— that is to say, by deposi- tion of fresh layers of inorganic substance upon that already laid doAvn— and if such accretion takes place at one end of a rod-shaped spicule, it may have the result that the opposite extremity of the spicule is pushed outwards by the continued growth, with the result that the oldest portion of the spicule projects freely far beyond the limits of the body. As regards material, spicules are usually either calcareous or siliceous— in the first case generally carbonate of lime, in the second Fig. 18. — Acanthocystis chcetophora, a Heliozoon with a skeleton of slender radiating siliceous spicules, each forked at the distal end. In the interior of the body- are seen numerous symbiotic algse (dark) and non-contractile vacuoles (clear) ; one vacuole of larger size is seen, probably the contractile vacuole, sp., sp., Spicules ; ps., ps., pseudopodia. After Leidy, magnified 750 diameters. case amorphous silica. In the family Acanthometridce among the Radiolaria the spicules are formed of a substance which was thought to be of organic nature, and was named " acanthin," but which has been found to consist of strontium sulphate. As regards their form and relation to the body, the spicules in the simplest cases are rod-shaped or needle-like elements disposed radially or tangentially, A simple type of spicular skeleton is seen in Acanthocystis (Fig. 18), in which elongated siliceous rods, fre- 38 THE PROTOZOA Fig. 19. — Olathndina clegans, a Heliozoon with a lattice-liko skeleton, attaclicd by a stalk. Two individuals are seen, the younger with its stalk attached to the head of the older ; in the younger the lattice-work is still very delicate. Both individuals are sending out numerous radiating pseudopodia, very delicate and slender. After Leidy, magnified 750 diameters. THE ORGANIZATION OF THE PROTOZOA 39 quently branched at their distal ends, are arranged like radii of the spherical body, projecting freely for some distance from the surface. In other cases the spicules may be disposed tangentially to the body, as in the family Collid(B amongst the Radiolaria, and m other forms belonging to this order. From a simple type of skeleton composed of separate spicules, more complicated types of skeletons are de- rived by fusion of the spicules to form a connected framework. The commonest type of this is a fusion of tangentially-disposed spicules to form a lattice- work ; an example of this is seen m Clathrulina (Fig. 19), in which a lattice-like skeleton is formed at the sm-face of the body, standing off from it like a shell. Skeletons of this type are especially characteristic of the Radiolaria, a group in which the architecture of the skeleton may reach a very high degree of complication and exhibits endless variety. The lattice- like framework, made up of tangentially-arranged spicules united together, may be further strengthened by radially-disposed beams. As the animal grows, it may outgrow the framework first laid down, and another lattice-work is formed concentric with the first, and connected with it by radial beams ; later on a third and a fourth such framework is formed, as the organism continues to grow in size. Skeletons formed in this way may be " homaxon " — that is to say, built up on the axes of a sphere ; or " monaxon," with one principal axis ; or may follow various plans of symmetry, or may be asymmetrical (p. 250, infra). BibliograpTiy. — For references, see p. 477. CHAPTER IV THE ORGANIZATION OP THE PROTOZOA {Goniinued)- THE PROTOPLASMIC BODY The substance composing the bodies of Protozoa was termed originally sarcode by Dujardin ; but after it had been shown to be Identical in nature with the living substance of the ceUs of animals and plants, the same term was employed universaUy for both and the word 'protoplasm, coined by von Mohl to designate the living substance of plant-cells, supplanted the older term sarcode, which has now quite dropped out of current use. It would be impossible within the limits of the present work to discuss in detail the various theories that have been put forward with regard to the nature and constitution of protoplasm ; they can only be summarized in brief outline here. Protoplasm,' when seen under the microscope with powers of moderate strength, presents itself as a viscid, semi-fluid substance, sometimes clear and hyaline in special regions, but ahvays showing, throughout at least the greater part of its substance, numerous granulations, which vary greatly in size, from relatively coarse grains to those of the minutest size visible with the power of the microscope used. The most important of these granulations are the so-called " chi-omatin- gi-ains," which are discussed fully in Chapter VI. ; in this chapter only non-chromatinic granules are dealt with. The coarser proto- plasmic grains may be present in greater or less quantity, or may be entirely absent ; they are to be regarded for the most part as so-called metaplastic bodies — that is to say, as stages in, or by- products of, the upward or downward metabolism of the organism. On the other hand, the minute, ultimate granules, or " microsomes," are never absent, except over limited areas, in any sample of proto- plasm. It is on the constant presence of granules that the so-called granular theory of protoplasm, especially connected with the name of Altmann, has been founded. On this view, each minute granule is regarded as an elementary organism, or " bioblast," capable in itself of all vital functions, and equivalent to a single free-living bacterium, just as a single ceU of a Metazoan body may be compared with a single Protozoan organism. Protoplasm, on this view, is re* 40 THE ORGANIZATION OF THE PROTOZOA 41 gcorded as ca colony of bioblasts, imbedded in a fluid matrix, com- parable in a general way to a zoogloea-colony of bacteria. A special and important class of metaplastic granules are the so-called " deutoplasmic " bodies, consisting of reserve food- materials stored tip in the protoplasmic substance. Examples of such are the yolk-granules of ova, the paraglj'cogen-grains of gregarines, the plastinoid bodies of coccidia, starch-grains in holophytic forms, etc. _ Amongst the granulations of the protoplasm, special mention must be made of the bodies known generally as choiidriosomes and mitoclionclria, but also by a variety of other names (cytomicrosomes, bioblasts, spherules or sphero- plasts, and, collectively, ergastoplasm). The chondriosomes are not to be classed with the temporary, metaplastic inclusions, but are permanent ele- ments of the cell- protoplasm. The chondriosomes of Protozoa have recently been the subject of detailed study by Faure-Fremiet (38-5). In the living condition they are small transparent bodies, feebly refractile, and of a pale grey tint. In shape they are generally spherical, and vary from 0-5 /x to 1-5 /X in diameter. In some cases the chondriosome appears homogeneous in structure ; in others it presents the appearance of a vacuole with fluid con- tents and a denser peripheral layer. In contact with water or with weak alkalis they swell up immediately. When the nucleus (in Infusoria the micronucleus) divides, the chondriosomes also divide simultaneously, and the daughter- chondriosomes are sorted out between the two daughter- cells ; they have, however, no direct relation with the nuclear apparatus. In the process of division each chondriosome becomes first rod-like, then dumb-bell- shaped, and is finally constricted directly into two halves. A purely chemical definition of the chondriosomes, according to Faure- Fremiet, cannot be given. They exhibit the reactions of a fatty acid, and can be considered as combinations of fatty acids or of phosphates of albumin. The physiological function of the chondriosomes is not clear, but Faure- Fremiet considers that they " play an important part in the life and evolu- tion of the sexual cell," in Protozoa or Metazoa, and are active in the elabora- tion of deutoplasmic substances of fatty nature, into which they may bo transformed directly. It has been shown, however, that the minute granules of proto- plasm do not lie isolated from one another, suspended freely in a matrix, but are seen in the microscopic image to be connected with one another by fine lines or darker streaks, the whole forming a delicate network, at the nodes of which the granules are lodged. In some cases the granule itself is perhaps only an optical effect produced by a node of the network. On these appearances has been founded the so-called reticular theory of protoplasm, connected especially with the names of Heitzmann, Schafer, and others. On this view protoplasm has been regarded as composed of an exceed- ingly fine reticulum, a network or feltwork ramifying in all planes, bearing the granulations at its nodal points, and bathed throughout by a fluid, more or less watery sap, or enchylema. The fibrillar theory of Flemming may be regarded as a modification of the reticular theory. Against the reticular theory of protoplasm, it may be urged that it leads to physical difficulties, in view of the generally fluid nature of protoplasm. For the reticulum must itself be either of a fluid or a solid nature ; if fluid, it presents the condition of one fluid 42 THE PROTOZOA suspended in the form of a network in another fluid with which it does not mix — a condition which could not exist for moi-e than an instant of time, since tlie fluid reticulum must break up immediately into minute droplets. If, on the other hand, the reticulum is of rigid consistence, the protoplasm as a whole could not be fluid, any more than a sponge soaked in water could behave as a fluid mass in the aggregate. The difiiculty can, however, be overcome by supposing the apparent reticulum to be the optical expression, not of a fine network of fibrils, but of delicate lamellae limiting minute closed chambers, or alveoli. Then the fine line seen with the microscope joining any two adjacent nodal points would be the optical section of the wall or lamella separating two contiguous alveoli, and protoplasm as a whole would possess a honeycombed structure comparable to that of a fine foam or lather — the fiuid lamellae of the foam represented by the apparent reticulum of the protoijlasm, and the air-contents of the individual bubbles repre- sented by the enchylema. Or, to express the state of things in a different manner, protoplasm could be regarded as an emulsion of very fine structure, composed of two fluids not miscible with one another — namely, the more fluid enchjdema, which is suspended in the form of minute droplets in the more viscid substance forming the alveolar framework. This is the so-called alveolar theory, especially connected with the name of Biitsclili ; by this conception of protoplasmic structure, not only are the necessary phj^sical con- ditions satisfied, but an explanation is given for many peculiarities of protoplasmic bodies, such as the radiate arrangement of the meshes of the reticulum commonly observed either at the surface of the body or around solid or fluid bodies contained in the proto- plasm, and so forth. The various theories that have been mentioned all assume tacitly that protoplasm is monomorphic — that is to say, that it possesses one fundamental type of minute structure. Fischer, on the other hand, seeks to unite all the different theories by supposing that protojDlasm is a polymorphic substance — that is to say, one that may exhibit a diversity of structure at different times and under different conditions, as the result of changes produced by its mherent vital activity. Thus, he supposes that a given mass of protoplasm may be at one time homogeneous, and at another time gramdar, reticular, fibrillar, or alveolar, as the result of a process of " vital precipitation," and that by reabsorption of the structural elements it may return to a homogeneous condition. Faure-Fremiet (38 and 38-5) also regards protoplasm as a homogeneous fluid, which is pre- cipitated by reagents, and which normally contains, in suspension, a certain number of granulations, some temporary, others per- manent in nature ; compare also Degen (154). THE ORGANIZATION OF THE PROTOZOA 43 Those investigators of the Protozoa who have expressed an opinion on the subject have been for the most part m favour of the alveolar theory of protoplasm, since it was first propounded by its author, BiitsclJi (see especially Rhumbler). Protozoa as a rule Ire very favourable objects in which to study the io-m-hke s^^^^^^ of the protoplasm (compare Schaudinn, 130, p. 188) But what- ever view be held as to the ultimate structure of protoplasm its essentially fluid nature is very apparent in these organisms and is a point upon which it is very important to be dear The fluid condition of the living substance is manifested directly by the streaming movements to be observed in it, and indirectly by a number of phenomena, such as the tendency, akeady mentioned of the body to round itself off when at rest, and the tendency of all vacuoles to assume a spherical form. A vacuole is a drop of fluid suspended in the protoplasmic body, and may be regarded as formed by the bursting and running together of many minute alveoli, just as a large bubble in a foam may arise by the union of many smaller ones ; or by the gradual enlargement of a single alveolus by diffusion of fluid into it from neighbouring alveoli, until it attains proportions relatively gigantic. Vacuoles assume uni- formly spherical contours, except when they are deformed by mutual pressure from crowding together or from other causes. In some cases the protoplasm may be so full of coarse vacuoles that it exhibits an obvious frothy structure, which must by no means be confounded with the ultimate alveolar structure of the protoplasm, a structure which is exceedingly delicate, only to be observed with high powers of the microscope and with careful attention to all details of microscopic technique. Examples of vacuolated bodies are seen especially in Heliozoa — e.g., Actinosphcerium (Fig. 3). The statement, however, that protoplasm generally is of fluid nature admits of its exhibiting many degrees of fluidity, and some samples of protoplasm are far more viscid than others. This is true both of different species of organisms, of the same species at different phases of its development, and of different parts of the same organism. In some cases portions of the protoplasm may be stiffened to a degree that perhaps oversteps the ill-defined boundary between the liquid and solid states of matter. In a great many Protozoa, perhaps the majority of them, the protoplasm of the body is divisible, more or less distinctly, into two regions — namely : 1. An external or cortical zone, termed ectoplasm or ectosarc ; in appearance and consistence typically clear, hyaline, more refringent, finely granular or without visible granulations, and of more viscid nature ; in function protective, kinetic, excretory, and sensory. 2. An internal or medullary region, the endoplasm or endosarc ; ^ THE PROTOZOA opaque, less refringent and coarsely granular ; the seat of trophic and reproductive functions. These two zones of the protoplasmic body are, in the more primi- tive forms, differentiations of the protoplasm more or less tem- porary and transient in nature. For instance, in an amoeba which IS ni active movement, fluid endoplasm is constantly flowing along the axes of the pseudopodia towards their tips, where it com'es into contact with the surrounding medium, the water or other fluid in Avhich the amoeba lives. Under the influence of the medium the endoiolasm is converted into ectoplasm, becomes of stiiier, less fluid consistence, and loses its coarse granulations. At the same time, at the hinder end of the amoeba, ectoplasm is continually passing into the interior of the body, where it becomes liquefied and granular in structure, and is converted into endoplasm (Rhumbler, 34). In Protozoa, however, which do not exhibit amoeboid movement, the ectoplasm and endoplasm may be two independent layers, well defined and perfectly separate the one from the other. The ecto- plasm is the seat of those functions which are connected with the relation of the organism to the outer world, to the environment in which it lives ; the endoplasm, on the other hand, is concerned specially with the internal affairs, so to speak, of the protoplasmic body. In the following two chapters the various organs of the Protozoa will be considered under the headings of the layer from which they are formed, and according to the functions they perform Bihliogra'phy. — For references, see p. 477. CHAPTER V THE ORGANIZATION OF THE PROTOZOA {Continued)— DIFFERENTIATIONS OF THE ECTOPLASM AND ENDOPLASM A. Ectoplasmic Organs. The various structures and organs produced from the ectoplasm are best classified by the functions they subserve, under the headings of protective, kinetic and locomotor, excretory, and sensory mechanisms. 1. The protective function of the ectoplasm is often seen in organisms m which no cuticle or envelope is present. It has been observed, for instance, that the species of Myxosporidia that iohabit the gall-bladders or urinary bladders of their hosts resist the effects of the medium in which they live so long as their ecto- plasm is intact, but succumb if it be injured. In most Protozoa other than those belonging to the class Sarco- dina, however, a special protective envelope or cortex is present at the surface of the body, and such forms are commonly said to be corticate. A cuticle may be formed in various ways, distinguished by the use of diiierent terms. It may represent the entire ecto- plasm, modified in its entirety to form an envelope, as in the perir plast of the FlageUata ; it may represent a transformation or modi- fication of only the most superficial layer of the ectoplasm, as in the pellicle of the Infusoria and of some amoebse — ^for instance, Amceba verrucosa, the epicyte of the gregarines, etc. ; or it may arise as a secreted layer deposited at the surface of the ectoplasm, and not derived from a modification of the substance of the ectoplasm itself, in which case it is termed a " ceU- membrane." Whatever its mode of origin, the cuticle may be developed to a very variable degree, from the thinnest possible membrane, some- times very difficult to discover, to a thick and tough investment which may be termed a " cuirass " or " lorica " (" Panzer "), when it is formed by thickening of a pellicle ; or a " house " or " shell," when it is a greatly thickened cell- membrane standing off from the body. In many cases the cuticle undergoes local thickenings to form spikes or hooks, which may serve as organs of attachment, as in the epimerite of gregarines (Pig. 142). 45 46 THE PROTOZOA In addition to the passive protection aflordcd by a cuticle, organs of active defence may be present in the ectoplasm in the form of bodies known as trichocysts, found commonly in many ciliate In- fusoria (p. 447, Fig. 187) ; they are little oval or spindle-shaped bodies which on suitable stimulation are converted explosively into a stiff tlu-ead which is shot out from the surface of the body. (For the nematocyst-like organs known as " polar capsules," in Myxo- sporidia and allied organisms, see p. 399, infra.) 2. The ectoplasm is shown to be the seat of movement both by the fact that motile organs arise from it and by the frequent presence in it of special contractile mechanisms. The motile organs which are found in the Protozoa are pseudopodia, flagella, cilia with their various modifications, and undulating membranes ; any of these structures may subserve the function of food capture in addition to, or instead of, that of locomotion. These organs wiU now be described in order, after which contractile mechanisms wUl be dealt with. (1) Pseudopodia are organs of temporary nature, extruded from the protoplasm when required, and retracted when no longer needed. They can be formed, probably, in all cases in wliich the body protoplasm is naked, or limited only by a cuticle not of sufficient thickness to inhibit the movements of the underlying protoplasm. They arise simply as an eruption of the protoplasm at some pomt at the surface of the body, forming an outgrowth or process which varies greatly in different cases as regards size, length, width, com- position, and activity. Pseudopodia always arise in the first instance from the ectoplasm, and may consist tliroughout of this layer alone, in which case they are relatively stiffer and more rigid ; or a core of endoplasm may flow into the pseudopodium when it has grown to a certain length, in which case the pseudopodium is more fluid and flexible. The formation of a pseudopodium is best studied in a common amoeba, such as Amoeba liroteus (Fig. 2) or ^. Umax (Fig. 20) ; it is then seen to arise as a protrusion of the ectoplasm, forming a shallow promi- nence at the surface of the body. The prominence continues to grow out from the body, and is at first hyaline, transparent, and free from granulations, since it consists of ectoplasm alone. In some cases the pseudopodium may grow to a relatively very large size, and still consist of clear ectoplasm alone, as in Ihitamosba histolytica (Fig. 90), a form rather exceptional in this respect ; more usually, so soon as the budding pseudopodium has reached a certain not very great size, a core of granular endoplasm flows into it and forms the axial part of the pseudopodium. It is then easier to study the formation of the pseudopodium, since the granules in the endo- plasm permit the characteristic flowing movements and currents to THE ORGANIZATION OF THE PROTOZOA 47 be followed. In the gi'owing pseudopodium a strong current can be observed flowing down the axis to the tip, and there sprcadnig out and breaking up into weaker currents which turn round and flow backwards along the surface of the pseudopodium. In amoebse with a very viscid surface layer the back-currents are very feeble, ceasing a short way from the tip of the pseudopodium, and often scarcely discernible, or even absent altogether ; in species with a fluid ectoplasm, however, the back - currents are distinctly seen and may even pass back and bend round again to join the forward axial current, as described by Rhumbler (34) in Amoeba blattce. While the extrusion of the pseudopodium is an active process, the retraction requires nothing but the action of purely physical forces of surface-tension to explain it. The protoplasm then flows back into the body of the animal, and may present some character- istic appearances in doing so. If one surface is in contact with the substratum on which the animal is creeping, the adhesion of the pseudopodium often causes the tip to be drawn out into slender processes like spikes or hairs. At the same time the surface of the Fig. 20. — Diagram to show the protoplasmic currents in a limax- amceba which is moving forward in the direction indicated by the large arrow on the left. The smaller arrows indicate the direction, and their length the intensity, of the currents in different parts of the body. A forwardly-directed " fountain current " starts from near the hinder end, and passes along the axis of the body to the extremity anterior in movement ; there it turns outwards and passes back along the sides of the body, diminishing rapidly in intensity, and finally dying out in the regions where the two dots are placed. After Rhumbler (34). pseudopodium may present a wrinkled appearance, as the viscid ectoplasm shrinks in consequence of the rapid withdrawal of the fluid endoplasm. The pseudopodia of difierent species of organisms, or even of the same species at different periods of the life-cycle, vary greatly in form, appearance, and structural characters, and the more im- portant variations require a special terminology. In the first place, the pseudopodia may be broad and thick relatively to their length, as in Amoeba proteus (Fig. 2) ; they are then termed " lobose " (" lobopodia "), and usually have a core of endoplasm. A typical lobose pseudopodium is, in fact, nothing more than an outgrowth of the body-protoplasm as a whole. In the most extreme cases of this type, the whole body flows forward in one direction, forming, as it were, a single pseudopodium. Such a mode of progression is characteristic of AfiK^ba Umax (Fig. 20) and other similar forms, in which the body glides forward like a slug as the animal creeps over substratum ; the end which is anterior in movement is rounded THE PROTOZOA sl!fulrt^ri^^ end commonly becomes drawn out into processes In othe. f . 'T" 'V P^^^^^«P°dium in process of retraction. out on al .7' '""'. r/- ^''^'T P^^^dopodia are sent out on all sides and balance each other, in which case there is very httle translation of the body as a whole, and the pseudopodia serve chiefly for food-capture. If, however, the outflow of the pseudo- podia is strongest on one side of the body, the organism moves in that direction as a whole, and the larger, more strongly developed pseudopodia counteract and overcome the pull exerted by those that are weaker. It wiU be readily understood, therefore, that the most rapid powers of progression are possessed by the slucr-like amoebae, m which a single pseudopodium drags the whole body along without opposition from others. Rhumbler (34) has drawn attention to the existence of two modes of progression exerted by amoebije of the lobose type. In the more fluid species which creep upon a substratum to which they adhere more or less firmly, like Amceba proteus, the animal pro- gresses by a flowing movement, such as has been described ; this is the commonest type of amoeboid locomotion. On the other hand, in species of the type of A. verrucosa and A. terricola the very slightly fluid body is limited by a thin pellicle, and does not adhere to the substratum ; then progression is effected by " rolling " movement. The animal throws out a number of pseudopodia on one side, which cause it ultimately to overbalance and roll over to that side ; by continued repetition of this procedure, a slow progres- sion in a particular direction is efiected. At other times, however, A. verrucosa may flow along like other amcebae. Contrasting with the lobose pseudopodia are the slender, thread- like, so-called " filose " pseudopodia, formed entirely of ectoplasm. Pseudopodia of this type can effect a slow creeping movement, but are not very effective for locomotion, and serve for food-capture principally, or even entirely, as in the radiate floatmg forms (Heliozoa and Radiolaria) ; food is entangled by them and drawn into the body. The filose pseudopodia may radiate from the body in all directions, remaining separate from one another, or they may anastomose to form networks, and are then termed " reticulose." Pseudopodia of the reticulose type are specially characteristic of the Foraminifera (Fig. 21). Radiate pseudopodia which do not form anastomoses, on the other hand, characterize the grou]DS of the Heliozoa and Radiolaria, organisms of floating habit. As noted above, pseudopodia of the radiate type are generally supported by an axial rod, a secreted structm'e of firm, elastic nature, and are hence known as axopodia. The actual rod reaches some way into the endoplasm, often to the centre of the body, as in Acantliocyslis (Fig. 18), Wagnerella (Fig. 48), etc. ; it THE ORGANIZATION OF THE PROTOZOA 49 X' ^'SVi. f'Ti* A'-v^. ' ■ y 'M^.s#'*^.----^--C>--... Fig. 21. — Gromia ovi- formis, M. Schultze (=6, ovoidea, Rhumbler), living specimen with out- stretched pseudo- podial network (ps.), in which a diatom («!.), Navi- ctda sp., is en- tangled and will be drawn into the shell (sh.). Other diatoms are seen inside the shell, and at its fundus several nuclei are seen as clear spheri- cal bodies in the protoplasm. The pseudopodial net- work is drawn at a magnification of about 200 linear, but for want of space is repre- sented extending over about one- third of the area over which it com- monly spreads. A part of the pseu- dopodial network is reflected back over the shell, and streams out back- wards from the pole opposite to the shell - mouth. After M. Schultze. 4 50 THE PROTOZOA is probably of endoplasmic origin, and is pushed out from it in a centrifugal direction. As it grows out, the ectoplasm forms a sheath over it, and extends usually some way beyond it. When the pseudopodium is retracted, the axial rod is liquefied and absorbed by the protoplasm. Food-capture is effected by the pseudopodia in various ways (see p. 189). In forms with lobose pseudopodia they flow round the body to be ingested, enclosing it on all sides, and finally imprisoning the prey in a closed chamber of the living substance, together with a drop of water which forms the food - vacuole (Fig. 2, P\ P^) in which the prey is digested (p. 192, infra). A very noticeable feature of pseudopodia of all kinds is their adhesive- ness, due to the secretion of a slimy substance at the surface of the ectoplasm. In DiffLugia, if the pseudo- podia be touched gently with a glass rod, the slime can be dra-wn out into threads, like the mucus of a snail (Rhumbler, 34). The adhesive power of the pseudopodia is of service both in adhering to the surface upon which they creep and in the capture of their food. The slow-flowing amoebae, such as A. verru- cosa, do not as a rule flow round the body to be in- gested, but draw it into their interior, as if by suction. In this manner^, verrucosa absorbs and devours filamentous algae (Fig. 23), which are " imported " into the interior of the body and there coiled up and digested. Rhumbler has shown that this process can be imitated by drops of fluid ; for instance, a drop of chloroform m water will draw in a thread of sheUac and coil it up in its interior m a manner similar to the ingestion of an algal filament by an amoeba. Fig. 22. — Portion of an Actinosphcerium, magni- fied about 660 linear, ect., Ectoplasm with larger vacuoles ; end., endoplasm with smaller vacuoles; N., nucleus; ps., pseudopodia; ax., delicate axial rod in the pseudopodia. After Leidy. THE ORGANIZATION OF THE PROTOZOA 51 The pseudopodia of the filosc type adhere firmly to organisms suitable for food with wliich they come in contact, and it can be observed that the prey is both held fast and killed by them, in- dicating that the pseudopodia secrete some toxic substance in addition to that of an adhesive nature. In the reticulose type, diatoms and organisms of various kinds are entangled in the pseudopodial network (Fig. 21), and are generally digested there also. In a few cases pseudopodia exhibit a peculiar form of movement known as mdation. An example of this is seen in the remarkable Heliozoon described by Schaudinn (43) under the name Gamptonema nutans (Fig. 47), which possesses slender axopodia in which the axial Fia. 23. — Four stages in the ingestion of an OscLllarian filament (/.) by Amoeba verrucosa. In A the amcBba has crept along the filament ; in B one end of the amoeba is bending up, and is about to fuse with the rest of the body, producing a twist in the filament ; in C two have been produced ; in Z) a considerable length of the filament has been drawn into the amoeba, and is twisted up into a stout coil. A, B, and 0, are drawn at intervals of quarter of an hour, D several hours later. After Rhumbler (34). filament does not extend the whole length of the pseudopodium. The pseudopodia perform a slow rotating movement, and "describe the mantle of a cone, sometimes acute, sometimes obtuse, remaining stretched out straight for their entire length, and bending only at their base." Similar movements are performed by the pseudopodia of Trichosph cerium (p. 229) and Wagnerella (p. 246). In Gamptonema the pseudopodia also have the power of bending suddenly when brought in contact with prey, which they capture like the tentacles of a polyp. The bending takes place beyond the point at which the axial filament ceases. Movements of tills kind are transitional to those seen in flageUa. (2) FlageDa are vibratile thread-like extensions of the protoplasm, capable of performing very complicated lashing movements in 52 THE PROTOZOA a/x every direction. A flagellum consists of an elastic axial core enclosed in a contractile sheath or envelope (Pig. 24), from the extremity of which the core protrudes freely in some cases, forming a so-called " end-piece." The flagellum takes origin from a more or less deeply-seated granule, the blepharoplast, or basal granule, which wiU be described in dealing with the nuclear apparatus (p. 82, infra). The elastic axis, arising from the blepharoplast, can be regarded as a form -determining element of endoplasmic origin, the sheath as an ectoplasmic motor substance. A flagellum is usually cylindrical in form, with the axial filament central in cross-section, but may be band-like, with the axial filament at or near one edge ; it is usually of even thicloiess throughout its whole length, but when the axial filament is exposed to form a terminal end - piece the flagellum tapers to a fine point. Like pseudopodia, flagella serve primarily for locomotion, and secondarily for food-capture, which is effected by causing food-particles to impinge on some point or aperture at the surface of the body, where they are ingested. In their relation to locomotion two types of flagella can be distinguished, termed by Lankester pulsella and tractella respectively. A pulsellum is situated at the end of the body which is posterior in movement — that is to say, it is a flagellum which by its activity propels the body forwards. Flagella of this type occ\xr in OxyrrJiis (p. 278) and m the Choanoflagellata (p. 271), but are comparatively rare in the Protozoa. In the majority of cases the flagella are tractella — that is to say, their action is such as to drag the body after them — hence they are situated at the end which is anterior in progression. Con- sidered generally, the movements performed by tracteUa are of two types. In some cases the entire flageUum is thi-own into even, sinuous undulations, and the body of the flagellate progresses with a smooth, gliding movement, which may be extremely rapid, and is then weU expressed by the French phrase " mouvement en fleche "; this type of movement is well seen in the trypanosomes and aUied genera, such as Leptomonas, etc In most free-living flageUates, however, the flagellum is held out stiff and straight for the proximal two-thirds or so of its length while the distal third performs peculiar whu-ling or pulsating Fig. 24. — Structure of the flagellum of Euglena. aa;., Axial filament; c.p., con- tractile protoplasm enveloping the axial filament ; e.p., end - piece of the flagellum, consist- ing of the axial fila- ment exposed ; r, root of the flagel- lum passing into the body (compare Fig. 84). After Biitschli (3). CM THE ORGANIZATION OF THE PROTOZOA 53 movements,* which drag the body along in a succession of more or ^^n'm^y Slated organisms, forwardly-direeted flagella may be combined with so-called "trailing fiagella" [' PP'f «f which are directed backwards, running along the side of the body, either quite free (Fig. 25) or united to the body by an undulating membrane (Fig. 5). In such cases the trailing flagellum is perhaps the chief organ of propulsion, acting as a iralsellum, while the forwardly-directed flagellum or flagella may function more as tactile organs or feelers than as locomotor organs. The flagellum may also serve as an organ of temporary attachment in some cases, especially in parasitic flagellates ; it then often exhibits at its distal extremity a distmct bead-like swelling or enlargement, doubtless of adhesive nature. Such terminal enlargements are sometimes seen, however, in free-swimming forms. There are many grounds for assuming the existence of a gradual transition from fiagella to pseudopodia, and especially to the slender axopodia seen in Heliozoa, etc. In organs of each kind the type of structure is essentially similar, an axis of firm elastic nature, which is pushed out from the endoplasm, in many cases from a basal granule of centrosomic nature (p. 82), and is covered over by a sheath of contractile fluid ectoplasm. The difference between them is one of degree, the axopodia being relatively shorter in proportion to their thickness, and consequently less flexible, but the nutating and bending movements seen in axopodia are essentially similar in type to those manifested by flagella. The Heliozoa are con- nected with the Flagellata by transitional forms which indicate that their pseudopodia have arisen as modifications of flagella (p. 248). Goldschmidt, who discusses the whole question (41, pp. 116-122), de- scribes in a Cercomonas-\\kQ flagellate the shorten- ing of the flagellum, and its transformation into a pseudopodium which swings to and fro. A flagellum may be considered as having arisen by modification and specialization of an axo podium, and as capable in many instances of reverting to that type of organ. (Compare also p. 465, infra.) (3) Cilia are slender, thread-like extensions of the ectoplasm which differ from flagella mainly in three points : they are as a rule much shorter relatively to the size of the body ; they are present usually in much greater numbers, and in their most primitive tj^pe Fig. 25. — Anisonema grande, ventral view, showing the " hetero- mastigote " arrange- ment of the flagella. a.f., Anterior flagel- lum ; p.f., posterior trailing flagellum ; S, oesophagus; c.v., contractile vacuole surrounded by a number of feeding vacuoles ; N., nucleus ; an., anus (cytoj)yge). After Stein. * For a detailed description and analysis of these movements, sec Delage and Herouard (6), pp. 305-312. 54 THE PROTOZOA ot arrangement form, as it were, a furry covering to the body • and their movements are different from those of flagcUa. A'c'ihum performs simple regular movements of alternate contraction and relaxation, whereby it is first bent like a bow, wth a slight spiral twist (Schuberg, 44), and then becomes straightened out again ; from this it may be inferred that the contractile substance is developed mainly on one side of the elastic axis— on that side, namely, which becomes concave during contraction— instead of ensheathing the axis completely, as in most flagella. Then the bending of the cilium would be the result of active contractility, acting against the elasticity of the axis, which is operative in causing the cilium to straighten out again when the contractile substance is relaxed. Cilia are usually implanted in rows on the surface of the body, and their movements are co-ordinated in such a way that the con- traction— or, as it may be better termed, the pulsation — of a given cilium takes place slightly after the one in front of it, and before the (me behind it (Fig. 26). On the other hand, the neighbouring cilia of adjacent rows pulsate in unison ; consequently, when a ciliated Fig. 26. — Diagram of ciliary movement, representing tlie successive phases of contraction and expansion in a row of cilia. After Verwom. surface is seen from above with sufficient magnification, the move- ments of the cilia produce an optical effect similar to that seen in a cornfield when the wind blowing across it gives rise to an appearance of waves following each other in a continuous succession. When, however, a row of cilia is seen in side-view, the successive beats of the cilia may produce the illusion of a rotating wheel ; hence the origin of such names as Rotifer, Trochophore, etc., applied to Metazoan organisms bearing rings or girdles of stout cilia. In spite of the appai'ent differences between cilia and flagella, there is no difficult}'^ in regarding cilia as derived ancestrally from flagella by a process of modification and specialization in structure, movement, number, arrangement, and co-ordination. Like pseudo- podia and flagella, cilia may serve both for locomotion and food- capture. In many cases the cilia specialized for these two functions may be sharply distinct ; the food-capturing cilia, found in connec- tion with the mouth and the peristomial region, are commonly much longer than the locomotor cilia, and show the tendency to form fusions ]Dresently to be described. In sedentary forms loco motor cilia may be absent in the ordinary state of the animal, and only developed temporarily during motile phases. On the other THE ORGANIZATION OF THE PROTOZOA 55 hcand in a purely parasitic form such as Ovalina (p. 439), in which a mouth is entirely absent, only locomotor cilia are present. The chief modifications of cilia, apart from variations m size and function, are the result of a tendency to adhere or fuse together ; thus arise various types of organs, of which the most common are the cirri, membranellce, and undulating membranes. Cim are organs resembling bristles, formed by fusion of a tuft of cilia, just as the hairs of an ordinary camel's-hair paint-brush adhere when moistened so as to form a flexible pencil. In many cases the cirri have frayed- out ends, m which the component cilia are distinct from one another ; and reagents often cause a cirrus to break up into separate cilia. Cirri have a locomotor function, and are especially characteristic of the ciliate Infusoria which are of creepmg habit (order Hypotricha, p. 440, infra). The cirri occur on the ventral sm-face of the body— that is to say, on the side of the body turned towards the substratum on which the organism creeps, using the cu-ri practically as legs. Membranellee are flapping or swinguig membranes formed by fusion of two or more transverse rows of cilia implanted side by side, and adhermg to form a flat membrane, the free edge of which often has a frmged or frayed border, representmg the free ends of the component cilia. Membranellae occur usually in the region of the peristome in spu-al rows, implanted one behind the other, and each membranella performs simple movements of alternate flexion and expansion, comparable to those of a smgle cilium. Both in structure, origm, and movements, the membranellae must be distmguished clearly from the undulating membranes presently to be described. Undulating membranes are sheet-like extensions of the ectoplasm, which perform rippling movements, comparable to those of a sail placed edgewise to the wmd ; or, better still, to the undulatmg movements performed by the dorsal fin of a sea-horse {Hippocampus) or a pipe-fish (Syngnathus) when swimmuig. The undulating mem- branes of Ciliata consist simply of a single row of cilia fused together. Such membranes are found commonly in the oesophagus of In- fusoria ; in the vestibule of Vorticellids there are two membranes of this kind. In some genera, such as Pleuronema (Fig. 27), they represent the princixDal food-capturing organ, and reach a great development. Pleuronema swims about by means of its cilia, and comes to rest sooner or later in a characteristic attitude, with the cilia projecting stiffly from the body ; the large undulatmg membrane is then protruded from the mouth, and serves by its movements to waft food-particles down the 03Sophagus. Undulating membranes are also of common occurrence in the Flagellata, where they are of a different type from those of Ciliata. The undulating membrane in this class is always found m connec- 66 THE PROTOZOA tion ,vith a flagellum, and is to be regarded as a web of the ecto- plasm (periplast) connecting the flagellum to the surface of the oocly feuch a condition may arise either by attachment of a back- wardly-dn-ected trailing flagellum to the side of the body, as in Inchomonas (Fig. 5) and Trypanoplasma (Fig. 36), or by the shiftmg backwards of the point of origin of an anterior flagellum as IS well seen in the transition from crithidial to trypanif orm phases an the development of trypanosomes (Fig. 131). As a rule, only the proximal portion of the flagellum is uavolved in the formation of Fig. 27. — Pleuronema chrysalis. M, The undulating membrane ; o, mouth ; N, macronucleus ; n, micronucleus ; c.v., contractile vacuole ; f.v., food vacuole ; a., anal pore. After SchewiakofF, magnified 660 diameters. the undulating membrane, and the distal portion projects freely beyond it ; but in some cases a distal free portion of the flagellum may be quite absent, and then flagellum and undulating membrane are co-extensive (Fig. 12, J). Undulating membranes in Flagellata appear to be specially related to the endoparasitic mode of life, and in free-living species they are found rarely, if ever ; they may be regarded as an adaptation to life in a broth-like medium, such as the intestinal contents, or the blood of a vertebrate, containing many suspended particles or corpuscles. In such cases the membrane may assist the organism to force its way between the solid bodies suspended in the fluid medium. Undulating membranes may, how- THE ORGANIZATION OF THE PROTOZOA 57 ever serve for other functions than that of locomotion, in flagel- lates' as well as in ciliates. In large, stout forms of trypanosomes, for example, the animal may remam perfectly still while its mem- brane is rippling actively, and in that case the function of the mem- brane is probably to cause currents in the fluid surroundmg the body, and to change and renew the liquid bathing the body-surface. In such a case it has been noted that the undulating membrane may from time to time reverse the direction of its movements the waves running for a time from the hinder end forwards, and then for a time in the opposite direction (Minchiii and Woodcock, p 150) It is probable that the undulatmg membranes which pass down the vestibule of Vorticellids can reverse their movements m a similar manner, since this passage serves both for passage of food- particles to the mouth and for the ejection of excreta from the anal pore and the contractile vacuoles. The only structures found in free-livmg Flagellata which can be compared at all with undulating membranes are the peculiar " collars " found m the Choanoflagellata (Fig. 110), and also m the collar-cells of sponges. Each collar is an extension of the ecto- plasm which grows up from the edge of a circular area round the insertion of the flagellum, formmg a membrane like a cufE or sleeve surrounding the basal portion of the flagellum, but quite distinct from the flagellum itself, and not formed in actual connection with it like the undulatmg membrane of a trypanosome. The collar differs further from a true undulating membrane in not being energetically motile, but only slowly protrusible and retractile. It has been stated, both for Choanoflagellates and for the collar-cells of sponges, that the collar is formed by a spirally-folded membrane. Their function appears to be that of assistuig m food-capture by a sessile, flagellated organism. (4) Contractile mechanisms in Protozoa, when they are visible, take the form of so-called myonemes, minute contractile fibrils run- ning in various directions in the ectoplasm, like an excessively minute system of muscle-fibres. Such elements are not found in Sarcodina or in the non-corticate forms of the other classes ; in naked forms with amcEboid movement the ectoplasm, as has been pointed out above, is only a temporary differentiation of the proto- plasmic body, which can arise by conversion of the endoplasm, and which can be changed back again into endoplasm. Myonemes occur commonly, however, in those Flagellata, Sporozoa, or Infusoria, which owe a definite body-form to the presence of a firm cuticle or cortex, representing a stable ectoplasm. The myonemes are often, however, extremely fine, and sometimes escape detection in cases in which we can infer their presence with certainty from the move- ments or contractions of the organism or of its ectoplasm. As a 68 THE PROTOZOA general rule they are visible more or lass clearly iii the larger but not in the more minute, species. Thus, in trypanosomes, myonemes can be made out m large forms as delicate lines running parallel to the undulatmg membrane (Fig. 28), but in small species of trypano- somes it may be impossible to discover them, although the nature ot their movements may leave no doubt as to the existence of con- tractile mechanisms in the ectoplasm. In other cases, both motile species possessmg myonemes and non-motile species lacking them may occur within the limits of a smgle group, as in Gregarmes, where the motile species show a very distinct layer of myonemes (Fig. 29); while the non-motile forms have a much thinner ecto- plasm, represented practically by the cuticle alone, with no trace of myonemes. In the non-motile trophozoites of the Coccidia myo- FiG. 28. — Trypanosoma jje cw, stout form stained with iron-haamatoxylin to show myonemes. After Minchin, X 2,000. Fig. 29. — Grcgarina munieri, showing the layer of myonemes at the surface of the body, slightly diagrammatic. After Schneider. nemes are similarly absent. In the ciliate Infusoria the myonemes run parallel to, and beneath, the rows of cilia, and in species of large size and great powers of contractility, such as Stentor, the myonemes are lodged in canals and show a transverse striation (Fig. 186, /). According to Schaudiim, these motile mechanisms, both flagella and myonemes, are derived from the achromatic spindle of a dividing nucleus. In the development of a trypanosome from a non-fiagellated condition, he describes the entire kinetic apparatus as arising from a nuclear spindle consistmg of two polar centro- somes connected by a centrodesmose (p. 103, injra), and by mantle THE ORGANIZATION OF THE PROTOZOA 59 fibres, but with chromosomes apparently rudimentary or ab ent Such a spindle is stated to persist and to grow greatly i length, one pole of it finally projecting beyond the anterior end of the body The centrosome at the proximal pole of the spmdle becomes the blephLplast or basal gJanule of the flagellum ; the centrod-^^^^^ itself becomes the flagellum, or at least its axial elastic filament , Fig. 30. — Development of the locomotor apparatus of trypanosomes. ^ A — F, De- velopment of Trypanosoma nocluw : A, the single nucleus of the "ookinete is dividing into two unequal halves ; in each half a centriole is seen, connected -with its twin by a centrodesmose ; B, the division of the nucleus complete ; the two sister-nuclei still connected by a centrodesmose uniting the centrioles ; C, the smaller nucleus (n. ) is dividing unequally to furnish a third nucleus (''.£7-) 5 D, E, the third nucleus is dividing to furnish a proximal and a distal {h.g.~) centriole, while the fibrils of the achromatic spindle become the myo- nemes (mij.);F, development of the trypanosome— iV, trophonucleus ; n, kineto- nucleus ; 6.7.^, basal granule (true blepharoplast) of the flagellum. In G the pigment (P) present in the earlier stages is being thrown off. After Schau- dinn (132). 0, stage in the development of the mcrozoite of Tri/fanosoma rolalorium into the trypanosome-form ; N, trophonucleus, still connected by a cen- trodesmose with M, the kinctonucleus, which has budded off h.g., the basal granule of the flagellum. After Machado (469). the distal centrosome is carried out on the tip of the flagellum ; and the mantle fibres form the myonemes, stated in this case to be eight in number, of the body, which are continued on into the contractile sheath of the flagellum (Fig. 30). However fascinating the views put forward by Schaudinn, with regard to these points, may be, it must be stated that the greatest doubt attaches to the correctness 60 THE PROTOZOA of the observations upon which they are founded, and that they lack confirmation entirely.* 3. Organs apparently of excretory function are present ui many Protozoa as the so-called " contractile vacuoles," one or more droplets of clear liquid which make their appearance in the ectoplasm, grow to a certain size, and then burst, emptying their contents to the exterior. When the contractile vacuole reaches its full size, it often bulges inwards far beyond the limits of the ectoplasm, and hence may appear to lie in the endoplasm ; but its first appearance is always in the ectoplasm, to which it strictly belongs. In non-corticate amoeboid forms the contractile vacuoles simply empty themselves to the exterior, and the changing form of the body does not pormit of determining whether the position of the vacuole is a constant one. It is common in amoebse for the vacuole to be lodged in the region of the body which is hindmost in progres- sion ; but this may be simply the mechanical consequence of the streaming movements in the protoplasm, whereby the vacuole is carried along to the hinder end of the body. In corticate forms, on the other hand, the contractile vacuoles are constant both in number and position, and void their contents through a definite pore in the cuticle, directly or indirectly ; in many Flagellata and Infusoria, for instance, the vacuoles do not discharge directly to the exterior, but into the oesophagus or into a reservoir-vacuole communicating with the oesophagus. The growth of the contractile vacuole is caused by fluid draming into it from the body-protoplasm. In amoebse and forms of simple structure no channels supplying the contractile vacuole are visible, and it must be supjDosed to be fed by a process of diffusion through the protoplasm from all parts of the body. In the highly-organized ciliate Infusoria, however, the deepest layer of the ectoplasm has a loose, spongy texture, and forms a definite excretory layer full of spaces contaming fluid, which drains into one or more main canals * It must be added further that, to judge from the figures left by Schaudinn and published on Plate xxix. of his collected works (" Fritz Schaudinn's Arbei- ten," Hamburg and Leipzig, 1911), the statements cited above appear to be founded on preparations made by a method of technique which is recognized generally as giving unsound cytological results — namely, the method of dried films stained by the Romanowsky stain. Schaudinn's statements are nevertheless cited above on account of the numerous theoretical discussions and speculations in modem protozoological and cytological literature of which they have been the foundation. For my part, I disbelieve entirely in the theory that the flagellum represents a centrodesmose between two centrosomes ; I regard it as a siraple outgrowth from a blepharoplast of a nature essentially similar to the axopodium of a Heliozoon. It is curious that no one has as yet extended Schaudinn's theory to the axopodia, the axial filament of which should also represent a centrodesmose, if that view is correct for the axial filament of the flagellum, a view that seems to me quite unthinkable from a phylogenetic standpoint. Is it to be supposed that the formation of each pseudopodium by a Heliozoon represents a rudimentary mitosis ? THE ORGANIZATION OF THE PROTOZOA 61 supplying the contractile vacuole or vacuoles. Thus, in Stentor 8) the single vacuole is fed by a canal running the length of he'body, and in Paramecium (Fig. 185) the two vacuoles are each surround^ by a number of canals forming a star-shaped figure. As regards the function of the contractile vacuoles, it should be noted i^ the first place that their contents are always fluid and watery, and never contain solid particles of any land. The fluid which a contractile vacuole drains from the body is doubtless replaced by water absorbed from the surrounding medmm by diffusion tlH-ough the superficial layer of the protoplasm, or it may be tlii-ough the mouth in some cases. The contractile vacuole is generally regarded as the organ of nitrogenous excretion, comparable functionally to the urinary organs of the Metazoa, but it is highly probable that the liquid discharged from it contains also the carbon dioxide pro- duced by the respiratory process. Hence the contractile vacuole may be regarded as both excretory and respiratory in function (see also p. 197, infra). 4. In the majority of Protozoa there are no organs for which a defi- nite sensory or nervous function can be claimed, although these organ- isms show by their reactions to the environment or to stimuli that they possess sensory and psychical func- 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 amoebse. Rhumbler (34) has shown that many amoebse 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 Fig. 31. — Pouchetia cornuta, one of the Dinoflagellata, to show the large stigma (st.), in front of which is a lens {L). 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 " Gregarmes (p. 326, infra), the body within the cuticle is entirely endoplasm. On the other hand, in Ciliata, in wliich 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 then- relative proportions are consequently more or less constant for a given phase of the life-history ; but in amceboid 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 pseud opodia, and least when it is in a restmg 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 of 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-vacuoleSy mmute droplets of fluid in which the solid particles This is one of a Series of Designs by the famous Artist, Walter Crane, specially prepared for tJie SCOTTISH WIDOWS' FUND LIFE ASSURANCE SOCIETY The Complete Set may be had free on application to Head Office: EDINBURCH-9 ST. ANDREW SQUARE. LONDON: 28 CORNHILL, E.G., and 5 WATERLOO PLAGE, S.W. SCOTTISH WIDOWS' FUND MUTUAL LIFE ASSURANCE SOCIETY ALL PROFITS DIVIDED AMONG THE POLICYHOLDERS A GREAT BONUS-YIELDING OFFICE FUNDS OVER £20,250,000 ANNUAL REVENUE £2,250,000 Prospectuses on application Head Office: EDINBURCH-9 ST. ANDREW SQUARE. LONDON: 28 CORNHILL, E.G., AND 5 WATERLOO PLACE, S.W. THE ORGANIZATION OF THE PROTOZOA 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 f^cal 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 stomodagum 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 defaecation, 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- THE PROTOZOA tion for developmental changes, especially in the female gamete m a manner analogous to yolk-grains in an ovum. Instances o^ bodies resultn.g from catabolic activity are waste-products of various kmds in the form of granules, crystals, pigment-grains, etc., often present m 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 m the mtracapsular 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 mternal 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 OF THE PROTOZOA {Continued) ^TUE NUCLEAR APPARATUS— CHROMATIN, 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 chromatia 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 chromatiti-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 contams, in addi- tion to one or more nuclei, extranuclear granules of chromatin, termed chromidia,* which may be scattered in the cytoplasm * The term " chromidia," ia 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 chromidia 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 of which the chromidial mass ia made up, and which grow and multiply by division like other elementary living bodies. It is clear, however, that tlie chromidiosomcs 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. THE PROTOZOA fheltTf '^i' -mre^-ted in certain regions of the body to form clu-omidial 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 bcmg then m the form of chromidia, from which nuclei arise by a process of condensation and organization of the chromatm in com- bmation with aclu-omatinic elements. Such a condition may be rega,rded 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, speakmg generally, a true nucleus, but only scattered grams of chromatin. Hence the chromidial condition of the chi-omatm 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 chi-omatm 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 mdividual opmions 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 chromatm from the nucleus, wliich either casts ofE a certain amount of chromatin mto the cyto- plasm, while preservhig its individuality, or may undergo complete fragmentation, becoming resolved entirely into chromidia, and ceasnig to exist as a definite nucleus. In other cases, cliromidia arise from pre-existing chromidia, by growth and multiplication of the chi'omidiosomes, thus keeping up a clrromidial mass or stock which is propagated from cell to cell tlirough 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 trabeculse, on a protoplasmic framework, and the mass is often vacuolated and contains substances other than chromatm. In Difflugia, Zuelzer (85) has shown that in the autumn the chromidial mass assumes a vacuolated or alveolar structure, and in each alveolus grams 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, Arcella (Fig. 32), the formation of so-called " secondary " nuclei (wliich, however, do not differ from other nuclei except in their mode of THE NUCLEUS 67 origin), by concentration of chromidia 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 m subsequent chapters. Those who regard the chromidial condition as the more primitive will see m 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 foregomg 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 Fig. 32.—Arcella vulgaris, 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 chromidia! ring. N>-, Primary nucleus ; N'^, secondary nucleus in process of formation ; chr., chromidial ring ; o, aperture of the shell. After R. Hertwig (65). is no evidence that chromatm, within or without the nucleus, can ever arise de novo or m any way except from pre-existmg 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 simjpler 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 chi'omatin in their properties and charac- teristics, representing stages in the buildmg-up of the complex material of the chromatm-substance. In one or the other of these two ways it is possible to account for bodies in the cell laiown by various names, such as " metachromatinic grains," " cliromatoid 68 THE PROTOZOA grains," and so forth— bodies which are often mistaken for true chromatin, 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), volutm is a nucleic acid combination which is to be regarded as a special reserve-material for the forma- tion of the nucleo-protems of the chromatin-substance ; during phases of the life-cycle in which the chromatm in the nucleus increases in quantity, the volutin in the cytoplasm diminishes, and, conversely, when the quantity of chi'omatin is stationary, the volutui-grains increase in number. Volutm-grams 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-grams, 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 chromatm. In some cases, however, the meta- chromatmic grams may represent chromidial extrusions from the nucleus which are breakmg down or being modified mto other substances ; compare, for example, the extrusion of vegetative ckromidia, 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 wliich resemble chromatin very closely may often render extremely difficult the task of identifying and distinguisliing the true chro- matin, esj)ecially when it is not concentrated into a definite nucleus, but is scattered in the cytoplasm m the form of chromidial grains. The test upon wliich reliance is most usually placed for the identi- fication of chi'omatm is its stainmg properties, and especially its readiness for combining' with basic aniline dyes and certain other colouruig 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 " stams more intensely than the true chromatin itself ; on the other hand, in cellular organisms Avhich jpossess true nuclei contammg undoubted chromatm, the staining reactions of the nuclei may be strikingly different in different cases. A good examiDle of each of * The iiamo " volutin " was coined by A. Meyer in 1904, and is derived from tlic fact that the substance was first studied by him in S'pirillum volutans. THE NUCLEUS 69 these statements is furnished by the trypanosomes parasitic m 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 ot 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 " 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 stam with certain dyes. As regards the chemical nature of chro- matin, it is characterized by containing protein-substances more complex in com- position than any other part of the cell ; it is not possible to say definitely, however, 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 chromatin 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 A clinosph cerium this .stain colours the plastin-framcwork, 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. Fig. 33. — Diagram to repre- sent in a graphic manner the action of colouring matters that stain chro- matin. The circle drawn with an uninterrupted lino 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, though notinvariably, but they will also stain other things which are not chro- matin. '^^ THE PROTOZOA or grain in the cell cannot be definitely identified as chromatin in an cases by .ny chemical or physical test, but only by its relation to the bfe anc development of the organism as a whole, and more especially to the function of reproduction and the phe'nome na o sex, as will be shown more fully by means of concrete instances in ubsequezit chapters. The sum of modern loaowledge with regard to the vital activities of living bodies and the life-liistories of organisms, whether plants or animals, Protozoa or Metazoa indicates that the chromatm 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 cuttmg Protozoa mto pieces some contammg 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 m- dividual of the same species — difEers 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 are followed by periods of depression, during which assimilation and reproduction are at a standstill, even in the midst of abundant nutriment (see especially Calldns, 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 trophochromatin. 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- THE PROTOZOA njatin 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 remainmg 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 Sarcodma 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 Arcella (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 chromatm. 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 chromatm (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 consistmg 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 enchylema or nuclear sap, filling THE NUCLEUS 73 the interstices of the linin-framework ; (3) pZasim, 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 Avhole nucleus is commonly enclosed in a memhrcine, 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 more 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- FiG. 34. — Cyclical vegetative changes in the resting nuclei of Tridiosflicerium 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 ; G, the same process 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 ; H, the network has become fine- meshed again ; /, a karyosome is being formed by condensation of the chro- matin at certain points, leading to the condition of A again. After Schaudinn, X 2,250. 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." THE PROTOZOA nuc e ^^.^.^^ ^^^^^^^^^ y , m a „ S actM^^^ of n T!f P^"^^ ^^^'^"g Periods actually undergoing the process of reproduction by division • but 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 ? ^«™gements of its chromatin-substance and other T)i?bn« "J^o; gregarine Porospora gigantea, Leger and -UUDoscq (72) have observed changes taking place rhythmically in o o rio 3o.— Successive stages of the kaiyosomo (sec p. 7G) of Porospora qinantea 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 SSscq (72) chromatin in front of the micropyle. After L6ger and the living condition (Fig. 35) ; compare also Chagas (48 5). Hert- wig (64) has shown that the structure of the nucleus of Adino- sphcerium 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 chromatui-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 Zmaa;-type, in which the THE NUCLEUS 75 nucleus consists of a Large mass of chromatin suspended m 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 ]ust such a nucleus as may be imagined to have arisen by a concentration of chromidiosomes at one spot in the cell-body, and m many cases such nuclei can be seen to be formed actually in this manner ihe kinetonucleus of trypanosomes may be considered as a nucleus of tliis type in which the single mass of dense cliromatm fills almost or quite completely the space in which it lies. In other eases there may be a clump of chromatin-grains more or less equal m 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, presentmg 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 difiicult 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 tlireads 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 reticulum 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 THE PROTOZOA I^inin framework as different in any essential point from the alveolar framework of the genera] protoplasm, with which, in nuclei tliat Jack a true membrane, it is perfectly continuous. Hertwig (66) regards the cytoplasmic 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 m 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 acliromatinic 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- sphcerium, according to Hertwig (64), the membrane is a super- ficial condensation of the acliromatinic 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, plastm is commonly, if not invariably, present in the form of masses or bodies which receive different names, according as they consist of pure plastrn 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 cases, 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 apjDlied 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 clu'omatin ; 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 hoemogregarine-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 cliromosomes of dividing nucleus (see next chapter). Goldschmidt (41) observed that the formation of generative chromidia in Maslirjdla (p. 265) was pre- ceded by the extrusion of plastin from the nucleus mto the cyto- plasm, to serve as a matrix for the chromatin which passed out from the nucleus subsequently. In AdinosvlKBrium, Hertwig has shown that a karyosome or chromatin-nucleolus, present during certain states or phases of nuclear activity, may give o£E its chro- matin-substance into the nuclear framework (reticulum), 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. Certam phases of the development of Actinosphceriwn are further characterized by the formation in the nucleus of numerous small plastm-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 achromatmic 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 plastin (nucleoli) or of plastm 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 strmg of beads (" moniliform "), or be branched 78 THE PROTOZOA radians a colony is formed by budding, which resembles super- hcia ly a hydroid colony, each hydranth being represented by the licad of an Acmetan mdividual with suctorial tentacles- the branched nucleus is continuous tlu-oughout the whole colony, 'pass- ing unmterruptedly from one mdividual to another. Typically the cell-body contams 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 difEerences in size, structure, and function. In many Sarcodina multiple nuclei without differentiation are found to occur constantly in certam species ; for mstance, two in Amoeba hinucleata and Arcella ; several, perhaps a dozen or so, in Difjiugia (Fig. 16) ; from twenty to forty up to some five hundred m ActinosphcBrium (Fig. 3) ; so also m Pelomyxa; and in the large plasmodia of Mycetozoa many thousands of nuclei are found. Differentiation of nuclei, when it occm-s, may be related to various causes. In trypano- Tnjixmo- somes and allied forms two nuclei occur con- stantly— a prmcipal nucleus, or trojjJionucleus, so called because it appears to regulate the general metabolism and trophic activities of the cell-body ; and a kineionucleus, which is m special relation tq the organs of movement, flagella, and undulatuig membrane. As a rule the kinetonucleus is smaller, in some cases very minute, and has a dense compact structure, while the tro]phonucleus has a vesicular struc- ture ; but in other cases {Trypanoplasma) 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 m relation to sexual phenomena and reproduction {vide p. 153, infra) shows that the macronucleus is composed of vegetative cliromatm, while the micronucleus contains the reserve generative chromatm. In some cases — for example, m 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 wantmg entirely ; but this statement Fig. 36. plasma (jurneyorum from the blood of the pike {Esox lucius). a.fl., Anterior flagel- lum ; n, kinetonu- cleus ; N, trophonu- cleus; ■p.fl., posterior flagellum f ormingthe edge of the undula- ting membrane, and continued beyond it as a very short free flagellum posteriorly. After Minchin (478), X 2,000. 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 Avhich 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 may 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 pah: of grams (" 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 arcJioplasm, 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 stnations themselves being the optical expression of a radiate arrangement of the protoplasmic alveoli (meshes of the "retic- ulum "), indicating Imes of force or tension centred in the centro- some. In some cases it is probable that archoplasm showing radiate striations may be present without any centrosome. In Actinosphasrium Hertvvig 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 retaui permanently durmg both the resting and dividmg conditions of the nucleus. The simple nuclei of the protokaryon-type probably contain in most cases a centro- somic grain 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 examjiles of the occurrence of extranuclear centrosomes in Protozoa ; but these are for the most part cases in which 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 mmute grain, 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 correspondmg to different phases of the activity of the nucleus. Thus, m ActinosphcErium, Hertwig (64) describes the centrosome at its first appearance as a relatively large body of spongy structure, formed at one ^Dole 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 famter and disappear (Fig. 37, F, O). 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 the 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 , .-.■^•t^ti'./j;.. Fig. Sl.—Achnosphoerium eichhorni: formation of the centrosome. A, Concentra- tion of the nuclear reticulum towards one pole of the nucleus, near which n appears free from granulations, forming the archoplasm ; B G,^ I), passage of a portion of the nuclear reticulum to the exterior to form the spongy centrosome " lying in the archoplasm ; B, spongy centrosome with striations passing from it through the archoplasm to the nucleus ; -f, Cx, the 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 wliich 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 may 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 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 comiection ; in other cases it repre- sents the remains of the nuclear spindle of the previous nuclear division, as in the swarm-spores of Stemonilis flaccida (Jahn, 69) and ABC Fig. SS.—MasUgina setosa. after Gold- Fig. 39.— Connection of the flagellum Schmidt (41). n., Nucleus from which and the nucleus m Mashgma setosa. the long flagellum arises; the body A and 5 As seen m the living is full of diatoms and other food- state ; 0 after fixation and staining, bodies. The surface of the body has a After Goldschmidt (41 ). covering of short bristle-like processes. the collar-cells of Heteroccela (Robertson, 79) ; wMle in some instances it may be formed by outgrowth of root-like processes, of no special cytological significance, from the 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 : , • r ^--^^o 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, which is a centro- THE NUCLEUS 83 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 maybe 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- tigella (Fig. 40), and by collar-cells of the Clathrinid type, the fiagellum takes origin quite independently of the resting nucleus. In both cases alike, the flagel- lum generally disappears before division of the nucleus begins ; the blepharoplast becomes the centrosome, 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 (j 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. ^.-S n. Tha. 40. — Mastigella vitrea, after Goldschmidt (41). n, Nucleus, almost obscured hy the mass of food-bodies of various kinds in the cytoplasm. 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 groAv 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 sflendida, to show different ways in which the daughter-flagella arise. Compare the stages of 8. uvdla (Fig. 42). A, Resting condition of the cell. B, Early stage of mitosis; the two flagella of the parent cell are in process of absorption, together with their blepharo- plasts. G, Daughter-flagella 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 blepharoplasts are still enclosed ; in the other daughter- nucleus the blepharoplasts have become distinct and the flagella are given off from them. E, Simflar 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 origm 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 centrosome, and subsequently a neAV flagellum ; 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 karyosome, in which the centriole is lodged, and a few irregvdar grains of peripheral chromatin in the nuclear cavity. B, Early stage of mitosis ; an achromatinic spindle is formed with the centrioles at the poles, one centriole (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 flagella have disappeared, together with their blepharoplasts. C, Similar stage in which the daughter-fiagella are growing out precociously from the centrioles, one on the left, two on the right. D, Later stage in which the equatorial plate 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, Division of the nucleus nearly complete ; no flagella. F, Nucleus completely divided, daughter-nuclei in process of reconstruction ; from each a pair of blepharoplasts has been budded off, still connected by a centrodesmose with the centriole contained in the karyosome ; a pair of daughter-flagella has arisen from one pair of blepharoplasts, but not as yet from the other. After Hartmann and Chagas (62), magnification about 2,400. 3. In certain flagellates — for example, trypanosomes and allied forms (" Binucleata ") — the cell-body contains two nuclei, as already noted : a trophonucleus and a kmetonucleus. To what extent these nuclei are provided with centrosomes is at present a little doubtful ; probably this 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 D C F Pig. 43.- — Stages in the division of Polylomdla agilis. A, Resting condition of the cell ; the four flagolla arise from four blepharoplasts which arc connected by a rhizoplast with the nucleus ; in the nucleus is seen a largo karyosome, which contains the centriolc 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 centrodesmose. 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 chromosomes have fused into conical masses ; centrioles and centrodesmose still visible. F, Division of body beginning. 0, Centrodesmose broken through, the two daughter-nuclei separate. H, I , J, Division of cell complete, one daughter-cell only represented, to show the reconstitution of the daughter-nucleus ; the polar cap becomes the Icaryosome, enclosing the centriole ; the conical mass formed in Stage E by fusion of tlie 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 agani 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 kinctonucleus 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)* ; (6) the trophonucleus may have a centrosome of its own, lodged in the karyosome, m addition to the centrosome-blepharoplast in connection with the kmeto- nucleus ; this is probably the most usual condition with two sub- ordinate variations, according as the centrosome-blepharoplast is lodged within the Idnetonucleus, a,B in Leishmania tropica (Wenyon, 84), or is situated close beside it, as in most trypanosomes ; in either case the kinctonucleus 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 kinctonucleus, and lastly the tropho- nucleus, divide. The various forms of flagellar 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 contaiiis a single centriole (Fig. 44, 0="), we may suppose the flagellum at its first origin to grow out from the centriole in the nucleus {0^). No such condition is actuaUy known amongst flagellates, though it may be compared to the origin of the axopodia from a central gr'ain 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 (1'^) or becoming quite independent of it (1''), the two variations of the first type. The second type 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 (2^) or become quite independent of it (2^). The third type may be supposed to arise from the hypothetical primitive condition (0") by supposing that, not the blepharoplast- centrosome alone, but the whole nucleus, divides to form two nuclei of unequal size and distmct function, the trophonucleus and kinctonucleus, each with its own centriole (3'^, 3^). The centriole of the kinetonucleus, which is at the same time the blepharoplast, may either remain within the kinetonucleus (3*^) or come out of it (3"=) ; its relations to the kinetonucleus are parallel to those of the centro- some-blepharoplast to the nucleus in types P and 1'^. 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 3-' or 3'' 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 tliis 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 trypanosorao or other " binu- cleate " exists which has but a single division-centre in the cell. 88 THE PROTOZOA form two or more grains when there are numerous flagella. Thus, in Lopho7nona% Avhich 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 Fig. 44. — Diagrammatic representation of the possible phylogenetic origin of the different tyi^es of flagellar attachment in flagellates. For the sake of sim- plicity it is supposed that the animal has but a single flagellum. 0", Non- flagellated cell with a centriole in the nucleus ; 0^, in a cell like the last a flagellum arises from the centriole ; condition with a flagellum arisuig close beside the nucleus ; 1'', condition with the blepharoplast quite separate from the nucleus ; 2", division of the single centriole into a definitive centro- some and a blepharoplast, which becomes quite independent (2'') of the nucleus ; 3*, division of both nucleus and centriole to form distinct kinetic and trophic nuclei, each with its own centriole ; the kinetonuclear centriole remains within the nucleus ; 3S the kinetonuclear centriole becomes distinct from the nucleus ; 3"', condition with a single centriole in the cell ; 3^ condition with a blepharoplast distinct from the centrioles 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 flagella takes origin from the distal granules of the ring. When the nucleus divides, the daughter-centrosomes give rise to new rmgs of blepharoplasts, THE NUCLEUS 89 from which daughtei-tufts of flagella grow out ; the old tufts, with then- rings of blepharoplasts, persist for some tjme after the new ones have been formed (Fig. 45, G), but ultimately they degenerate and disappear. The ring of blepharoplasts m 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 TrichonympJddce ; 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 LopJiomonas— namely, a double rmg of blepharoplasts, which, however, fuse together to form a ring of homogeneous appearance. The blepharoplast-grains 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. 40. — Actinojilmjs sol, showing the axial filaments of the i^seudopodia centred on the nucleus. N , Nucleus ; fs., jiseudopodia ; 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 m Masligina and Mastigella respectively, and which may be explamed by supposmg that in the one case the kinetic centre lies within, in the other case without, the nucleus itself. Thus, in Actinophrys (Pig. 46) the numerous pseudopodia are all centred on the smgle nucleus, in which the centrosome is contained. A variation of this type is described by Schaudinn (43), in the peculiar multmucleate form THE NUCLEUS 91 Camvlonema nutans, in Avhich a pseudopodium 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 Acantlwcyshs a centriole is contained in the karyosome of the nucleus, but during the growth a ^ ^ . .2 05 o ut 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 mto as many daughter-individuals as there are nuclei or pairs of dif- ferentiated nuclei ; this method is known as muUiple fission (Fig. 127) ; or it may be — , . , • . a Ih) Permanent, givmg rise to a multmucleate body which is termed a vlasmodmm. Then division of the body may take place at any time by cleavage of the body into two or more multumcleate narts • this process is known as plasmolomy. Ultimately, however, in all 'cases a plasmodium breaks up by multiple fission mto 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 m more detail, beginning mth — 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 " cliromidial fragmentation." It is of comparatively rare occurrence, but examples of it are found among Sarcodina and Sporozoa. In EcUno^oyxis 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 o£E into the cytoplasm a fine dvist 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 cyto]3lasm, must be distinguished clearly from the above- mentioned process of chromidial fragmentation. In 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 nucleus divides simultaneously into a number of portions by multiple 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 m 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 A B Fig. 50. — Formation of microgamotes 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 chromidia have collected at the periphery of the body ; the karyosome is left at the centre, and has become jjale 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 packed. 0, H. The patches of chromatin take on a definite form as the future nuclei of the microgamotes. /, Two flagella grow out from close to each microgamete-nucleus, and by their activity the microgamotes, 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 eind ; 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. Nucleus, k karyosome, of the microgametocyte ; n', n'. nuclei of the microgamete^. After Schaudinn (99); A—E magnified 1,000, F—I magnified l,oOO, 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 acliromatinic spindle is formed by the centrodesmose, if it persists, and the remamder of the spindle is constituted by the so- called ' ' mantle-fibres ' ' running from pole to pole. The mantle-fibres are derived from the achromatmic reticulum of the nucleus and the archoplasm ; they are probably in 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 in line with one another, while the cross-junctions are the transverse walls between consecutive alveoli. On tliis 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 ]Drobably 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 karyoldnesis 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 acliromatinic 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 series 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 nuclccar division is found, from the most simple cand direct to karyokinesis as perfect as that seen in the Metazoa. The nuclear division-processes of Protozoa arc 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. Spealdng generally, the mdirect 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 remammg 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 Avhich 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 comj)lication, is exemplified by the division of the nucleus in the schizogony of Goccidium (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 acliromatinic 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 karyosome. F, Nucleus of full-grown schizont. G — 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 or 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). k^, Karyosome ; F, F, daughter- karyosomes ; 1, intermediate body. After Schaudinn (99), magnified 2,250. jPio 59 —Direct division of the nuclei in the oocyst of Coccidium sclmhergi. A, The it^ting nucleus; B, 0, D, clumping together of the ehromatin-granuies preparatory to division ; E, F, G, the nucleus elongates and becomes dumb- bell shaped ; H, the nucleus has just divided into two hahes. After Schaudinn (99), magnified 2,250. also does not become difierentiated. As the daughter-karyosomes connected by a centrodesmose, travel apart, half the periphera 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 arc seen m 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 hfemogregarines (Fig. 53). In these two cases the presence of a centriole in the nucleus is doubtful, but is affirmed by Hartmann and Chagas (89) for hsemo- gregarines ; a true nuclear membrane. Fig. 53. — Direct division of the nucleus in the zygote of Hcemogregarina stepanowi. 3 , Degenerating male elements attached to the zygote ; N., divid- ing nucleus of the zygote, two successive stages (A and B). After Reichenow (78). C.V.- 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 present (Nagler). When centrioles are 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 ofE a smaller daughter-nucleus from the main mass. Remark- able instances of 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- FiG. 54. — Paramecium cauda- tum : division showing the macronucleus (N) dividing without mitosis, the micro- nucleus (n) dividing mito- tically. c.v.^, Old, and c.v.^, new, contractile vacuoles. After Biitschli and Sclie- wiakofE, in Lcuckart and Nitsche's Zoologische Wa7id- tafeln, No. Ixv. 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 ccntrodesmose 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 entirelj') from the peripheral zone of chromatin, partly from the chromatin contained in the karyosome. A good example of tliis mode of division has been described by Aragao (87) in an amoeba named by him A. diflomitotica from the fact that two types of mitosis occur in this species. In the first type (Fig. m,A—G), the little rod -like chromo- somes are not arranged in a definite equatorial plate, but are scattered irregularly along the 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, representmg 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, ^). _ In the second type of mitosis seen in A. dijjlomitoitca (J^ig. 5b, H—K), the chromosomes arrange themselves in a definite equatorial Fig. 55. — Budding in Podophrya gemmipara. The macronucleus of the parent has sent off a number of outgrowths, which extend into the buds and give rise to the nuclei of the daughter-individuals about to be budded off. N^, Parent-nucleus ; N^, nuclei of buds. After Hertwig. THE REPRODUCTION OF THE PROTOZOA 109 plate, which divides into two equally definite daughter-plates com- posed of distmct chromosomes ; Avhether 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, 95) Fig. 56. — The two methods of Duclear division in Amaiba diplomitolica. A, Resting nucleus ; B—G, first method ; H—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, hoAvever, the entire mitosis goes on within the nuclear space. The chromosomes may show every possible condition in different cases, from complete irregu- 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 brealcs 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 elements — namely, plastin, chromatin, and centriole Fig. 57. — Nuolcar division in Arcella vvlgaris: karyokinesis of one of the two principal nuclei. A, Spireme-stage, resulting from disruption of the karyosome ; Ji — D, formation of an equatorial plate of minute chromosomes (?) which split; E, anaphase ; F, the two daughter-nuclei shortly after division. After Swarczcwsky (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 plastm-substance at the same time fiu-nishing the required ground-substance of the chromosomes (Fig. 58). On the other hand the karyosome may disappear from the restmg nucleus also •' Chatton (49) has brought together a number of mstances of nuclei showing a gradual reduction of the karyosome m different species, and the evolution of a granular type of nucleus in which the chi'omatin is scattered through the aclu-omatinx 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 karyokmetic THE REPRODUCTION OF THE PROTOZOA 111 figure may be produced, as in Euglypha (Figs. 59, 60), only differing from that of Metazoa in that the whole mitosis takes place withni 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 karyokmetic mmmm Fig. 58. — Division of Hcematococcus pluvialis. A, Resting condition, the nucleus with a conspicuous karyosome and fine grains of chromatin in an achromatin'c reti- culum ; B, G, preparations for nuclear division, the chromatin jDassing from the karyosome into the nuclear reticulum ; D, further stage, the kaiyosome in disruption and chromosomes beginning to bo formed ; E, nuclear spindle ; F, division of the nucleus comjDlete, the karyosomes reconstituted in the daughter -nuclei, the cell-body beginning to divide ; G, 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 Fig. 59.— Division of Eughjfha alveolata, as seen in the living animal. q ' 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 of hvaline contrictile vacuole (c.v.) ; (3) near the mouth of the shell is a zone of hyaline protoplasm from which the pseudopodia {ps.) are given ott. n Earlv stase of division, about twenty minutes later than A. xne pioto t,lasm is stiSng out of the shell-mouth to form the body of a daughter- the division of the karyosome, no longer visible m the "^'^^^Z^, from a centriole contained in the karyosome). [GonUnued at foot of p. 113. J THE REPRODUCTION OF THE PROTOZOA 113 Fig. 60. — Details of the structural changes of the nucleus of Euglypha alveolata during karyoldnesis, 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. B^, Some of the fibrils of this stage more highly magnified. C, Later stage ; the fibrils have become smoother and more parallel in arrangement. C^, Fibrils more highly magnified ; they consist, as in the last stage of darker and lighter parts (the former chromatin, 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 Schewiakolf (100) ; magnification of A, B 0, 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 Fig. 59 — continued: D About fifteen minutes later than Q. The daughter-shell is now com- pletely formed, and the middle granular zone of the parent is passing over mto 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 ot tfie 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 ' fi^?. minutes later than E. The daughter-plates have travelled apart, and the division of the nucleus is beginning vc tid-veiiea 0 About five minutes later than F. The division of the nucleus is com- tXX^''' d-i^gbter-nucleus has passed over into the body of the daughtS- be^i^n'in^r 1''*^'' ^ ^25 minutes from the thf Trl^' V. n 1°^ P™t«Pl^sm of the middle zone flows back into £?.?hfr f"' """^ ^^'^ individual has its own contractile vacuole ; the two daughter-nuclei are reconstituted, and the karyosome has reannearorl in each ; pseudopodia are being protruded from thj mouths o the TeSs the division IS complete and the animals are beginning to separate. ' After SchowiakofE (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, whi,ch arrange themselves at the equator. At the same time the achromatinic framework shows a longitudinal fibrillation or striation, the apparent fibrillse 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 fibrillatcd final than F e Fig. 61. — Stages in the division of the micronucleus of Para- mecium. A, B, Early stages ; C, spindlc-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 ; /. reconstitu- tion of the daughter-nuclei, which are widely separated, but still connected by the greatly elongated separation- spindle the central part of which shows a dilatation prior to its absorption. After Hertwig. Figs A—E are drawn on a larger scale 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 contmues 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 bendmg or twisting of its fibrils, as the result of the inert resistance of the sur- rounding cytoplasm. Finally, however, a limit of growth is attamed ; the daughter-nuclei become constricted oS 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 difierent modes of karyokmesis, which, however, for present purposes may be classified under two heads : karyokinesis without and with centro- somes. In the ordinary nuclear division during the vegetative life of the organism, and also ui the divisions by which the primary -r G Fig, &2.—Achnos'phcerium eichhorni :_sta,ges of the ordinary, vegetative nuclear division, without centrosomes, ^^of free-living individuals (not encysted) A ii, i^ormation 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 (r.p.), and equatorial plate of chromosomes (e.r)- V, stage with daughter -plates of chromosomes which have travelled towards the polar plates ; E~G, division of the nucleus, reconstitution of tne daughter-nuclei, 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 o£E from each seoondarv cyst. In the ordinary karyokinesis of Actinosphcerium (Pig 62) an equatorial plate is formed composed of a large number of small, rod-hke 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 mm ir « '■ > « Fig. «3.-^c«mosp7i«riMm cicWiorni ; first reduction-division, with centrosomes (the stages here shown follow those of the ccntrosomc-formation ^ ^.g- 3 )• T ccntrosome with radiations in a mass of archoplasm at one pole of the nucleus ; J two centrosomes and archoplasmic cones, teknig up posi^^ions nn oOTOsite sides of the nucleus, in which chromosomes are begmning to appear , formatSn o the nuclear spmdlo and equatorial plate of chromosomes ; 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 archoplasm 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 Acantliocystis (Fig. 64), however, the nuclear membrane disappears completely from the karyokinetic figure, and it is no longer possible, m 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 move reasonable to expect that in some cases at least other distinct types of division-mechanisms would have been evolved — side- IS. B A F G Fia. 64.— Division of Acmithocystis aculcaia. A, Resting state of the animal. N Nucleus ; c, central grain ; a.f., axial filaments of the pseudopodia, ps. ; sv' spicules. B, Pseudopodia withdrawn; nucleus in the spiieme-stage ; central grain dividing. G, 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 tlic membrane is becoming dissolved, the reticulum is becoming modifaod in arrangement to form the achromatin^c spindle (or a part of it), and the chromo- somes arc taking up their position in the equatorial plate. E (^tnpicte nuclear spindle, with centrosomes, achromatmic spind e, and equatorial plat«. TLater^stago with daughter-plates and division of the cell-body beginning^ G. Division of the nucleus and of the cell-body nearly complete. After Schaudinn (82). THE REPRODUCTION OF THE PROTOZOA 119 branches, cas it were, of the stem which culminates in the Metazoan type. An example of this is seen in the peculiar karyolcinesis of Nodiluca (Fig. G5), in which the division is directed by a large " sphere," consistmg of a mass of archoplasm containmg the cen- trioles. The sphere divides and forms the axis of the karyokinetic figure, of which 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 oflEends as being too teleological, we may alter the plirase- ology, and inquire. What is the result of the process 1 The answer is perfectly obvious. The result egected by equating karyokinesis Fia. G5. — Stages in the nuclear division of Noctiluca miliaris. A, Early stage, the "sphere" (sph.) beginning to divide, the nucleus wrapping round it; B, later stage, the sphere nearly divided, the two poles of the nuclear spindle in section attached to the two daughter-spheres ; G, 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, smce 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 multieellular 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 tliis condition could only be brought about, prob- ^b^Y' karyoldnetic division of the nuclei in the process of cell- multiplication. In the foregomg 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 Trachelocerca (Fig. 66 ; see also j). 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, Fm. 66.— Four stages of direct multiple fission in „„rUliPiTmltinlpfission the nuclei of Trachelocerca fhcenico'pterus. After ana tne multiple nssion 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 of£ singly from it, or by the nucleus as a whole breaking up ; then each karyosome becomes the foundation of a new nucleus. Division of this type, which may be termed a multiiDle promitosis, has been described by Zuelzer (86) m Wa^- 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 m the male sporont of Aggregata (Fig. 67), as described by MorofE (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. 255, infra). In conclusion, men- tion must be made of the theory of cell-divi- sion and of the causes which bring it about, put forward by Hertwig (91, 92). This theory is based on the sup- ]Dosition, of which men- tion was made in the previous chapter (p. 70, supra) — that for the normal performance of Fig. 67. — Multijjle nuclear division in the male vital functions a cer- sporont of Aggregata jacquenieti. The nucleus, of . . which tlie outline has become irregular but is still tarn quantitative re- visible, is surrounded by eight centrioles, from lation must be main- each of which striations pass towards and into , • 1 1 J ,1 the nucleus. After Morolf (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." Hertwig (GG) has criticized this conception, and has shown its untenability in the case of Protozoa, which behave as single individuals whether they possess one nucleus or many. Hartmann, considermg 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 karyokinosis as the division of a polykaiyon, in which each separate chromosome represents a distmct nuclear element or monokaryon — a conclusion which appears to lead rather to a reductio ad ahaurdum of the theory. 122 THE PROTOZOA m 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 cand 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, restormg the standard balance of nucleus and cytoplasm. Relative increase of the nuclear substance retards the cell-division, and brmgs about increase in the size of the cell ; relative decrease of the nuclear mass has the opposite efiect. 2. Division of the Cell.— A distmction has been drawn above between binary fission, or division of the body mto two, and mul- tiple fission into many parts simultaneously. The daughter-indi- viduals produced in 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 jDarts 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 gemmation, 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 Icb? amount of residual protoplasm, which degene- ates and dies off ; budding of this land is termed sporulation. 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, ua 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-mdividuals, 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 Fi(}. 68.— Budding of Acanthocyslis aculaata (compare Fig. 64, A). A, B, Division of the nucleus, in which the central grain takes no part ; G, extrusion of a bud ; D, three buds in process of extrusion, the nucleus of the jmrent dividing again; E, free bud; F, flagcllula, and G, aracebula, produced from buds; II 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- tudmal. In any case, the two products of fission may be equal or subequal m size, without perceptible difEerence 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 question 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-mdividual, 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, al one 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 buddmg, and in Arcella (Fig. 80) and some amoebfe, 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 Kvwpi. vefj-effo-ard, Kvirpi dvaroiaiv a.irexO-t)S. 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, Cojyromonas (Fig. 111). 125 126 THE PROTOZOA Sexual union of similar gametes is termed isogamy ; of dissimilar amsogamy. 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 kmd, 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 lands of cell-individuals, the one rich in motile or kinetic protoplasm but poor in trophic substance, 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 mdividuality. 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 (Pig. 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, S3Tigamy is essentially nothing more than an intermingling of chromatin-substance derived from two distinct cell-individuals. Plus ga change, plus c'est la meme chose. The chromatm 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 ImoAvn cases amongst Metazoa and plants, syngamy takes the form of karyogamy. The nuclei of the gametes are termed pronuclei, and the nucleus that results from fusion of the pronuclei in the zygote is termed a synkaryon. In many Protozoa {e.g. Coccidmm, Fig. 69) the fusion of the two pronuclei is effected by moans of a peculiar mechanism termed a " fertilization- spmdle." 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 cliromatin 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 pronuclei, 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 microgametes ; 0, 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 and fertfiization is complete, c? , Microgametes ; ? , macrogamete ; w c? , male pronucleus; w?, female pronucleus; f.s., fertilization-spindle; c, oocvst • no synkaryon. After Schaudinn (99), magnified 2,250. ^ ' True sjTigaray, as defined above, must be distinguished carefully from certam 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 the mother-cells of the gametes the gametocytes, enter mto a more or less close association prior to the formation ot gametes, which are produced in due course and then perform the act of syngamy m the normal manner. An example of such association is seen m gregannes (p. 3.30), where association between adult gametocytes is the rule. Sometimes the two gametocytes associate in the earliest stages of their growth, as m Diplocystis (Fig. 70, A), and their bodies may then fuse ^w!^^7n^ ^/ ''i ^^^^^ ^^'^^in distinct, as in Cystobia (^ig. 70, Ji) and give rise in due course to the pronuclei of distinct gametes LTr!!!^"'" P''f,''?,T''^ ''association of this kind occurs are described as bemg 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 plastogamy (or plasmogamv) from true syngaray. Plastogaraic union may be temporary or permanent • in the latter case It loads to the formation of plasmodia, as in the Mycetozoa (p. ^6J). iho signihcance of plastogamy is obscure in many cases but in some It may perhaps be comparable to the association of gametes 'already described, and m 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 m 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 aflagellar 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 blood of the host. Similar phenomena are well known in bacteria as agglutination, since in this case the agglutinated individuals are unable to separate, while in Fig. 70. — Precocious association and noogamy of gametocytes in gregarines. A, Diplocystis minor, parasite of the cricket: m., common membrane uniting the two associates ; g., grains of albuminoid reserve-material. B, Cijslohia liolothurice, parasite of Hdolhuria tuhiUosa, showing the two nuclei in an undivided body. A after Cucnot, 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 s3Tigamy 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 mdividual 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 8EX IN THE PROTOZOA 129 reached the organism tends to reproduce itself. In Protista, as described in the last chapter, two principal types of rejiroduction 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 in their 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 ; contuauous, 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-liistory is a recurrent cycle, of which an act of sjmgamy 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 livmg things ; secondly, that it is an acquisition at some period or stage in the evolution of organisms, and not a primary characteristic of livmg beings. The sex-philosopher Weminger* 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), standmg in fundamental antithesis to one another, and combmed 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 hvmg bemgs 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 chromatm, 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, iHghly doubtful. In Protozoa, syngamy has been observed to occur m a vast number of forms, but by no means in all. In the Weiningcr, 0., " Sex and Character," chapter ii. London : W. Heinemann, 0 130 THE PROTOZOA case of those species in which syngamy has not been observed, there arc three abstract possibihties : 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 plienomena 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 m 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 constitutmg the germen ; they are lodged in the soma and dependent upon it — parasitic upon it, so to speak Ibut in a sense distmct 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 mdividual, 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 iii every species, though ion-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 stitutiiig the complex body of a Metazoan individual ; the other represented by the single cells of which both soma and germon alike are built up. The phrase " reproduction," whether sexual or non-sexual, as apijlied 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- mdividual 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 remam separate from one another and independent, so that no multicellular body is produced, except in the colony-building species, nor is there any distuaction of somatic and germinal cells, save in rare cases, such as Volvox (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 r 1 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 m Protozoa, any more than in Metazoa ^ ^ It has been pointed out above that the life-history of a Protist Tr'' 1"™'^ and Voductt 1 1 those Piotozoa m which syngamy has been observed, it is found a period of reproduction, sometimes at the end of a period cf may be a difference between the two sexes of the same species ll l owT!n1 ''''' '^^^^'^y ^'^kes place be ween uU grown individuals of the species, as in Aclmophrys (Fig 71)- second case, syngamy is between the smallest individuals produced 132 THE PROTOZOA by fission or gemination, as in Eoraminifcia (p. 235), Arcella (Fig. 80), etc. — so-called microgamy, which may be isogamous or slightly anisogamous. 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 beiiag 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 Tig. 71.-Copulation of Actinophrys sol. A. Two associated free-s vimmmg individuals. B, The two individuals arc beginning to encyst tliemselves ; then nuclei (N., arc preparing for kaiyokinesis ; an outer gelatinous envelope {g) rs ier^fed round ^hc\wo |anietes;and -l-/--;^-^^ mcLvidu.^^ cyst-envelopc (c. ), incomplete at the surface of contact. G. The i^u^lcus o e^^^ jrLiotc is div ding by karyokincsis (first polar spindle, 2^.52^. . D, i'oimation of he polar odief or reduction-nuclei {r.«.) ; the reduced pronuclei (pn.) tike a cSral position in the body of the gamete ; the ^oj^^.;^^^^^^^^^ arc beeiiminc to fuse. E. The pronuclei are fusing ; the rcduction-nuciei lia\ e rsse^tSl tl e wall of the inner cyst. F, The synkaryon (s/:.) is bcginnmg frdivicb by karyokinesis ; the degenerating reduction-nuclei h^ve passed out of the iniS- cys^? N., N., Nuclei of the gametes before reduction. After • Schaudinu, magnified about 850. to all vital functions of the organism, and therefore only indirectly to reproduction-that is to say, only hi so far as reproduction may result from renewed and mvigorated 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 ^y-^-^^ - ^^^^^f into relation with the production of a higher ^^^^uali y, the body, comparable to the whole Protozoan life-cycle, and i is m this ense . hat the phrase " sexual reproduction must be under- SYNGAMY AND SEX IN THE PROTOZOA 133 stood ; as already pointed out, sjaigamy 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 distmct. 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 tauglit. It is, I believe, the pre- vailing view among zoologists, and has boon enunciated clearly by Calkins (5). It has, however, been attacked vigorously by Doboll (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 aro not to be regarded as vmicellular, 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 wliich have reached what may be considered as the truly cellular type of organization. I am not, therefore, concerned with Dobell's 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 oialy cases m which the ovum breaks up into cells by 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 ordinary textbooks of embryology. I must content myself with a single instance, that, namely, Avith 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 u., p. 68), the ontogeny may be divided into four phases or perfod's, which mdicate clearly, in my opinion, the general Imes 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 bmary 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 remam connected to form a colony as for example, in many Phytomonadina. 2. Of the blastomeres thus formed, a certain number, variable in different species, biit relatively few, retain their original characteristics, while the rest become differentiated into columnar flagellated cells forming the wall of a cavity (blastocoelo). The undifferentiated blastomeres give rise to the archajocy OS, from which ultimately the germ-cells and gametes arise. The tlagollatcd col s are the ancestors of the tissue-cells (histocytes) in tho future sponge. At this stage, in which tho embryo is hatched out as a freo-swhnmintr 134 THE PROTOZOA U^u Porfoctly comparable to a colony of flagellates such as Volvoz, in which the ordinary individuals have lost the .power of becoming, or giving rise to, gametes, which can only arise from certain special individuals. 3. The free-swimming larva, composed mainly of flagellated cells, with the archaaocytos cither at the hinder pole or in the mteriial cavity, undergoes changes as it swims about, which consist in some of the flagellated' cells losing their flagellum, becoming modified in structure, and migrating mto the interior of the larva ; in this manner the two gorm-layers arc 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 sufiieient 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 colls undergo a comiilote rearrange- ment, which shows clearly that every coll 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 colls from one another. I am unable, therefore, to accept the standpoint of Dobell with regard to the relations of Protozoa and Metazoa, but consider that the comparison of a Protozoan individual to a single cell in a Motazoan 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 Plagellata 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 ?" it may bo rephcd that in many Volvocinea? 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 Motazoan embryology. In Protozoa the iilasraodial phase is always temporary, and ends sooner or later by breaking up into separate uninucleate individuals. 4. TJie Effects of Syngamy — (1) wpon the Individual, (2) wpon the Species. — 1. Of all Protozoa, the ciliate Infusoria are the group in Avhich sjmgamy 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 envkonment, becomes less vigorous SYNGAMY AND SEX IN THE PROTOZOA 135 and shows signs of enfeeblement 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 sjoigamy 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 Cihata 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 m abeyance are not wanting even in higher organisms. An instance ^u*Ju ^'^ '^^^^'^ "'^ Paramecium, Woodruff (142) expresses the view that most, )f not all, normal individxials have, under suitable environmental conclitions, unlimited power of reproduction without conjugation or artificial stimulation." Compare also Woodruff and Baitsell (14.3). 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 floAvers are sterile and incapable of fertilization, consequently the tree bears fruit which are entirelj^^ seedless. Hence the cultivated banana- tree is propagated entirely by a non-sexual method — namelj^ 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 sjrngamy can be entirely dispensed with m an organism rela- tively so high in the scale of life as a flowermg plant, it seems probable in the highest degree that the same may be true m 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 Euro]De and propagated for many years from one infected animal to another by artificial uaoculation, without the natural agency of an inverte- brate host. If it be true, as is generally believed, that in trj'-pano- somes syngamy takes place in the invertebrate host, then in the long- continued artificial propagation of pathogenic trjrpanosomes 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 examj)le 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. 305, infra). But it is certain that in trypanosomes generally, 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 distmguish 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 occm's 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- cycle, from one act of syngamy to the next, may comprise, ap- parentl}^ 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 iadefiiaitely, in some in- stances or under special circumstances. Two other phenomena of apparently Avidespread 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 C D Metazoa of various classes Fig. 72.— Parthenogenesis of Plasmodmm vivax. that it is unnecessary to ^. A female gametocyte, of vvhich the nucleus cite instances of it here ; it may be defined briefly as the power to develop without syngamy possessed by a sexually-dilf erentiated 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 mherent 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 m Trypanosoma noctuce, and by Prowazek for Herpetomonas muscm-domesticce ; none of these cases, however, are entirely free is dividing into a darker jjortion {n^) and a lighter portion [n^); B, the separation of the two parts 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.f.) containing the melanin-pigment. After Schaudinn (130). 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 m 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 Entamoeba coli, made Icnown by R. Hertwig (64) and Schaudinn (131) re- spectively. In Actinosphcerium an ordinary indi- vidual (Fig. 3) be- comes encysted as a multinucleate " m o ther - cy s t , " 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 distmct 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 viuscm-domesticce a process interpreted by him as parthenogenesis (" etheogenesis ") of male individuals, but the correctness both of his observations and of Ms 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 distnict organism named by Flu Odosporea muscce-domesticce, and now referred to tlie Microsporidia. It is greatly to be deprecated that interpretations of such un- certain validity should bo used, as has been done, to support general theories nx the discussion of tlie problem of syngamy. Fig. 73. — Autogamy in Entamceha coli. A, The amoeba at the beginning of encystation with a single nucleus ; B, the nucleus dividing ; G, the two daughter-nuclei throwing off chromidia ; a space has appeared be- tween them ; D, each nucleus has formed two re- duction-nuclei, winch are being absorbed ; E, a resistant cyst-membrane has been secreted ; the partial division in the protoplasm has disapi^cared, and the two reduced nuclei arc 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 synkaiya. After Hartmann (116), drawn by him from the de- scription given by Schaudinn (131). SYNGAMY AND SEX IN THE PROTOZOA 139- In Entamoeba 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 stainmg 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 case 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 j)air 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 amoebul£e 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 Polystomella crispa would only copulate when a couple came together in which each gamete was of distmct 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 {AcUnosphoerium) the parent cell divides mto two complete cells, each of which, after a process of matura- tion, becomes a gamete. In the more advanced stage {Entamoeba coll), the division of the parent-cell is checked, and only its nucleus divides, each daughter-nucleus becoming apronucleus after reduction. Tho occurrence of autogamy lias been asserted in a number of cases which arc, to say tho least, extremely doubtful, as, for example, the Myxosporidia 140 THE PROTOZOA (p. 407) and allied organisms, whoro it is far from certain that the two nuclei or colls, 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 hoterogamous, 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 autogamy, is whether there is a union of cliromatin derived from distinct s,txa,im—am'pUmixis—ov from a common parentage— autom.i.ris. Thus, it has been pointed out above that in gregarines two gametocytos may associate, and even fuse into one body, but witli 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 gamotocyte-nuclous, those of the opposite sex from the other, with subsequent syngamy of a truly heterogamous typo. If the fusion of the gametocytos 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 gamoto-formation took place, the syngamy would be, to all intents and purposes, a typical case of autogamy, and would certamly be so described if it were not Icno-mi that the single gametocyte-nucleus had arisen by fusion of two distmct nuclei. If, however, in each couple of copulatmg gametes, one pronucleus contained chromatin derived from one of the two original gametocyto-nucloi, the other pronucleus, similarly, chromatin derived from the other nucleus, the result would bo a true amphimixis, just as in ordinary heterogamy. In Actinosphceriuin, 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 Entamaiba coli and Amoeba albida (Fig. 87), thoro seems little reason to doubt that the autogamy is a true aiitomixis. Analogous cases of seLE-fertilization are well know in flowering plants, where they are sometimes the rulo, sometimes an alternative to cross-fertilization. In free-living Ciliata, also, sjmgamy has been observed between cousins, the descendants of an ex-conjugant after but four divisions (Jennings, 121), which is not far removed from automictic autogamy. The conclusion j)ut forward above, on experimental grounds, that syngamy has a strengthening or invigoratmg effect on the cell-organism, receives further support from the many instances in which it is observed to occur as a preliminary to the ]oroduction of resistant stages destuaed to endure unfavourable conditions of life. 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 remaui dormant until the sprmg. 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 aud ciiclure the vicissitudes of tlie 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 pomted 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 then- 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 mter mingling of distmct 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 ; sjoigamy levels up such diver- gencies and keeps the tendency to variation withm the specific limits (compare Enriques, 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 distmct 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 briefly as follows : Syngamy is a process of inter- mingling, in a single cell-individual, of chromatm derived from two distinct individuals, gametes, which may exhibit differentiation into " male " individuals, characterized by preponderance of kinetic activity, and " female," in which trophic activities are more i^ronounced. 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 1"^^ THE PROTOZOA it does not occur amongst organisms of the bacterial grade Svn- gamy is related to the life-cycle as ^ whole, and not specially to eell-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 para,sitism syngamy may be deferred for a very long time, and may even be completely m 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 i^rocess 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 ordurary cell-individuals of the species m 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, mider normal conditions so long as they i-emain single. In some cases among plants, however, the cells that have undergone nuclear reduction may multiply by fission and produce a multicellular organism (gametophytc) 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 elimiiaation 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 hfe-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 dui'ing 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 Coelenterates 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 mto four gamete-cells of equal size, the spermatids, each of which becomes a spermatozoon. In the female sex the oocyte-divisions are very unequal, producmg the ovum, ripe for fertihzation, and three minute sister-cells of the ovum which, as the so-called " polar bodies," are cast off and die away. By syiigamy 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- mme their number. Many cases in which a fixed number of chromosomes is alleged to occur, as ui Trypanosoma noclum (Schau- dmn, 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 fm-ther, 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 reducmg divisions in Protozoa suggest forcibly a comparison Avith 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 AdinosjJhoenum, 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. Moi'eover, 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 diAdded, 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 dii'ect, 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 chi-omosomes permits of their beitig accurately counted, and in which they are seen to be reduced to half the usual number in the maturation-divisions of the gametes. In Pelomyxa the first division reduces the chromosomes from eight to four ; the second division, however, is equating, and no fui-ther 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 rcgaixl 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 Opercularia (Enriques, 112), Ghilodon (Enriques, 113), Carchesium (Popoff, 125), Didinium (Prandtl, 126)', and Ano'plo'phrya (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 ® © * * ® ® L M N 0^ K Fia. 74.— Behaviour of the micronucleus during successive stages of the con- jugation of Anoplophrya [Collinia) hranchiarum. A. Micronucleus of one conjugant preparing for division ; B, later stage, with six chromosomes dis- tinct ; G, nuclear spindle, with an equatorial plate of six chromosomes • D, diaster-stage, with six daughter-chromosomes at each pole of the spindle • A, later stage, with the chromosomes at each pole fused into one mass • i\ ' ^•,f<^«iif. action of the daughter-nuclei ; the remains of the spindle between them disappears gradually; /, the two micronuclei prepariX for infirm ''-^ chromosomes in each (one nucleus Fs seel in profile, the other from one pole ; J, diaster-stages, showing three chromo somes at each pole o the spindle (reducing division) ; K. !ater stage The chromosomes fused into masses of chromatin ; L. four granddaughter Sicro nudei ; M, one of them grows in size, the other three begin to delenSe N. division of the persistent micronucleus to form the two pronuclef 0 two aTor^iordif^^^^^^^^^ (?0):^mtnificaS 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 ot syngamy, and regard reduction as a criterion whereby true syngamic union of gametes and pronuclei can be distinguished trom plastogamic and nuclear fusions which have nothing to do ^vith 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^chromS 10 l46 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 consj)icuous 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. 75.— 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 ; C, the karyosome expelled from the nucleus of the macrogametocyte has reached the surface of the body and broken up mto a number of fragments, which lie scattered in the body of the host-cell or are extruded from it ; D, the macrogametocyte has now become a ripo macro- eamete having rounded itself oS, eliminated the karyosome from its nucleus. Ind divested itself entirely of the host-cell, n Nucleus of the gametocyte ; k its karyosome ; n.'. nucleus of the host-cell ; Ic.'. k.'. fragments of extraded karyosome. After Schaudinn (99), magnified 1,000. the phrase, occur but have been overl6oked, or that the method of reduction is one that can only be brought mto 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 cliromatm 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 ehmination of half of its mass ; and that from thifj 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 in 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 cultiire, 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 conpequ-i:oes of antecedent events* in the life-history —as processes which i.i 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 m order to illustrate the salient features of these processes. The most convenient method of classification ot the examples chosen is to distinguish those cases in which chro- midia are present in addition to nuclei from those in which nuclei alone are present. 1. Syngamy and Reduction with Nuclei and Chromidia —In a great many Sarcodina, especially those belonging to the orders Amoaba^a (p 218) and Foraminifera (p. 231), chromidia may be present m the gamete-forming individuals as a permanent con- renre^^^^^^ . n body-structure. In such cases the chromidia rise bv f ' T P^'*' g^^er^tive chromatin, and give nse, by forma ion of secondary nuclei, to the nuclei of the gametes tm oftn ' '^"P*^^- '"^^ distinct lorms of syngamy have been described. the%^l^in1ueT; wK'eCrr.Th^ point out that starvation is by no means 148 THE PROTOZOA (a) Karyogamy.—T\iQ body of an Arcella gives rise by multiple gemmation to a number of amoebulae, pach 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 sjnikaryon. 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 Arcella. In Foraminifera (p. 235), in which the sjoigamy 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 Arcella, by formation of secondary nuclei ; but in some broods each amoebula divides into four micramoebae (micro- gametes), while in other broods the amoebulae remain undivided as macramoebfe (macrogametes) ; copulation then takes place between two gametes of different size. (&) Chromidiogamy (Fig. 80, M—Q).—1vfo ordinary adult ylrceMoe come together and apply the mouths of their shells. The proto- plasm of one individual flows over almost entirely into the other shell, takhig 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 chromatm-particles and become intimately commmgled. When this process is complete, the protoplasm with the chromidia becomes again distributed between the two shells, and the two conjugants separate. Then in 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 Arcella. 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. Cliromidiogamy is a rar(^ but very interestiiig 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 ca.se 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 B Fig. 76. — Gamete-formation and syngamy in Plasviodiophora hrassicce. A, Normal vegetative nuclei of the myxamcEbsa ; B, C, 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 ; 0, spore (zygote) containing two nuclei, one of which is going through a' further reduction-division ; H, fusion of the two pronuclei within the s])ore ; /, ripe spore with synkaiyon and two centriolcs. After Prowazek (127)' magnified about 2,250 diameters. it produces a disease Imown as " fingers and toes " (Kohlhernie). According to the investigations of Prowazek (127) and others, Plasmodiophora goes through a development which may be 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 number of " myxamoebse," aanoeboid individuals (Plasmodia) each with many nuclei contaming distinct karyosomes (Fig. 76, ^). From the nuclei chromidia are given off into the cell, and during this process the karyosomes disappear and centro3omes 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 myxamooba then undergoes multiple fission into as many cells as there are nuclei m 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, G). The syngamy in Plasmodiophora is stated to be a case of autogamy, but this allegation assumes that the nuclei of the myxamoebge are sister- nuclei derived all from the division of one original nucleus ; they may equally well be nuclei of different origuis brought together by plastogamic fusions. The two examples selected, Arcella and Plasmodiophora, show that the chromidia may represent generative chromatm in one case, vegetative in another. Goldschmidt (57) has proposed to distinguish these two conditions by a special terminology, retainmg 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 retaua the term " chromidia " in its original significance, to denote simply extranuclear 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 jDurely 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. Tho account given by Bott (103) of gamete -formation in the common Pelomyxa {AmcBbma 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 tho secondary nuclei have been purified in tliis way of their vegetative chromatin, the generative chromatin remaining in eacli of them forms a karyokinetic spindle with eight cliromo- 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, formmg a spindle with four cliromosomes which spht, 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 wlien the process is complete. When formed the gametes wander out as Heliozoon-like indi- SYNGAMY AND SEX IN THE PROTOZOA ISl viduals, wliich copulate in pairs, and the uninucleate zygote grows up into the multinucleate PeZomyxd. . .,. , .1 The conception of vegetative and generative chromidia has not been accepted universally or without criticism. Hartraann, as pouited out above, considers that many cases of generative chromidia are really the result ot 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 lile- cyclo generative chromidia are to be considered as a new acquisition, a hasten- ing of the process of the formation of numerous gamete-nuclei ; Dobell (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 recrard 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 sjnagamy 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 162 I'HE PROTOZOA 77^Diagram showing the successive stages of conjugation in Infusoria. A, The two conjugants attached, each with a macionucleus (N) and a micro- nucleus (w) ; 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 ; 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 stationaiy 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 sjoikaryon [8.) 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 ua 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 mitiates a fresh cycle. It has been observed that the two ex-conjugants sometimes differ markedly in their capacities, one of them multiplymg much faster than the other. In the syngamy of Ciliata it is seen clearly that the macronucleus represents effete vegetative or " somatic " chromatm, which is ehmmated 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 m which the conjugants remain distmct, and merely exchange pronuclei (so-called "partial karyogamy "). Versluys (137), foUowmg 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, alter which the zygote divided into two individuals ; on this view the final division of the micronucleus which gives rise to the two 154 THE PROTOZOA pronuclei is to ho 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 S3aigamic 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 prm- 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 uidividual, 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 ampJd- 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 jDrocess 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 lorobable that the develop- mental stimulus is supplied by the greatly-developed centrosome of the spermatozoon, that of the ovum having completely atrophied, apparently, after the completion of its maturative j)rocesses. The introduction of a male pronucleus— that is to say, the process of amphimixis —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 sjmgamy, a necessity for the evolution of livmg 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 withia a multicellular body. Apart from the question, however, whether any such innate variations exist in the Protozoa, where all cells 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 sjoigamy first arose must be sought. It has been pomted 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 - increasmg 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 more precise meaning to the terms "youth" and "old age," terms that certainly stand in need of further explanation, since a 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 is 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 begmning, 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 tlue 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 ui Protozoa — namely, the elimina- tion of a large amount of chromatin which appears to have been regulating the vital activities durmg 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 macronucleus 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 chromatm, 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 rejpresent 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 retamed 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 nuclear substance or by plastogamy, 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 in 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 Biitschli originally suggested, from inequalities in 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- atmgly male show greater kinetic and motile energy, those that have more female qualities show greater trophic activity. With con - tinned cell-division these opposite tendencies tend to accumulate m certam cells which in consequence become altogether one-sided m their vital activities. Thus a want of balance m 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 rein vigor ated. No gametes, however, whatever their degree of specialization, 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. Schaudiim, whose work on Protozoa must secure full considera- tion for any statement of his observations, however inherently improbable the facts or the uiterpretations based upon them may seem, founded his theory chiefly on data alleged to have been observed by him in the development of Trypanosoma noctuce (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 mterpretation (see p. 176, injra). 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 sjmgamy in any organisms of the bacterial grade. The processes that have been interpreted by Schaudiim 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 remain meaningless, connoting merely unknown, mystic properties, not 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 m 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 pomt 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 m 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 ki 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, ia 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 the 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 does not 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! PR0T020A 161 minute bodies reproduction by fission does not ijroduce 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 uiternal rearrangements of the livuig substance such as liave been described in. the larger Bacteria. Not until the process of natm-al 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 suflficient 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 exertmg different physiological activities, as suggested by Biitschli, Geddes and Thomson, Schaudimi 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 havmg a high degree of structural differentiation than m 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 tor, 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 m parasitism, seems to dimhiish the necessity for syngamy. It can hardly be supposed that intensive culture can dimmish consequences arismg from defective cell-division ; but P^rS T"'^":^^ P'^"^""" ^ strengthenmg effect equal to, and ZttfmTl"''''^.^'^^ ^^-i f^<^-^ amphimixis, nroeeed t j • !• '^"''^^ ex-conjugants may and the 1 T!^^' of syngamy In tht on? .7 ""^V ^ cell-division mteLnrng"^ d?vi tLanLTh ^'^y consequences of eell- eXiation ' A ^^^""^ ^^^^^^^ ^^me other S.amv Tl i ^"'^^"^^ ^^^^'^i^^^^ the function syngamy m Infusoria is to maintain the fixity of the species. Bihliographj.—Fov references see p. 479. II 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 livmg 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 origmally 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 differmg from one another and from the parent- species to a gi-eater or less degree. The problem of the origin of species is one that it is not necessary to discuss here ; it is suflacient 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 6mcn- group, probably to be regarded, as pointed out above (p 27), as instances of species in an incipient or nascent conditiom 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 thmgs perfectly intelligible if existing species are regarded as havmg 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 mdividuals 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-lcnown 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 m 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 mdividuals of a species related to differences in the functions wliich they perform, not merely at successive phases in the life-history, as m some cases of ontogenetic differentiation already mentioned' but even at correspondmg phases of the life-history— a phenomenon best seen m 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 correspondmg 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 durmg 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 cihate Infusoria can be cited. Other Protozoa, on the other hand are extremely volymorpUc~t}xs.i is to say, they occur under a variety of widely-differing forms at differenLtages in the life cycle or m res|onse 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 unravellmg of the complicated life lycles of Protozoa is attended by far greater difficulties than in Metazoa ha? Zr:rT' 1'™ ''^"^ - the Proto oa that, namely, of sexual maturity. A naturalist has no hesitation in pronouncing a trochophore to be a larval form, and a roti^e to be an adult organism, from the fact that the former I sexual v immature, while the latter produces ripe generative cells In S 164 THE PROTOZOA Protozoa, however, there is no visible criterion of any similar state of maturity or the opposite which might bo a guide in estimating the significance of a particular form. It is certain that with in- creasing Imowledge many sj)ecies of Protozoa now regarded as distinct Avill 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 mcluded 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 bodv-form. In the case of naked amoeboid forms such a change of form follows naturally, as pomted 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 mternal form-givmg elements must be dissolved, so that the protoplasm is free to conform to the natural physical tendencies of a fluid body. In the great maiority of cases, an individual in process of encystment becomes perfectly spherical, whatever may have been the form of its body m the active state, but m some cases the spherical form ^ not fully attamed, and the body becomes ovoid or pear-shaped During tlie process of roundmg off, any food-particles or foreign bodies contamed hi the cytoplasm are rejected or absorbed, as a rule ; the contiactile vacuoles, if there be any, cease to be formed and vanish ; and all locomotor organs, such as cilia, fiagella, and of course pseudo- poZ are absorbed or cast off. At the same time the protoi^asm of the organism becomes less fluid and more opaque, aiid usually d minishes appreciably in bulk, probably through loss of watei , 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 whiter 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 m 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 syorocyst, and the entire body a spore.* * The word " spore " has come to be used in two distinct senses, as applied to -Protozoa thereby producing a regrettable confusion and ambiguity. The word itselt IS denved from the Greek crwdpos, a seed, and was applied by botanists to those cases where plants produce seed-like bodies which are not true seeds • for mstance, the seed of an ordinary flowermg plant is a complete embryo, with 'root and shoot distmct, encapsuled in protective envelopes, but the " seed " of a fern is merely a smgle cell enclosed in a protective membrane. Consequently the SseeSflorri^rpirnt?*"^^^' " ''''' " rr,innn^?f-r''t ^^7- T'^^ ^^^^^ parasitic Protozoa produced M- ^^^'''I- 7^°^ conveyed the infection ; for those of Myxosporidia was usTd h' P««™«P^"-B." in general the te/m " s^po^'' 7s ave^!h Jt, \ T'T"^ ^'°^P ^'^^^^ production of such spores ^„TW characteristic feature was named the Sporozoa. cas^ the essSlT^^ f^.^^"" investigation, it was found that in a great many .Sfbv rZipJ/ f spore-namely, the encapsuled protoplasmic body- arose by a process of multiple fission, hence termed " sporulation " from a larcrpr S'rsenre t'o'drT^'''^*'^*'^ " " beenS trmany S a sS! of fheTalariarm^^^^^ formed by multiple fission, as in the merozoites ox me malarial parasites. It is preferable to retain the word "snore" in ;<-a c7sr:ndTo trtho'^ " r^'''"' ^P.^^ - resi^taTenvelJS'or spo S thr^r^t 1 • u"". ^'^'''^ Sorra " equivalent to the Gci-man word Keim) for riioro 18 no ossmtW dittcronoo bchvcon a »y,t a'nd » sporo, osccpt their relation 166 THE PROTOZOA Sporocysts are often simply rounded or oval bodies, like cysts, but in some cases they exhibit special foi-ms, and may be prolonged into spikes, tails, or processes of various kinds. In many cases the purely i)rotective uses of the cyst may be combined with the performance of some special function within it. The contained organism may remain merely in a resting state within the cyst (hypnocyst) ; or it may utilize its leisure for the digestion of large quantities of ingested food - material, or for carrying on i^rocesses of reproduction or syngamy. As a process of similar nature to encystment, the formation of " sclerotia " in the Mycetozoa must be noted (see p. 240, infra). Active adaptation to changed conditions is seen in those forms in which the mode of life is bound up with changes of environment during different periods of the- life-history — that is to say, more especially in parasitic forms, m which a change of hosts is necessary for the continuance of the species. In such forms there are in general two functions for which provision must be made : the first is that of multiplying in the host itself and keeping up a stock of the parasites in it ; the second is that of infecting a new host sooner or later (see p. 20, supra). In the most primitive types of para- sitic Protozoa there is no differentiation of form or structure corre- sponding to these two distinct functions ; but as a general rule a given parasite in a given host exhibits usually two forms or series of forms, which may be termed " multiplicative " and " propagative " respectively (Doflein). Multiplicative forms may be wantmg in some cases, as in the Eugregarines, but propagative forms are always found, being an absolute necessity for the continuance of the species. As examples of multiplicative and propagative forms, we may consider first species which are parasitic only on a single host in the course of the entire life-cycle. A typical example is seen in Coccidium (p. 342, Fig. 152), in which adult forms, " schizonts," multiply rapidly in the host by a process of multiple fission, " schi- zogony," a process which takes place unaccompanied by any sexual phenomena, and in which no resistant cysts are formed, since they are quite unnecessary. Sooner or later, however, generations of individuals, " sporonts," appear which do not multiply like the schizonts, but which, as gametocytes, give rise to the gametes. After a process of syngamy the zygote forms a resistant cyst within to a developmental cycle ; the " spores " of Bacteria are for the most part simply cysts, but are called spores on account of their small size. ^In this book the word "spore," when not qualified by any prefix, will be used to denote a resistant seed-like body protected by a tough envelope or sporocyst, Ind the production or development of such bodies mil be termed spore-forma- tbn." On the other hand, the production of numerous small cells or germs by multiple fission will be termed " sporulation." POLYMORPHISM AND LIFE-CYCLES 167 which it multiplies to form a number of germs, which may or may not be enclosed in sporocysts, in different species. Cysts and spores pass out of the host, and do not develop further unless they are devoured by a second host of a species in which they are able to estabhsh themselves ; if this event takes place, the spores germinate in the new host and produce a fresh cycle of infection, each germ when set free growing up into a schizont. In this case it is seen that the schizonts 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 fife-cycle of the malarial parasites shows that a given phase of a parasite is only to be regarded as multipHcative or propagative in relation to a particular host. In the vertebrate blood the schizont is the multipficative, the sporont the propagative, phase. As soon, however, as the sporont passes into the mosquito, it becomes there the multipficative 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 m another, according to circumstances. It should be noted further that m the fife-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 Hfe- 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 Aggregata (p. 353). Here the schizonts are parasitic in crabs, and reproduce themselves by- multiple fission without encystmcnt 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 fseces, 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 multipUcative phase, but only a propagative phase with resistant cysts. 2. PolymorpMsm 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 shghtly 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 lewisi of rats, in wJiich two types of suoli fission are seen : eitlier 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 by fission. In the subclass Telosporidia of the Sporozoa, the contents of the spore may divide up in this way to form a variable number of slender sickle-shaped germs, for which Aime Schneider coined the term sporozoites, a term which has since been frequently appHed in senses quite diSerent to its original meaning. An active germ produced by sporulation is termed a swarm-spore or zoospore, whether or not the active phase is preceded by a resting spore-stage. The swarm-spores of Protozoa may be of various types in different cases. The swarm-spore may be amoeboid and creep about by the aid of pseudopodia ; it is then termed an amosbula (or pseudopodiospore). It may be provided with one or more flageUa as organs of locomotion, and is then termed a flagelMa (or flagelUspore). It may have a coat of ciha, as in the young stages of Acinetaria, and may then be termed a ciliospore. Lastly, the swarm-spore may be without organs of locomotion, whether perma- nent or temporary, and may progress by twisting and wrigghng movements of the body as a whole, or by ghding forwards on its long axis in a manner similar to the ghding movements of gregarines ; swarm-spores of this type are speciaUy characteristic of the Telo- sporidia amongst the Sporozoa, arising either by sporulation of a schizont (merozoites) or in the process of spore-formation after syngamy (sporozoites), and may be termed gregariniform swarm- spores or gregarinulce comprehensively. In some cases the swarm-spore may pass through more than one active phase, and exhibit different modes of locomotion in each ihis IS well seen in the Mycetozoa (p. 239), where the germination 01 the spore produces an amoebula, which may acquire a flagellum 170 THE PROTOZOA and become a flagellula ; after a time the flagellula settles down and becomes an amoebula again after loss of the fiagellum. A very interesting point, in connection with the question of young and adult forms of Protozoa, is the occurrence of stages in the developn\ent which may be interpreted as recapitulative in the phylogenctic sense — ^that is to say, as representing past stages in the evolution of the species, in a manner comparable to the recapitu- lative larval or embryonic stages in the development of Metazoa. It is probable that such recapitulative stages are commoner in the development of Protozoa than has been generally supposed (compare Awerinzew, 47). The best-lmown instance is furnished by the cihated larvae of Acinetaria (p. 459), indicating that this order is descended from a ciliate ancestor of the order Peritricha, a relation- ship fully confirmed by the similarity of their reproductive processes to those of other Infusoria. The crithidial phase that occurs so constantly in the development of trypanosomes (p. 299) is probably to be regarded as a recapitulative form representing a type of structure antecedent in evolution to that of the typical trypanosome- form. The frequent occurrence of flagellated swarm-spores in the development of Sarcodina (Foraminifera, p. 235 ; Radiolaria, p. 254) probably has a phylogenctic significance, as pointed out by Biitschli. Finally attention may be drawn to the remarkable series of forms in the ontogeny of Arcella described in the next chapter ; first the amoebula, then the Nuclearia-stage, followed by the Pseudoclilamys- stage, which grows finally into the adult Arcdla-iovm. In the many cases where young forms are markedly different from the adult, it may be a difficult matter, as it often is in the case of Metazoa, to decide whether a given larval form is to be interpreted as recapitu- lative or merely adaptive ; but even in cases where the characters of a larval form have an obvious adaptive importance, as in the cihated larvae of Acinetaria, atavism may be nevertheless a factor determining the particular form taken by the adaptive characters in question— that is to say, by the organs of locomotion in the example chosen. 3. Polymorphism in Relation to Sex.— The phenomena of sexual differentiation consist primarily of differences in size, structure, and other characteristics between the gametes, the cells which are con- cerned in the act of syngamy. Secondarily such differences may extend to other cell-individuals, both in the Ufe-cycle of a Protozoon or in the body of a Metazoon. In the previous chapter it has been pointed out that, while in Metazoa the gametes at least are sharply differentiated in all cases, in the Protozoa every condition is found from perfect isogamy to a differentiation nearly as pronounced as that in the Metazoa. The question has been discussed in the last chapter whether or no sexual differentiation is to be regarded as POLYMORPHISM AND LIFE-CYCLES 171 an inherent property of all living beings, as maintained by many high authorities. Whatever view be held with regard to the existence or non- existence of inherent, intrinsic sexual diiferences in living organisms, it seems clear that the apparent sexual differentiation of the gametes is largely, perhaps purely, adaptive, and furnishes good examples of the principle of morphological differentiation of structure in relation to physiological division of labour. One gamete, termed "female," tends to be bulky and inert, storing up reserve- material in greater or less quantity, a provision (sit venia verbo !) for future requirements ; it is economical of substance, and but few are produced. The other gamete, termed " male," develops in the opposite direction in every respect ; it tends to be small and active, not weighted with superfluous material of any kind, but with motor mechanisms strongly developed ; it is prodigal of substance, and many are produced, but few are favoured by destiny. In extreme cases the female gamete is a relatively huge, inert cell, incapable of movement, crammed with foodstuffs ; the male is excessively minute, and is practically nothing but a nucleus which has its constituent parts packed into the smallest possible space, and with motor mechanisms attached to it. In reviewing the progressive differentiation of the gametes in Protozoa, it is convenient to treat separately those forms in which there is little or no ontogenetic differentiation from those in which there is a more or less pronounced difference between the young and adult forms. An example of the first type is seen in Copromonas (Fig. Ill), in which the gametes are ordinary individuals of the species, only differing in that their nuclei have undergone a process of reduction. Good examples of monomorphic forms are furnished also by the Infusoria, a group in which a species may be free-swim- ming, or may be more or less permanently attached and sessile in habit. In the free-swimming ciHate Infusoria, sexual differences in the conjugants are frequently not discernible ; if they exist, they can only be inferred from the fact that syngamy takes place, or from subsequent behaviour of the individuals after conjugation, as, for instance, the fact observed by Calkins, that in Paramecium one ex- conjugant multiplies much more rapidly than the other. In other cases differences of size more or less pronounced are exhibited by the conjugants (Doflein, 111). As pointed out above, differences of structure have also been noted in some cases between the stationary and migratory pronuclei produced by a conjugant Colhn (50), however, was unable to find the shghtest morphological diflerentiation of the conjugating pronuclei of Anoplophrya. In the sedentary Infusoria, sexual differentiation may be as little 172 THE PROTOZOA me: apparent as in the free-swimming species, as, for instance, in Acinetaria, where conjugation can take place between two adjacent individuals each on its own stalk. But in the Vorticellids special free-swimming individuals, microconjugants, are developed which are budded off from a sedentary individual, and then acquire cilia, swim off, and conjugate with another sedentary individual (Fig. 78). It seems obvious that this state of affairs is an adaptation to the exigencies of a sedentary life to insure cross- fertilization analogous to the formation of complemental males in the Cirripedes. The free - swimming microconjugants of Vorti- ceUids are commonly termed " males," but it is open to question whether, strictlj^ speaking, they deserve that title. It is in species with marked differences between young and adult forms that the greatest differentiation of the gametes occurs, though by no means universally even in such forms. In polymorphic species of this type, three different conditions can be distinguished, to which reference has been made in the previous chapter. 1. Macrogamy — that is to say, syngamy between full-grown individuals of the species. In this ty3)e the gametes appear to be always gant) with two microcon- p ^^jy similar, SO far as is known; ex- lugants (m.c.) attached ^ , . ^ . 7 /tt mi \ amples are seen m Achncpirys (Jbig. 7i), the chromidiogamy of Arcella (Fig. 80), and possibly Noctiluca (p. 279). 2. Microgamy — sjTigamy between the youngest individuals, products of the rapid multipHcation of an adult. Conjugation of swarm-spores is by far the commonest tjTpe of syngamy in Protozoa, and may be re- garded as the normal type. In this case there is usually complete iscgamy, as in Foraminifera (p. 235), sometimes sUght anisogamy, as in Radiolaria (p. 254, Fig. 108). 3. Mixed microgamy and macrogamy — that is to say, syngamy between a fuU-sized adult individual on the one hand and a minute individual, a swarm-spore, on the other hand. Tliis type may bo regarded as derived from microgamy by progressive, and finally complete, inhibition of the divisions that produce the swarm-spores in one sex— possibly also with an enhanced tendency to such divisions in the other sex. Thus in Arcella, as described in the previous Fig. 78. — Vorlicella micro- stoma, Ehrb. On the left an ordinary, sedentary individual (macroconju to it, one of which (to the left) is in the act of conjugation. On the right is an individual with the stalk contracted and the body enclosed in a cyst. N , Macro- nucleus ; P, peristome and adoral ciliaiy spiral. After Hickson. POLYMORPHISM AND LIFE-CYCLES 173 chapter, the macramoebse proclucod arc fewer than the micraiuoibaj, showing that the tendency to division is more restricted in the former case than in the latter. Again, in the development of C'enlro- pyxis, as described by Schaudinn (131), formation of gametes is initiated by a process of multiple fission combined with formation of secondary nuclei from chromidia, as in Arcdla, and in this way a number of amoebulae are produced. The amoebulae from one Centropyxis remain undivided, as macramoebse, while those pro- duced from another adult divide each into four micramoebse ; syngamy takes place later between a micramoeba and a macramoeba, after each has secreted for itseK a shell. When the inhibition of the gamete-forming divisions is quite complete in one sex, the result is the most pronounced type of anisogamy occurring in Protozoa ; and, conversely, it may be said that aU cases of extreme anisogamy in Protozoa are of this type. In Metazoa the disproportion in the size of the gametes is mainly due to the relatively enormous growth of the gametocytOi partly also to the inequahty of the four cells produced by the reducing divisions, in the female sex. In Protozoa with extreme differen- tiation of gametes, on the other hand, such as the Coccidia and Ha3mosporidia, the gametocytes do not differ greatly, sometimes not at all, in size, though the female gametocyte may contain more reserve food - material, and consequently less protoplasm. The disproportion of the gametes is due almost entirely to the fact that in the female sex the gametocyte does not divide, but becomes a single macrogamete, while the male gametocyte sporulates to produce a larger or smaller number of microgametes. Very instructive in this respect is the comparison of the formation of the gametes in the gregarmes (p. 331) and the coccidia (p. 346) respectively, two groups of Protozoa which are certainly closely alhed to one another. In such a form as Coccidium (Fig. 152), the gametocytes remain separate one from the other, and the male gametocyte forms numerous minute microgametes which swarm away ; the female gametocyte, on the other hand, becomes a macro- gamete after going through a process of reduction, and is fertilized by a single microgamete. In gregarines, however, the gametocytes associate in couples, either before or after attaining then* full size, and become surrounded by a common cyst, within which each gametocyte sporulates to produce a large number of small gametes. The gametes of gregarines can be arranged in a series, showing marked anisogamy at one end, complete isogamy at the other. Thus in PterocepMlus (Fig. 79, A, B) the gametes are very unequal m size, and the microgametes are motile, the macrogametes not so. In Stylorhyndms the gametes of opposite sexes are equal in size, but m one sex the gametes are motile, in the other not (Fig. 79, 174 THE PROTOZOA G, D). In Monocystis (Fig. 79, G—L) the gametes differ sUghtly in size in tlio two sexes, but have no organs of locomotion in eitlicr case. In Urospora (Fig. 79, E, F) the gametes are not appreciably different in size, but in those of one sex the nuclei are slightly smaller than in those of the other. Finally, in Gregarina, Diplodina, and many other genera, no difference whatever is perceptible between the two gametes that perform syngamy. In those gre- garines which have dimorphic gametes, syngamy is alwaj^s between two dissimilar individuals of distinct parentage, and it may be inferred, therefore, that in all cases aUke the gametes that unite are derived from distinct gametocytes. Fia. 79. — Gametes of different species of gregarines. A, Male, B, female, gamete of PterocepJudus (Nina) gracilis. 0 and D, Stylorhynchus longicollis : G, male gamete ; D, male gamete attaching itself to a female. E, Male, F, female, gamete of Urospora lagidis, showing differences in the size of the nuclei. 0 — L, Monocystis sp. : 0, male gamete ; //, female ; /, union of the two gametes, the nuclei still separate ; J, the two nuclei fusing ; K, the zygote becoming elongated ; L, the zygote has taken the form of the spore, and in the synkaryon a centrosome has appeared, preparatory to division. A and B after Leger and Duboscq ; G and JD after Leger ; E — L after Brasil. From a comparison of the life-cycles of the Coccidia and the Gregarines respectively (see p. 354, i7ifra), it is highly probable that in the common ancestor of the two groups the gametocytes were separate, as in Coccidium, and each produced numerous gametes, as in Gregarines. Since the gametes had to find each other, by a process of adaptation, those of one sex became smaller and more motile (microgametes), while those of the other sex were more bulky and inert (macrogametes). In the course of their evolution from this primitive ancestral type, the Coccidia, with some exceptions presently to be noted, retained the habit of the gametocytes, rcmainmg separate, and the Bpeciahzation of the gametes became greatly increased, as an adap- POLYMORPHISM AND LIFE-CYCLES 175 tation to this condition, tlic female gametocyte ceasing to divide and becoming a single macrogamcto, while the male gametocyte produced a swarm of minute, motile microgametes. Only in a few Coccidia, exemphfied by the genus Adelea (Fig. 154), did the gametocytes acquire the habit of association before forming gametes, a habit which led in this case to a reduction of the number of micro- gametes produced to four, of which one fertilizes the macrogamete, while the other three perish. It is clear that the formation of microgametes in close proximity to the macrogamete increases vastly the chance of the gametes finding each other, and renders unnecessary the production of a swarm of microgametes. In the gregarines, on the other hand, the gametocytes acquired the habit of associating and forming their gametes in a common cyst. Under these circumstances it becomes a certainty that a gamete of either sex wiU find a partner if the gametes of each sex are in equal numbers. Consequently there is seen in gregarines a progressive tendency, illustrated by the examples cited above, to disappearance of those characters of the gametes which are an adaptation to the necessity of the sexes coming together, culminating in production of gametes of opposite sexes which are perfectly similar. On this view the isogamy seen in many gregarines is a secondary condition brought about by the gradual obHteration of adaptive differences between the gametes of opposite sexes, under circumstances which render such differences unnecessary. The comparison of the gamete -formation in different species of gregarines furnishes an instance of a progressive levelluig-down of structural differentia- tion of gametes, under conditions in wloich no such differentiation is required until an anisogamy undoubtedly primitive has been reduced secondarily to a perfect isogamy. This has led to the view expressed in many quarters that anisogamy is m all cases a primitive, isogamy a secondary, condition ' The case of the gregarines is by no means adequate, however, to support so sweeping a generalization ; the only conclusion that can be drawn from it is that adaptive differences tend to disappear when the conditions to which thev are an adaptation no longer exist ; and the very fact that the obvious structural differentiation between the gametes vanishes in such a case is of itself a proof that such differentiation is not the expression of intrinsic constitutional differences between the gametes, for such differences could not be annihilated merely by changed conditions of environment ciuuinuatea rlJiir """"l ^ '''' anisogamy in the form of visible structural t^l lZT I f^' gametes of opposite sexes must have been acquSTd very early by gametes as an adaptation to their functions. On the other hand, It IS highly improbable, to say the least, that the earliest gLetes aiiterentiated. it must, of course, be postulated that the gametes nosses^ such m rinsic constitutional differences as would account f or thSehav our- that IS to say then: mutual attraction and union ; and in this sense aSsoSw may be considered as a universal and primitive phenomenon. But the numEe^ hlTn gametes are perfectly isogamous, as regards Srstrue tural or other differences, is a sufficient proof that purely consti^tu?i^n^l :?Sgitr "^^^^^^^^'^ ^^^^^^^ - l-4tibt dXtlnS 176 THE PROTOZOA So far only primary soxual differences — that is to say, those between the actual gametes — have beep, discussed ; but, as lias been stated above, the sexual diilerontiation may be thrown back, as it wore, into generations preceding the gametes. Thus, it is by no means uncommon, especially in Coccidia and Hsemosporidia, for the gamotocytes to be clearly distinguishable according to sex, the female gametocyte having the cytoplasm loaded with reserve food- material, and usually with a smaller nucleus, while the male gameto- cyte has the cytoplasm clear and free from inclusions, and the nucleus is relatively large. In Adelea the male gametocyte is very much smaller than the female (Fig. 154). In Cydospora caryolytica, parasitic in the mole, the sexual differentiation is carried back through generations antecedent to the gametocytes, and, according to Schaudinn (147), male and female merozoites can be distinguished. The various types of polymorphism that have been discussed in this chapter may be classified as f oUows : 1. Adaptive i)olymorphism. (1) Passive. (2) Active. 2. Ontogenetic polymorphism. (1) In size alone. (2) In structure also. (a) Recaj)itulative. (6) Adaptive. 3. Sexual polymorphism. (1) Primary (of gametes). (2) Secondary. (a) Of gametocjdies alone. (6) Of other generations also. In the task of unravelhng the compUcated life-cycles of Protozoa, it is of the greatest importance to distinguish clearly the significance of the various forms that are seen, and there can be no doubt that failure to do so has often been a source of error. With some writers it is an obsession to ascribe aU difierences to sex, and to interpret, for instance, in the development of trypanosomes, aU bulky forms as females, and aU slender, active forms as males, quite regarcUess of the behaviour of the forms thus designated. It is far more probable that in the majority, at least, of such cases the bulky forms are related to the multipHcative, the slender, active forms to the propagative function, respectively, and that the differences between them have no relation whatever to sexual functions, either in the forms themselves or in their descendants. POLYxMORPHISM AND LIFE-CYCLES 177 B. Life-Cycles. In the foregoing section the various forms have been described under which one and the same species of Protozoon may occur in the course of its hfe-history, and in response to the conditions of its particular mode of hfe. In some species it has been seen that the changes of form and structure are so shght that the species are practically monomorphic, in the sense that they can be identified without difficulty in any active phase of hfe ; no species is absolutely monomorphic, since, in addition to resting states, differences in size due to gro^vth, at least, will always be found. Other species, on the other hand, are polymorphic to such an extent that their specific identity in different phases can only be determined by tracing their development in a continuous sequence ; and in extreme cases of polymorphism the life-history becomes a varied pageant of dis- similar forms succeeding each other in more or less regular order, determined largely, if not entirely, by the conditions of the environ- ment. In a former chapter the distinction has been drawn between a developmental cycle, consisting of a recurrent series of different forms, and the complete Hfe-cycle, consisting of the whole series of forms or phases which appear between one act of syngamy and the next. The complete hfe-cycle may comprise many develop- mental cycles. As a concrete example of a hfe-cycle comprising a great number of different forms, and in which also the development may follow more than one course, the hfe-cycle of Arcella vulgaris may be selected pig. 80). The hfe-history of this form has now been made known m detail by the combined labours of many investigators, amongst whom Hertwig (65), Elpatiewsky (144), Swarczewsky (101) and Khamsky (145), must be speciaUy mentioned. The form which may be taken as the starting-point of the hfe- cycle is a minute, amoeba-hke form, with a single nucleus (Fig 80 A) The amcBbula, when set free, feeds, grows, and becomes aft^r a time spherical m form with radiate pseudopodia (Fig 80 B) ■ in this stage it resembles a species of the genus Nuclearia. Mtev a time the A^^cZeana-form secretes a shell, and now resembles an example of the genus PseudocMamys (Fig. 80, C). With further growth chromidia are given off from the nucleus into the cytoplasm, the ch rt ^ 'J^^' -i-al thus assumes^duali; the chitinous shell, circular in outline, flattened in profile- view and shgh ]y eoncave on the under-side, in the centre of which is a We ZT:rTT' "'^^^ pseudopodia stream out The body-protoplasm contains two nuclei situated approximately at 12 178 THE PROTOZOA Fig 80 -Combined diagram to show the different methods of reproduction and syngamy in the life-cycle of Arcella. A-D The four stages in the ontogeny: ^, the amcebula ; B,t\x& NucUaria-io^m ; C the PseudocMamys-tovm ; D, the adult Arcdla. „ . . ^ ^^tonlasm n o Rtastes in the vegetative reproduction by fission : -B, the protoplasm POLYMORPHISM AND LIFE-CYCLES 179 the opposite ends of a diameter of the circular body, and an irregular ring of clu-omidia forming a dense chromidial net. Under certain conditions Arcella becomes encysted, forming a spherical cyst with a tough impervious membrane within the shell, closing the mouth of it. The adult Arcella reproduces itself by a variety of methods, Avhich, however, may be reduced to two principal types : binary fission, producing daughter-individuals (Arcellce) of approximately equal size ; and gemmation, producing small amoebulse such as have been described above as the starting-point of the ontogeny. The production of the amoebulse may or may not be in relation to syngamy, which, when it occurs, may be of one or the other of two distinct types — karyogamy between amoebulse, or chromidiogamy between adult Arcellce. Binary fission (Fig. 80, B—O) is the ordinary type of reproduction during the " vegetative " life in the summer months, when the animal is actively feeding, growing, and reproducing itself. In the process of binary fission, the two nuclei divide by a form of karyokinesis (Fig. 57, p. 110). A quantity of the body-protoplasm streams out through the mouth of the shell, together with some of the chromidia, and one of the two daughter-nuclei of each pair also passes out of the sheU. The daughter-^rceZ^a thus formed secretes for itself a new sheU, and separates from the parent-individual which retains the old sheU. Thus in binary fission both nuclei and chromidia take part, the former dividing by mitosis, while the latter are subjected to a roughly equal partition. The ordinary binucleate form of Arcella may become multi- FiG. 80~conlinued : ih^l f ^fig'^fes l)elow the level of D represent reproduction by gemmation • those to the left are reproductive processes not combined h =7^ * those on the right show the methods of syngamy '"'"'''''^^ syngamy; from^hrratTas^irX^far^'^^ ^^^^ - ^i'^-ated singly V^renf£^X't:X?:r^^^^ *° P-*«Pl-- of the J, Bud-formation external to the shell • the nrnlnnlnoTr, h^c 4- he^^th°"'^' ^^f'r^ P-«on.:oAtltiS SfpTmarrnS sheH ; the extruded protoplasm producing buds tith fLmat[roftoondai;; the shell and copulate (?) to produi ti; zyX llLbl^/) ^ "'^^ plafnr?v'itMS anl'^ ^^r }, the proto- over into the shell of the other O a^fr ^^t^ ^ nuclei of the one passes protoplasm becomes eTuX distribnt/d h ^ °* ^^'^ chromidii. the Modifiedjrom a diagram by Swarczewsky. ISO THE PROTOZOA nucleate by formation of secondary nuclei from the chromidia, as described above (Fig. 32, p. 67). The secondary nuclei are entirely distinct in their origin from the primary nuclei, which degenerate Avhen the secondary nuclei are formed. A multinucleate Arcella may reproduce itself by binary fission after division of each secon- dary nucleus by karyokinesis ; of each pair of secondary daughter- nuclei, one goes to one daughter-^ rceMa, the other to the other, so that each daughter-ylrceWa has the same number of nuclei exactlj'' (Hertwig, 65). Gemmation takes place in multinucleate forms containing a number of secondary nuclei. A portion of the body-protoplasm becomes centred round each secondary nucleus, and thus a small cell is formed, which becomes amoeboid, quits the parent-body, and either grows directly into an adult Arcella by the successive stages described above, or before doing so performs an act of syngamy. Gemmation, as above described, takes place in three difierent ways, as follows : 1. The buds are formed one at a time, and the parent-individual persists and continues to reproduce itself (simple gemmation, Fig. 80, H). 2. The whole body of the Arcella breaks up into numerous buds which swarm out of the shell, leaving behind in it the two primary nuclei, with a small quantity of residual protoplasm. The parent- individual then dies ofi, apparently, but it is possible that it may in some cases regenerate the body again. This process of multiple gemmation differs only from the simple gemmation described in the previous paragraph in being, as it were, greatly intensified, taking place with such rapidity as to use up almost the entire protoplasm at once (Fig. 80, 7). 3. The protoplasm of the Arcella, with the chromidia, streams out of the shell, leaving in it only the degenerating primary nuclei. Outside the shell the amoeboid body forms secondary nuclei, and breaks up by multiple fission into a number of amoebulse. This process differs from that described in the foregoing paragraph only in taking place outside the shell (Fig. 80, J). As akeady stated, the amoebulaj formed by multiple gemmation may either be agametes, which develop directly into the adult form, or gametes, which first go through a process of syngamy which has been described in the previous chapter (Fig. 80, K, L). Both agametes and gametes arise in the same manner ; the gametes, however show sexual differentiation as regards size. The zygote is an am'oebula which develops into the adult form in the same way as an agamete. In addition to syngamy (karyogamy) between amoebulse, chromidiogamy between adult Arcelloi also occurs, as akeady described ; the result in this case also is the formation of a POLYMORPHISM AND LIFE-CYCLES 181 number of amcsbulse which develop into the adult in the usual way (Fig. 80, M—Q). Arcella thus furnishes a surprising example of diversity both in the courses taken by the development and in the methods of syngamy. We may now consider some further complications of the life-cycle, which in other Protozoa takes usually a more definite and stereotyped course, less liable to the variations in one and the same species seen in Arcella. One of the commonest compHcations introduced into the hfe- cycles of Protozoa is the differentiation of sexual and non-sexual cycles. In the account given above of the life-cycle of Arcella, it has been seen that an adult may produce amoebulae which as agametes can grow up directly into the adult form without syngamy, or which as gametes copulate before developing further. The adult Arcellce, however, do not, so far as is Imown, exhibit any differentiation in relation to these developmental differences, the form that produces gametes being perfectly similar to that which produces agametes. But in other cases there may be two distinct forms of the adult individuals : the one, laiown as the sporont or gamont, which gives rise to gametes ; the other, termed the schizont or agamont, which produces agametes.* In this way an alternation of generations is brought about in which the life-cycle as a whole becomes a combination of two distinct types of developmental cycle — one known as schizogony, in which no sexual processes occur ; the other as sporogony, in which at one stage gamete-formation is followed by syngamy. An example of alternation of generations in a free-living form is seen in the life-cycle of Trichosphcerium (Fig. 81), as described by Schaudinn (146). The adult phase is a relatively large amoeboid form, approximately spherical in contour, and having the body surrounded by a gelatinous envelope in which at intervals there are apertures through which the lobose pseudopodia are extruded ; the " Ui^l-T^^'^ZTT^ ^ modification suggested by Butschli for the term fn^^na nl,'. ""S^^^^^ T'^^'^ ^'"^^ Schneider to denote the adult spore- eXrlC W ^\^°P^1"^° Gregarines (p. 339), and to distinguish it from the Shnolwr%^ bears the epimerite, known as a cevhalont ("cephalin." o?ders such P of resistant spores in Gregarines and allied ''snor;,nt ^"''^■^/^J^ accompanied by sexual phenomena, the word sporont has undergone both an extension and a change in its original meaning and has come to be used to denote a gamete-producing form. In E ™mo h on fhetl rcS/?o'r'''J\?^^ '''' "sporont" in this sens" aTd come worcL™scht/o?nnv ' " agamete-producing form, and further coined the rvccsrcsrotivflv^ ^ ^PO'-ogo'^y to denote the non-sexual and sexual Jvcn to t ha ^L^^^^^ " secondary meaning thereby tbn of res stanf 1^^^^^^^^ the occurrence of syngamy and not to the fornuv ouswoias agamogony and "gamogony " for "schizogony " and " sporogony." 182 THE PROTOZOA jfja 81 —General life-cycle of Trichosphcerium sieboldi, as an example of dimor- pliism in the adult condition combined with alternation of generations A Schizont or non-sexiial form, distingui8hcd by the possession of rod-like fodL in the envelope (compare FU this form^may ^^^'Ply, ^^^^^^ multiple fission (plasmotomy) in a " vegetative manner, or by the piocess S siSat on (schizogony) seen in B and 0. in order to give rise to tlie garao^e- pro udng form ; B, division of the body of the schizont into as many ccUs r sporSionr^ ") as there are nuclei ; G. rupture o the envelope and escape if r:|orogon\a as active am.bul.. each of wh.^^^^^^^^^^^^^ POLYMORPHISM AND LIFE-CYCLES 183 protoplasmic body is a multinucleate Plasmodium. There are two forms of the adult — the schizonts (agamonts), which are dis- tinguished by the presence of rod-like spicules in the envelope (Fig. 81, A) ; and the sporonts (gamonts), which have no spicules (Fig. 81, F). The schizonts reproduce themselves either in the free state or after encystment. In the free state the reproduction is by simple or multiple plasmotomy — ^that is to say, by division of the Plasmodium into two or more portions. In the encysted con- dition the schizonts divide by multiple fission into as many daughter- cells as there are nuclei in the Plasmodium (Fig. 81, B), and each daughter-ceU is set free as an amoebula (agamete), which may either grow up into a sporont, or into a schizont which repeats the process of multiphcation by schizogony. The sporont may reproduce itself in the free state in the same manner as the schizont, by plasmotomy, or it may become encysted, and then it multiplies in a manner totally different from that seen in the corresponding phase of the schizont. The nuclei of the encysted sporont multiply rapidly by karyokinesis (Fig. 81, G) until there are a very large number of minute nuclei ; very probably the final divisions in this process of multiplication are reducing divisions. The protoplasmic body then becomes divided up into as many minute ceUs as there are nuclei, and each of the daughter-cells acquires two flageha, and is set free as a flagellula or gamete (Fig. 81, H). The gametes, which are not differentiated in any way, copulate with those derived from another sporont, and lose their flagella (Fig. 81, 1—L) ; the zygote is a small amoebula which grows up into a schizont (Fig. 81, L, M, N, A). An alternation of generations similar to that of Tnchosphcerium occurs also in the Foraminifera (p. 234). Here the schizont contains numerous nuclei, which multiply by fission as the animal grows, and also chromidia ; it reproduces itself by a process of multiple fission, breakmg up into a number of amoebulse (agametes), each with a! nucleus and chxomidia. The amoebulae creep out of the old shell, which is abandoned, and each amoebula secretes a sheU for itself' Fig. 81 conlinuei : itself and grows, with multiplication of the nuclei {D and E) Into the gamete- but without rods in the envelope ; the sporont may also multiply in a veseta tive manner by simple or multiple fission, or it may form gametes^n the tTKr^at^'numV' f ''''''' ^if iP^-*-^ oi tL nuclef7tt s^orJlft ,iT. In^^ ^ number of very small nuclei, after which the body divides up into as many mmute cells as there are nuclei ; these cells are the gametes and each gamete acquires two flagella ; //, rupturaof the envelopTto sS fieo morf f!!'. .T' -hich swarm out and conjugate 7, conjugatSrof ?vvo gamete moic highly magnified ; , 7, after fusion of the bodies of the gametes the flagella are thrown off ; K, fusion of the two pronuclei ; L. compkto zygote hio th^Zr'' "P",'^'^^ ^itl^ nndtiplication of the nuclei TaIN) StertchtZiMifl)."'"' ^'^'"^ ^ starting-point of the lifi-cycfel 184 THE PROTOZOA and grows up either into a sporont or into a schizont again. The sporont possesses only a single large nucleus, the primary nucleus originally present in the amoebula, and a great number of chromidia. When the sporont enters upon the reproductive phase, the primary nucleus degenerates, and an immense number of secondary nuclei are formed from the chromidia. Ihen the protoplasmic body divides up to form as many cells as there are secondary nuclei. The cells thus produced are the gametocytes, each of which divides by mitosis to form four small cells, the gametes, which acquire flagella, swim off, and copulate with gametes produced from another sporont ; there appear, however, to be no differences exhibited by the gametes of opposite sexes. The zygote forms a shell and grows into a sporont. Since the zygote is very much smaller than the amoebula produced by schizogony, the shell formed by it is also smaller. This shell is later the initial chamber of the polythalamous adult, and thus leads to a dimorphism in the adult shells, so-called " micro- sphaaric " and " megalospha3ric " forms (p. 235) — a dimorphism related, in this case, not to the manner in which the adult individuals reproduce themselves, but to the manner in which they have been reproduced. In free-living forms the alternation of generations is related to external conditions of the environment, as, for example, seasonal changes ; the sexual generation may appear in the autumn, while the non-sexual generations are found in the spring and summer. In parasitic forms, on the other hand, alternation of generations is of common occurrence in relation to a change of hosts. Thus, in the life-cycle of the Coccidia (Fig. 152), described above, the multi- plicative phases reproduce non-sexually by schizogony, as the so- called " endogenous cycle " ; the propagative phases are preceded b}^ gamete-formation, leading to spore-formation, the so-called " exogenous cycle." In Haamosporidia, such as the malarial parasites, for example (Fig. 156), the alternation of generations is related to an alternation of hosts ; the non-sexual, schizogonous generations take their course in the blood of the vertebrate host, in which the gamonts are produced, but do not develop further unless taken up by the invertebrate host, in which alone gametes are formed and sporogony takes place. The phrase " alternation of generations " must not be construed into meaning that the sexual and non-sexual generations succeed each other in a regular alternation. On the contrary, such regular alternation, if it occurs at all, is rare, and as a rule a single sexual generation is followed by several, or it may be by an immense number, of non-sexual generations before the sexual cycle recurs. The malarial parasite can multiply non-sexually in the blood for many years without dying out ; and if propagated artificially from one POLYMORPHISM AND LIFE-CYCLES 185 vertebrate host to another, it is probable that it could dispense alto- gether with the sexual cycle, which occurs only in the invertebrate host, so far as is Icnown. In the suborder Eugregarinse of the Gregarinoidea an opposite condition occurs, since these forms possess only the sexual cycle, sporogony, and there is no non-sexual schizogony. Whether this condition is to be regarded as a primitive state of things, or whether the Eugregarines are to be regarded as having dispensed with the non-sexual process of schizogony seen in the alUed suborder Schizogregarinse, must remain an open question. A further caution is also necessary with regard to the alternation of generations in Protozoa. From the known facts of the malarial hfe-cycle, in which an alternation of sexual and non-sexual cycles is correlated with an alternation of hosts, it has often been assumed, imphcitly or exphcitly, that a similar alternation of sexual and non- sexual cycles must occur in other cases where there is an alternation of hosts, as in the case of trypanosomes, and in particular that the sexual cycle must occur in the invertebrate host. This assumption is by no means justified, however, and has been the cause of much unsound or unwarranted interpretation of the facts, especially as regards the significance of the various forms of trypanosomes, which are continually ascribed to sexual differentiation on no other ground than the bare fact of form-differentiation, as pointed out in the previous chapters. Up to the present there is not a single case m which sexual phenomena in trypanosomes have been described m a perfectly satisfactory manner, free from all doubt ; and, on the other hand, it has been asserted that the syngamy occurs in the vertebrate host in these parasites (Ottolenghi, 492). Bibliographj.—For references see p. 480. CHAPTER X THE GENERAL PHYSIOLOGY OF THE PROTOZOA The Protozoa, as has been seen in the previous chapters, exhibit a wide range of structural differentiation, from forms which exemplify a ceU reduced to its simplest essential parts, nucleus and cytoplasm, to others in which the cytoplasmic elements give rise in different parts of the body to a great variety of structures and organs, each subservient to some special function. In the Protozoa of simplest structure, therefore, the study of the physiological activities of the organism coincides, more or less, with that of the elementary properties of the living substance, protoplasm, its pecuhar powers of metabohsm and transmutation of energy ; while in Protozoa of comphcated organization the mechanism and mode of action of the various cell-organs must be considered in relation to their structure, so far as it can be made out. It is not possible to discuss adequately, in the limited space of a chapter, the intricate problems, for the most part still very obscure, of the vital mechanisms of elementary organisms. The matter can only be dealt with here on broad general lines, and those desirous of studying the subject fm-ther must consult the references given to special works or memoirs .* On the other hand, the special functions and mechanisms of the various ceU-organs (" organelW) have been considered in describing the structure of the organs themselves. In this chapter, therefore, it is intended rather to fiU the gaps left in previous chapters ; and the physiological problems presented by the Protozoa will be sketched in brief outhne under the foUowmg headings • (1) Nutrition and Assimilation ; (2) Respiration ; (3) Secre- tion and Excretion ; (4) Transmutation of Energy ; (5) Reactions to Stimuh and to Changes of Medium or Environment ; (6) Degenera- tion and Regeneration. * For works dealing with the physiology of Protozoa iii a i^I^^I^i J„t should consult especially Verworn, " Allgememe ^y^^-}'^^'^^^^^^^^ ^^Zs THE GENEEAL PHYSIOLOGY OF THE PROTOZOA 187 ]. Nutrition and Assimilation. — Living organisms, considered generally, exhibit a great variety of methods of nutrition, which may bo classified into two main groups ; bearing in mind, however, that in aU classifications of living beings, or of their vital properties, any groups or classes that can be distinguished are always connected by gradual and imperceptible transitions, and that consequently forms will present themselves which, owing either to their transi- tional nature or to the imperfect state of our knowledge concerning them, can only be assigned to one or the other group in a manner as arbitrary as the statement that the 21st of June is the first day of summer — a difiiculty which in no way invahdates the distinction between spring and summer. In the first place, many organisms can build up the complex protein-substances, of which the living protoplasm is composed, from simpler chemical materials. Of this type there are found among Protozoa, as already stated, two types of nutrition : first, the holo'plnjtiG, or plant-like, in which the organism is able, by means of special cell-organs, to utiMze the energy of the sunlight in order to synthesize its body-substance from the simplest chemical materials, such as water, carbon dioxide, and mineral salts, through a series of substances in an ascending scale of chemical complexity ; secondly, the saprophytic type, in which the body contains no visible organs subserving the function of nutrition, but the organism is able to build up its protoplasm from food-materials consisting of organic substances in solution which are far less complex chemically than the body-proteins. In the second place, many organisms cannot build up their body- substance from materials of simpler chemical constitution, but are entn-ely dependent on a supply of protein-substance ready -made, which they obtain either by ingesting and digesting other Hving organisms m the holozoic method, or by Hving as parasites at the expense of other creatures. These two methods graduate into one another, since many parasites simply devour portions of the bodies ot their hosts m a holozoic manner, but the majority of parasites absorb fluid nutriment from their hosts in an osmotic manner • Hence it is convenient to distinguish holozoic and osmotic parasites'. Considering these various methods of nutrition, it is seen that Irom the pomt of view of the nature of the food, those which ingest It 1 ^""^ ^^l^f^' ^^'^^^'^^^ be distinguished from those which absorb then- food in a diffused or dissolved condition (holo- phytic and saprophytic forms and osmotic parasites). From the pomt of view of the structure of the organism, those which possess special organs of nutrition (holozoic and holophytic forms) can be 188 THE PROTOZOA (a) Holophytic Nutrition. — ^The characteristic of this type of nutrition is that the organism contains rpecial pigments by means of which it is able to decompose COg in the sunlight, setting free the oxygen and retaining the carbon^ which is built up in union with other elements derived from water and mineral inorganic salts. The pigments, termed comprehensively chromophyll, are contained in bodies termed " chromatophores," which occur in diverse forms and varying numbers in different species, and which multiply hy division when the cell divides. The chromophyU-pigments are of various tints — yellow, brown, green, blue-green, etc. — but the commonest tint is the green chlorophyll, similar to that character- istic of plant-cells. A blood-red pigment, termed hcematochrome, occurs in some flagellates — e.g., Hcematococcus ; it appears to be a modification of chlorophyll produced under certain conditions (see Reichenow, 97 '5). For the details of the complicated process of the synthesis of chemical substances in the holophytic mode of nutrition, the student is referred to botanical textbooks dealing with plant-physiology. There appears to be no essential difference between the assimilative processes of holophytic Protozoa and of ordinary plant-cells. A characteristic product of holophytic nutrition is seen in the forma- tion of amyloid substances, the most important of which are starch (amylum), and an aUied substance known as " paramylum," which differs from starch in some of its reactions, notably in that it is not coloured blue with iodine. Paramylum is of more frequent occur- rence in Protozoa than true starch. The amyloid substances occur in characteristic masses in the cytoplasm (see especially Biitschli, 153). The chromatophores of Protozoa contain usually small refringent bodies termed yyrenoids, which also multiply by division. The pyrenoids are often surrounded by a coat or envelope of paramylum, and appear to be the centres of the production of amyloid substance. Many flagellates with green chromatophores combine holophytic with saprophytic nutrition. Examples of such " mixotrophic " forms are seen in the genus Euglena (Zumstein, 223), the species of which flourish best m a medium containing organic substances, and cannot maintain themse ves in pure water. Euglena viridis was shown by Khawkine to be able to nve for a considerable period in the dark in media containing organic substances, but did not lose its green colour and did not multiply. E. gracilis, on the other hand, in Zumstein's experiments, lost its green colour and passed mto TistaM. phase in the da^rk, or even in the light when placed ui so utions very rich in organic substances, nourishing itself as a saprophyte. When the Astasia-iov^n was exposed to the light, in solutions ^^f "J^g ^^^y^S- of organic matter, it became green agam and passed back 'J'^^J^'T.^'^^^ T>hase The degree to which the species of Euglena can adapt themselves to a purely saprophytic life would appear to vary in different cases. In the ^oloudess JEorms thJ chromatophores lose their chlorophyU, and remam as ^tl^'c^mrnltf of holozoic and holophytic nutrition has been noted above (p. 15). THE GENERAL PHYSIOLOGY OF THE PROTOZOA 189 (b) Holozoic Nutrition. — In this type of assimilation three series of events must be distinguished, each of which may be effected by means of special organs : the capture and ingestion of the prey ; its digestion ; and lastly the rejection from the body of the non- nutritive residue (defsecation). The methods of food-capture and ingestion have been dealt vnth above in a general way. As regards food - capture, methods of prehension by means of pseudoj)odia, or by special adhesive organs, such as the suctorial and raptorial tentacles of Acinetaria (p. 457), the tongue of Didinium (p. 442), etc., must be distinguished from methods whereby the food is wafted towards the body in currents produced by special vibratile organs such as flagella and cilia. As regards ingestion of food, a distinction is imposed by the nature of the outer surface of the body-protoplasm, whether naked or invested by a firm cortex or cuticle. In naked forms the food is ingested at any point, by methods which vary in different forms. In Amceba proteus the hinder end of the body is most active in ingestion ; in Actinosphcerium all points on the surface are equally active. Rhumbler (204) distinguishes four methods of food-ingestion in amoebae : (1) By " import," when the food is drawn into the protoplasmic body as soon as it comes into contact with it, and with scarcely any movements on the part of the amoeba (Fig. 23) ; (2) by flowing round, " circumfluence," in Avhich the protoplasm, as soon as it comes into contact with the food-particle, flows round it on all sides and engulfs it ; (3) by " circumvallation," when the amoeba, while stiU at some distance from the object, sends out pseudopodia which flow towards each side of the prey, and ultimately meet round it and surround it com- pletely, without ever having been in actual contact with it ; (4) by " mvagmation," in which the amoeba touches and adheres' to the object, and the portion of the ectoplasm in contact with it is ■ invagmated mto the endoplasm like a tube, the waUs of which become hquefied and fused together, so that the food-particle is as It were, sucked into the endoplasm (Fig. 82). Of these various methods, the process of circumvaUation is most suggestive of a conscious and purposeful act on the part of the amoeba ; but a remarkable parallel to it is seen in the penetration of Lankesterella mto a red blood-corpuscle, as described by Neresheimer (seep 378 tnjra}. In this case, as soon as the parasite comes within a certain distance of the corpuscle, the latter opens its arms, as it were, to Lir'^'t'. it in a manner very similar to the Tn CircumvaUation on the part of an amoeba. which ovo^'' / ^^^'"'"^ '^"^^ «^b«*'^^c^ 190 THE PROTOZOA According to Rhumblor (204), with a more fluid condition of the ectoplasm, the food is ingested by import or circumfluence ; when the ectoplasm is stiffened to a membrane-hke consistence, the ingestion is effected by cii'cumvallation or invagination. Rhumbler maintains that all known methods of food-ingestion by amoebae, as well as their movements, can be explained mechanically by differ- ences of surface-tension in colloidal limiting membranes, and can be imitated artificially in substances that arc not living. Fig 82 —Ingestion of ca food-particle by " invagination in Amaba terncola. A-E Five stages of tiie process, semi-diagrammatic; diagrammatic fiaure'to show the direction of the currents on the surface of the body of tlio amceba during the process of ingestion. After Grosse-AUermann (245). In corticate forms the ingestion of food is hmited to one or more special openings or organs, in which a direct communication is estabhshed between the fluid endoplasm and the surromidmg medium, as in the cytostomes of FlageUata and CiUata and the suctorial tentacles of Acinetaria. The dic^estion of the food is effected within the protoplasmic body, and as a'nile the prey is taken bodily into the cytoplasm ; but the Acinetaria have the power, not fully explained, of sucking out THE GENERAL PHYSIOLOGY OF THE PROTOZOA 191 the body-substanco of their prey, probably by the aid of secreted ferments. Together with the food a certain amount of water is ingested, forming a drop or food-vacuole in which the actual digestion takes place. The quantity of water ingested with the food varies considei'ably, and, speaking generally, is inversely pro- portional to the size of the object that is devoured ; that is to say, small food-particles, such as bacteria, lie as a rule in a very distinct vacuole, but large bodies, such as diatoms, usually appear as if imbedded in the cytoplasm, with no liquid vacuole visible around them. AmoebaB not infrequently devour organisms larger than themselves, so that the cytoplasm of the amoeba appears hke a thin sldn or envelope over the surface of the prey. According to Greenwood (161), Amceba proteus takes in but little fluid when it ingests quiescent sohd matter, such as starch-grains or yeast-cells, but when actively-moving prey is dealt with an area of water not inconsiderable surrounds it; on the other hand, non-nutritious particles are not surrounded by fluid when they lie in the endoplasm. In forms in which food is ingested through a cytostome, as in Ciliata, the food-particles, usually of smaU size, are wafted down the oesophagus and coUect at its proximal blind end, where a depres- sion arises in the endoplasm, which gradually deepens, and finally closes over and separates from the oesophagus as a closed vacuole containing the food. According to Nirenstein (181), the food- vacuole is detached from the oesophagus by suction of the'endoplasm, hke a process of swaUowing (" Schlingvorgang "). The vacuole is at first immured in a thin layer of less fluid protoplasm, doubtless as the effect of contact with water (see p. 44) ; consequently the vacuole is not at first circular, but often spindle-shaped in its contours ; it soon, however, assumes a spherical form, indicating that its protoplasmic envelope has become liquefied. ^ In cases where actively- motile organisms are devoured— as for example flagellates by amoebae-the prey can often be seen to per- form violent movements within the vacuole ; but soon the — ■ — , '^KA.v avujxi. (jiio move- ments become feebler and cease entirely. Bacteria ingested bv Parameaum become immobUe about thirty seconds after the vacuole has become detached from the oesophagus. In many cases however, the prey is kiUed when seized by the pseudopocMa, anci before being mgested, as in Hehozoa and Foraminifera. After the prey is killed it is slowly digested within the food-vacuole l)urmg the process of digestion the food-vacuole may perform defimte migrations within the body of the animal. In amoeba the without, however, pursuing any definite course, and they tend to t TvlnlTnTlV".''^^ '''' ^^e animll IS moving m a definite cbrection. In the Infusoria, on the other 192 THE PROTOZOA hand, the endoplasm shows a constant rotating movement, known as " cyclosis." In Paramecium the vacuoles are carried round by the current of the cyclosis, and each vacuole may either do a short course or a long course ; the short course is simply round the nucleus, keeping close to it, while the long course travels the whole length of the body, up one side and down the other. As a rule a vacuole goes a short course two or three times, and then does a long course (Nirenstein, 181). The path of the vacuole varies, according to the nature of the contents ; but the tendency is to keep them in the region posterior to the nucleus, where the contents are either cast out through the anal pore, or the vacuole circulates again in the cyclosis. In Carchesium the food- vacuoles, when formed at the base of the oesophagus, pass down to one end of the horseshoe-shaped nucleus, and then ghde close along its concave margin, passing round and up to the opposite end of the horseshoe into the region near the upper end of the vestibule, from whence the vacuole is finally emptied through an anal pore into the vestibule itself (Greenwood, 162). The process of digestion within the food- vacuole has been studied by a number of investigators, amongst whom Le Dantec, Greenwood (162), MetschnikofE (180), MetalnikofE (179), Nirenstein (181), and Khainsky (170-5), must be speciaUy mentioned. Their results are not always in agreement, indicating that the process of digestion is not always the same in different cases, even in the food- vacuoles of one and the same species. According to Nirenstein (181), the food-vacuoles of Infusoria exhibit changes which can be divided nto two periods : in the first the vacuole shows an acid reaction, and the ingested organisms are IdUed ; in the second the vacuole has an alkahne reaction, and the albumens are digested. According to Khainsky (170-5), however, the reaction of the food-vacuoles of Paramecium is acid during the entire period o the Proteolytic process, and only becomes neutral and finally alkahne when the solution of the food-substance is at an end. In the first or acid period, according to Nirenstem (181), the ingested f ood-particles-e.g., bacteria-after being rendered im- SL are clumped together, enveloped in a turbid substance whth makes their outhnes indistinct. The reaction of the vacuole Ts strongly acid, due to the presence of mineral acid m the vacuole DiTng fhl per od, which lasts from four to six minutes, the vacuole di^infshes in size, iU it is not more than one-third of its original size Wh n the vacuole was first formed, its wall was — ded by a Imber of granules which stain very distinctly with neutral-red number oi gi „^ .^^^Iv into the interior of the vacuole after Thtsir dS'S tnsidera bly in si.e. Ni— .oga^s Tho red-staining granules as bearers of a trypUc ferment. THE GENERAL PHYSIOLOGY OF THE PROTOZOA 193 In the second or alkaline period the vacuole enlarges rapidly to more than its original volume. The red colour produced by staining with neutral-red disappears. The clumped food- mass breaks up into smaller particles again. From the red-staining granules of the first period deeply-staining spheres arise, homogeneous, refractile, and apparently fluid (Nirenstein, 181). According to Khainsky (170-5) the grains or droplets which are formed gather at the surface and pass out into the endoplasm ; they represent the first products of the assimilatory process in the vacuole, and their further chemical transformation takes place in the endoplasm itself (compare the refringent bodies formed^ in the process of digestion in acinetans, p. 458). According to Nirenstein, however, thespheres become smaller and smaller, bemg reduced to tiny grains which vanish completely, dissolved in the vacuole-contents. The vacuole now diminishes in size a second time, and passes to the anal region, where it fuses with other similar vacuoles, and is finaUy rejected from the anal pore. In other cases, however, no acid reaction has been demonstrated in the vacuoles at any time, as, for example, in Actinosphcenum—a. pecuHarity which is perhaps to be correlated with the fact that in this form the prey is kiUed when seized by the pseudopodia. It may be supposed that the processes which, in Infusoria, etc., go on during the first or acid period of the food-vacuole, take place in Actino- sphosnum and some other forms before the vacuole is formed in which case the vacuole itseH shows only the second or alkahne phase of the digestion. the^"ecS of ?oirrr."?.^f ^^T"^'"' '^^^ ^"S^^^^^ particles excite tne secretion of acid, but the true digestive vacuole is only foiled under the s nnulus supplied by nutritive matter. Metalnikoff (179) Cwever fjund £lW \T'A^^'^^^^"\f '^"^ food-vacuoles are Lt acSd then la^Vt^rZ^7 ^"""^^ throughout in their reactions and others S ceU has the'cn^/-? """f T'f''' ^^^^g^out ; he concludes that the EL the p^^^^^^^^^^ adaptmg Itself to the food supplied, and of menf? Th/rf.? i ^ digestive juices in accordance with its require- Tuf pVod^ceSeTent SSrT''!' -^-^ ^i^'blool patho^genirorgatrortw^^^ ^^^--^^ Proftn?"^^ 'V^'™"''? ^^^^ from different fnZZlf"" the digestion takes a different course m difierent cases. In the plasmodia of Mycetozoa, a peptic ferment which when acidulated dissolves fibrin, hL been i olaZ butTnee M^nZu ^l^,^rf " ^ ^^^-^^^^ alkai;;Lae Sn MeTschni^ol ,1^ that the ferment must be without function in +r r ^\ ^ however, that the food-vacuoles formed ^rZ^Z^'^'^.t^ ; - contra:* "he fertntL th^^^^^^^^ ^-^^-^ the peptic isolated r aLtStase,"^ ''''' '~ 13 194 THE PROTOZOA Some doubt has existed as to the power possessed by Protozoa of digesting fats, and, according to Staniewicz (208), no digestion of fat takes place in Infusoria. According to the recent investiga- tions of Nirenstein (182), however, Paramecia under natirral con- ditions contain fat in more or less considerable quantities. By choice of suitable food, the quantity of fat in the endoplasm can be increased greatly. The fat-granules serve as reserve-nutriment, and disappear under starvation. Paramecia which have lost their fat in this way, if then fed with milk, oU-emulsion, or yolk of egg rubbed up in water, show in a few hours the endoplasm fuU of fat- granules ; if fed with starch or particles of egg-albumen, the same result is obtained, but not to anything like the same extent. Experiments on fatty substances ingested by the animals showed that the fat remains unaltered during the first (acid) period of the digestion in the food-vacuole, and is digested during the second (alkaMne) period. Feeding with fatty acid and glycerine also leads to storage of fat in the endoplasm. If fed with oil-globules stained with Soudan III., unstained oil-globules appear in the endoplasm. Nirenstein concludes from his observations that the fat is broken up into its soluble components in the vacuole, and synthesized again to neutral fat in the endoplasm. The indigestible residues of the food are ejected from the body either at any point on the surface, in amoeboid forms, or through a definite aperture, in corticate forms. A great accumulation of fgecal matter may take place in some cases, as in the " stercome " of Foraminifera (p. 233), of Avhich the animal purges itself periodically. (c) Saprophytic and Parasitic Nutrition. — In this type the organism absorbs its nourishment by diffusion through the surface of the body without the aid of any visible organs or structural differentiations of any kind. Practically nothing is loiown of the mechanism by which this is effected or of the chemical processes involved, but it is probable that enzymes secreted by the organism reduce the nutritive particles to a soluble form prior to absorption. There is reason to behove that the nucleus is specially concerned in the production of enzymes, and in many species, parasitic or otherwise, the behaviour of the nucleus indicates a relationship between it and the process of absorption of food-substance. In Carchesium, as akeady stated, the path along which the food- vacuoles travel runs close along the inner edge of the horseshoe-shaped macronucleus (Greenwood, 162) ; in Euplotes, similarly, the large macronucleus encloses an area containmg aU the food-vacuoles (Fig. 182). Accordmg to Wallengren (214), the reactions of the food-vacuoles of Paramecium change as they pass the nucleus and the function of the cyclosis in the endoplasm is to bring the food- THE GENERAL PHYSIOLOGY OF THE PROTOZOA 195 vacuoles near, and under the influence of, the nucleus. In the coccidian partisite Caryotro'pha (p. 352), the nucleus of the parasite is connected by a Idnd of protoplasmic canal with the nucleus of the host-cell (Siedlecki, 653). In the astomatous Ciliata (p. 451) a diffuse nucleus is very commonly found, probably in relation to absorption of nutriment by the osmotic method. The process of nutrition in Protozoa may lead in some cases, not to growth of the protoplasmic body dh-ectly, but to the produc- tion and storage of reserve food-substances, which are precipitated ia the cytoplasm, and are utihzed at a later period for rapid growth dm-ing reproductive phases. The reserve- materials deposited in this way vary considerably in nature in different cases. Examples are the paramylum-grains of many flagellates; the paraglycogen- grains of gregarmes and cihates, similar in nature to glycogen, but with certain distinctive reactions ; the plastinoid granules of coccidia (p. 346) ; and other similar substances. In Radiolaria oil- globules and albumen - spheres occur. An important substance, acting apparently as reserve- material for the growth of the nucleus is volutin (p. 68). ' The effects of starvation on Protozoa have been studied by a number of investigators, most recently by Lipska (173), who gives a complete bibliog- raphy and resume of previous work on the subject. Lipska found that P^rameomm died after hye to seven days, a much shorter period than allowed by WaUengren (214) and others, indicating that Lipska's methods were more drastic and sources of food were more thoroughly excluded in her exjerimrt? fJ ^^y^ f ^T""'^ ° starvation the reserves ^ the endoplasm are^useTup Snu es mZ^^:1'' :T1 *^,f t^^^ «"^aUer endoplasm^ granules. After the fourth day the animal becomes deformed. Its dimensions voTume ^TT:;r'7' ""^-fr*^ -Pervenes when it has lost half ^ 3 volume. The ectoplasm with its cilia and trichocysts undereo no chanjro but the endoplasm loses its food-vacuoles and a part of iS^crvstals and ZT::7z\'T'''^'^t —nucleus becomes ei^vg:r:Ti>r:^, up into two halves. The micronucleus undergoes no chanel of aiiv kind Death IS preceded by a progressive enfeeblement of all Sions sucrf; movements of the ciha and pulsation of the contractile vacS AccoJcW Paramecia placed in™ ch tub^ wn« ^^P™ft«' liowever, the number of containing symbiotic aw iro "^""^ Paramecia without theS ^ t« starvation than those sto!,d^aTtn"°"'"~^^ respiration in its widest sense must be under- tored uV" b • "'^^'"^"^ "^^^'^^y Potential energy fu™ sh compounds of high complexity is set free to TTobilet ma?bVTrf -^-tie- object may be effected m two ways-by processes of oxidation 196 THE PROTOZOA or by the splitting up of complex chemical substances ; the result in either case is the production of energy in various forms and of simj)le chemical substances, such as water and carbon dioxide (compare Barratt, 148). For the processes of oxidation the organism may either absorb free molecular oxygen from its environ- ment, or may produce it by internal molecular changes of substances contained in its own body, as in anaerobic organisms hving in a medium in which free oxygen is lacking. Many free-hving Protozoa require oxygen, and are visibly and rapidly afiected by the lack of it, especially in theh powers of movement. No special organs of respiration are found in any Protozoa, being unnecessary in animals of such small bulk, and in which, consequently, the surface of the body is considerable in proportion to the mass. The contractile vacuoles, when present, are doubtless a means of eliminating carbon dioxide, together Avith other waste products, from the body. It must be supposed, there- fore, that as a general rule oxygen is taken up from the surrounding water by the protoplasm, of which the Umiting membranes are freely permeable, and that the carbon dioxide is given off in a similar manner. The experiments of Verworn (211) on Spirostomum show that the respiratory processes take place in the cytoplasm, independently of the nucleus, which takes no share in respkation. On the other hand, many sapropehc (p. 14) and parasitic forms inhabit media lacldng in free oxygen, and are anaerobic ; in such forms the respiratory processes of the protoplasm can only take place by intramolecular changes, in which the stored-up reserve-materials are probably spht up to supply the required oxygen. The experiments of Putter (201) on a number of species of Ciliata, both free-living and parasitic, showed that, when these animals were placed ni an anaerobic environment, different Individuals of the same species reacted very differently to the conditions, some dying very rapidly, others being quite unaffected for a long time. It was shown further that this difference was related to the amomit of reserve -materials present in the body (proteins and elvcoeen), which can be observed to vary greatly m different mdividuals from the same culture. If Paramecia were first starved for some days and then placed In anaerobic conditions, they succumbed much more rapidly than normal individuals. Moreover, under anaerobic conditions the reserve- materials were used up much more rapidly than under normal conditions Sid without resultmg in increased production of energy. Opahn^, when Xced in a culture-medium to which albumen was added by boiling up ch- ed wMte of egg in salt-solution, was able to make use of the energy of the albumen without the help of free oxygen, and so to live for a much longer time. The dhates were fo^id to succumb much more rapidly to the effects of anaerobu. conditions in smaller than in larger quantities of water, as the result of auto- intoxication in consequence of the defective excretion of the Products of anaerobic metaboHsm Svirostomum wb., found to be more affected by THE GENERAL PHYSIOLOGY OF THE PROTOZOA 197 products of metabolism, a primaiy necessity of anaerobic life. The question of size is also a factor, since deleterious substances may diffuse from the surface of the body, and in a small body tJie surface is greater in proportion than in a larger one. Consequently the conditions are more favourable for a smaller species, such as Paramecium, than for a largo form, such as Spirostomum. Excess of oxygen was found by Putter (198) to have an injurious effect on Spirostomum, affecting, however, only the cytoplasm, and not the nucleus, in the first instance. On the current view that the sjonbiotic vegetable organisms present in many Protozoa aid in the respiratory processes by absorbing the carbon dioxide, breaking it up, and setting free the oxygen, the experiments of Lipska (173) on a culture of Paramecia which contamed green algte (Proto- coccacese) in their endoplasm are of considerable interest. In two glass vessels of equal size there were placed, in the one Paramecia with, in the other without, the algse in their body. Hydrogen was circulated tlirough the vessels to di-ive out the air, after which they were hermetically sealed and exposed to the same conditions of light and temperature. After fifty hours the vessels were opened. The Paramecia without algaj were dead, but those contammg algse were stiU alive, though feeble in their movements, and they revived completely in about twenty-four hours after air had access to them. In another experiment two batches of Paramecia were kept in the dark • after eight days those without algse were dead, while those containing alga^ were perfectly normal. Old cultures of Paramecia containing algse showed no conjugation ; Lipska explains this as due to the influence of the algaj since, by settmg free oxygen, they prevent the development of anaerobic bacteria which produce substances toxic to the Infusoria According to Popoff (185), the depression-periods of Protozoa (p. 208) are partly due to derangements of the respiratory processes and to accumulation 01 products of metabohsm in the cell. 3. Excretion and Secretion.— The waste substances excreted from the protoplasm may be either soluble or insoluble in nature If soluble, they may either pass out of the protoplasmic body by diffusion from the surface, or may be removed by the agency of the contractile vacuoles. ContractUe vacuoles are of common occurrence in free-living fresh-water Protozoa, but are usuaUy wantmg in marine forms, or, if Jhe^fcurln them differentiatfon (CO "pare kTIsCT o's"?' ^f^' T"^^ ^V"^*"^^^ believed at one time to Pmrvf^r^' V " . ^ contractile vacuoles were as circulatory organs bXhi^nlAr^ ^'^ '^^-^^f y' ^^^^ ^^^^t^^^^ «™Ply fully, it harLTrov'ed th ?thir J" Tu'"^ they have been studied drl Jennings, m, lCZkl m-l^"^ '"^^'^ themselves to the exterior (compare pre^ssSrfmakel t'llf 'iZTr^K^^^^^ Forms assumed after addition of potash-solution : i), contracted, beginning to throw out pseudopodia ; E, F, radiosa-torms. After Verworn. cases the life-cycle appears to be of comparatively simple type, and the species is monomorphic or nearly so, as in AclinosphcBrium ; in other cases there is a well-marked alternation of generations, with dimorphism in the adult condition, as in TrichospJmrmm (p. 182), the Foraminifera, etc. Classification.— The Sarcodina are subdivided into a number of orders, the distinctions between which are based prmcipally 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, tlie 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 Ammha 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 limax-torm the whole body flows forward as a single pseudopodium, gliding along like a slug ; in the radiosa-iorm the spherical body becomes star-like, sending out sharp-pointed pseudopodia on 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 proteus. 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 Biitschli (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 Amoebo5a. —Amoshoid forms of simple structure ; skeleton lacking or in the form of a simple shell. 1. Suborder Reiiculosa {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 XenophyopJiora.— With skeleton of foreign bodies and a peculiar internal structure. IV. Order Mycetozoa. — Semi-terrestrial forms witli 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. Amgeb^a. 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 reticulose 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 tjrpes of organisms are referred to this suborder : (a) Large marine plasmodial forms ; an example is Pontomyxa flava, described by Topsent from the Mediterranean and British Channel. Pontomyxa is a multinucleate 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 s'ngle nucleus, marine or fresh-water, which reproduce by process of multiple fission forming swarm- spores. These forms have been subdivided into two famiUes, according to the type of swarm-spore found — Zoosporidce, pro- ducing flagellulse ; and Azoosporidce, producing amoebulse. An example of the Zoosporidce is furnished by the genus Pseudospora, which preys upon algse, diatoms, Volvocineae, etc. The adult phase is amoeboid, flagellate, or even Heliozoon-like. It feeds on the cell- substance and chlorophyll of the prey, and multipUes 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 flagellulse thus formed, which are therefore gametes; in other cases the flagellulse 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 smaU amoeboid form which, like Pseudospora, preys upon algse (Fig. 86), devouring the contents of the cell, and multiplying m the free state by binary fission. It also encysts and breaks up within the cyst by multiple fission to form a number of amoebulse, 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. Lohosa.— This suborder comprises a great number of organisms, which it is convenient to subdivide into— (a) Nuda (Gymnamoebse), Fig. 86. — Vampyrdla lateritia : various forms. A, Free Heliozoon-like phase ; B, creeping amoeboid phase ; C, amceboid form attached to a Gonferva-Giei- 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 ; G and D after Cash and Hopkinson. with no shell ; and (&) Testacea (Thecamoebse, Thalamophora), with a shell or house. General Characters.— Fam.ilia,Y examples of the Lobosa Nuda are furnished by the species of the genus Amosha and allied forms. A very large number of free-Hving 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 Am(x:ha 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 m but few free-hving amoebae, but in such protean organisms it IS quite unsafe 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 amoebulee 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-Hving amoebae are aquatic in habitat. A certain number, however, are semi-terrestrial, inhabiting damp earth, moss, etc. Such is Amoeba terricola {vide Grosse-AUermann). 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 bo:!ies of animals of all kinds, for the most part in the digestive tract. The entozoic amoebae are commonly placed in a distinct genus, Entamoeba, 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 Entamoeba blattce, from the intestine of the common cockroach ; others are E. ranarum of the frog (Dobell, 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 Labridce, 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 Arcella, 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- bulse, 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 Arcella. This type of life-cycle is probably very common in many amoebae, Fia. 87. — AmcBba 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 ; C, incomplete division of the generative nucleus ; p, 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 ; G*, 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 mto 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 ot ail amoebae of the Umax-gvoni^ ; 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 itseH 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 flageUa 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 Paramoeba (Fig. 49). Fig. 88. — Amceba 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 amcebaj, each with its two nuclei, encysted together, the nuclei beginning to give off chromidia ; 0, the two nuclei of each amoeba fused, numerous vegetative chromidia in the cytoplasm ; D, the bodies of the amoeba; 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 " gemmules " budded from small free amcebss of the vroteus-tjve, each gemmule becoming detached and developing into a flagellated gamete of a cercomonad type. The flageUulae were observed frequently to lose their flagella and become amoeboid. Copulation of two fla^ellulEe took place to form an amoeboid zygote. Metcalf s observations uwn the syngamy in this case recaU strongly the observations of Jahn (294) on the sexual processes of Mycetozoa (p. 242). It is possible that the sjaigamy observed by him did not form a part of the life-cycle of the amosba, but of some other organism. . , , t • x The sexual process described by Nagler (95) m Amoeba diploidea is of a TPmarkible kind (Fie. 88). In the ordinary vegetative condition the ama3ba poresis L^^^^^^^^ nuclei, which divide simultaneously each tmxe the THE SARCODINA 223 animal reproduces itself by fission. Tho sexual process begins by two such amcebffi commg together and surrounding themselves \vith a cyst in common. Within tho cyst their nuclei first give off vegetative chromidia, which are absorbed, after which the two nuclei in each separate amoeba fuse together to form a single nucleus, a synkaryon. The protoplasmic bodies of the two amcsbte 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 imdergoing this process of reduction, approach each other, but remain separate, and the amosba 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 hinucleata, 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 blattce 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 amoebulae are gametes which copulate after being set free, and the zygote grows into the ordinary vegetative form of the amoeba. E. blattce 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 coU was given by Losch to such organisms (synonym, Entamoeba hominis, Casagrandi and Barba- gallo). It is, however, certain that more than one species of amoeba occurs in the human bowel, and Losch's name must therefore be restricted to one of these. An epoch in the study of human entozoic amoeba? was marked by the researches of Schaudinn (131), who distinguished two species. The first, to wiiich Jie restricted the name Entamoeba coli, occurs commonly in Europe and elsewhere as a harmless inhabitant of the intestine— that is to say, like ^. blattcB 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 lltS'.Z ""Tl'Tt '^nd rectum. The second species, to which Schaudinn gave tho name E. histolytica* is, on the contrary 224 THE PROTOZOA reSn? .n.? kmd, which occurs in tropical and subtropical regions and is the pathogenic agent of amoebic dysentery and liver-abscoss : t 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 ot amcebse 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. E. histolytica ■n Fig. 89. — Entamoeba coli. A and B, Living amoebae showing changes of form and vacuolation in the endoplasm ; G, D, E, amoebse 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 ; H, an encysted amoeba containing eight nuclei ; /, a cyst from which young amoebsQ (al) are escaping; J, K, young amoebse 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 ectoplasmic layer. The life-cycles of these two species are also very different, as described by Schaudinn. ,r u- E. coli, ill the amoeboid multiplicative phase, reproduces itself by binary THE SARCODINA 225 fission of the ordinary typo, and also by a process of multiple fission is which the nucleus divides until there are eight nuclei in the body; the characteristic 8-nucloate 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 roimd 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 gelatuaous enVelope, and each of the two S3mkarya 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 amcebulae, 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 bmary 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 Dociy, ot which the greater part remains as residual protoplasm and dies off. ine mmute spores are the means of infecting a new host, as shown by ocnaudmn 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 toregomg paragraphs, first introduced clear ideas into the problem of the numan entozoic amoebae. Many of the works of subsequent investigators nave tended, however, rather to confuse and perplex the question, for various reasons. In the first place, in cultures made from human faeces, free-living fTrnn!i^''fl v''^''!-^ °^ amoebac make their appearance, which have passed tnrougn the digestive tract in an encysted condition, and emerge from their 15 Fig. 90. — Entamoeba 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 pseudopodia : n., nucleus ; h.c, blood-corpuscles. After Jiirgens. 226 THE PROTOZOA cysts in the cultures ;* such amcebse, for the most part of the limax-ty^, have been confused with the true ontozoic amoeba, and have given rise to erroneous ideas. Secondly, it is certain now that the two species of amoebte recognized by Sehaudinn does not exhaust the list of human entozoic amoebae. Thirdly, it is possible that Sehaudmn did not see the entire life-cycle of the forms studied by him, or tliat 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 amoebse occur in man. Hartmann recognizes two dysenteric amoebse, in addition to the harmless E. coli : E. histolytica, Sehaudinn, 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 amoebse descrilDed 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 amoebulse containing secondary nuclei formed from chromidia. In the propagative phase Noc's amoeba encysts and breaks up into amoebulse. Greig and Wells, in Bombay, obtained results very similar to those of Noc. In cultures 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 bmary 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 restmg 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 amoebaj remains to be worked out. From the foregoing it is clear that, with regard to the human pathogenic amoebffi, 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 life-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 restmg cysts or spores, which produce infection through being accidentally swaUowed with food or water (compare Walker, 276-5). In that case unfiltered water, uncooked vegetables * Whether this also applies to cultures made from the pus of liver-abscesses, as asserted by Whitmore (279) and Hartmann (247), may well be doubted ; it is Sot easy to understand how an encysted amoeba could be transported passively from the intestine into a Uver-abscess. THE SARCODINA 227 and herbs, or fruit that grows near the ground, are likely sources of infection by becoming contammated with the resting stage of the amcebo3 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 from the excreta of other animals also. From general considerations of parasitism m 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 Pelomyxa and Paramceba. The species of Pelomyxa (Fig. 91) are fresh- water amoebae of large size and " sapropelic " habit of fife (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 havmg 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 vacuolated, and contains a number of peculiar refringent bodies ("Glanzkorper ") of spherical form, with an envelope m which bacterial organisms {Cladothrix pelomyxce, Veley) occur constantly. The bacteria naultiply by fission in a hnear series in the form of jointed rods, which may branch ; as a rule they have five or six jomts, or less, but at least two. The refringent bodies are of albu- mmous nature (Veley). According to Gold- schmidt (57), the refrmgent bodies arise from the nuclei when they give off chromidia ; in this r^X.tl. lL^S^S^Z^Zi tZT,"*'' Y f''V« ol the nucC Fig. 91. — Pelomyxa palus- tris : a specimen in which the body is transparent owing to the absence of food-particles and foreign bodies, showing the vacuolated cytoplasm and the numerous nuclei and refringent bodies (the 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 cjrtoplasm 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. Pelomyxa 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 clu-omidia, 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, causmg the parent-individual to break up completely. Each free gamete is Heliozoon-like, vnih 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 Paramosba (Fig. 49) was founded by Schaudmn (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 /x in diameter, of rather flattened form and with lobose pseudo- podia. It contains a single nucleus, and near it a peculiar body, the " Nebenkern " of Schaudinn (see p. 95). In binary fission accompanied by It also becomes encysted and Yia, 92. — Portion of a section through the body of Pelomyxa. N., Nucleus ; r.b., refringent bodies ; b., bacteria on the refringent bodies ; 5., sand and debris in the protoplasm Gould. After this phase the amoeba multiplies by division both of nucleus and Nebenkern, , . , , ^ - • ^ process of multiple fission, which shows three stages ; in the ffoes through a process of multiple Ussion, wnicn snows tmee aui^gco , n. &st the Nebenkern multiplies by repeated division, the nucleus remammg unchanged ; in the second the nucleus divides repeatedly to form as many small nuclei as there are Nebenkerne present, and each nucleus attaches itseU Ta 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 flageUa. * The amoeba from the human intestine described by Craig * J^. J^^^f Pararnwla hominis certainly does not belong to this genus. See Dofle.n (7). pp. C02, G03. THE SARCODINA 229 The swarm-spores arc liberated from the cyst and live freely, fcedmg and multiplvin'^ by binary fission, in which the nucleus divides by mitosis and the Ncbcnkern°acts like a centrosome. After a time, however, the swarm-spores lose their flagella, and become amoebula; which develop mto the adult phase. SyjK^amy was not observed, but probably takes place between the flagellulae. Two°new parasitic species of Paramosba have been described recently by Janicki (71-5) ; see p. 95. , t 4.1, To the order Amcsbaia should be referred, probably, the parasite ot the Malpighian tubules of the rat-flea {Ceratophyllus fasciatus), described by Minchin under the name Malpighidla refringens, and the parasite of Ptychodera minuta, described by Sun under the name Protoentospora ptychoderce. The section Lobo3a Testacea or Thecamoebae contains a number of free-living forms familiar to every microscopist, such as the genera Difflugia (Fig. 16), Gentropyxis, 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 {TrichospTicerium), 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 Trichosphcerium, 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 envelope 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 Difflugia 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 Trichosphcerium 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, Gentropyxis, 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 ^ transversely (Pig. 50). In Difflugiaurceolata, Zuelzer (85) has described a process of chromidiosamv. iwo animals come together with the mouths of the shells in contact, and the entire contents of one shell flow over into the other, the empty sheU being cast ott 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 begmning of November. Prior to encystment the pscudopodia are retracted, all 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 refractile. 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 chromidial 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 occurs in this species. In Gentropyxis aculeata, 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 maximum 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 of the shell, and divides into a number of amoebulae, each containing chromidia which condense into a single nucleus. Some amcebulaj form a shell at once ; others before doing so divide into four smaller amoebulfe, and then form a shell. The larger are macrogamctes, 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 Cryptodifjlugia {" Allogromia") a remarkable type of life-cycle has been described by Prandtl (265) ; see also Doflein (7), p. 310, Pig. 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 Gryptodifflugia 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 Arcdla, Difflugia, Gentropyxis, 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 contam, the generative chromatin. The marine TrichospJmrim?i, 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 ; 23i II. FOBAMINIFERA. General Characters— Shell-Structure.— The characteristic features of this group are the possession of reticulosc pseudopodia and of a sheU 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 itseK, and then is usually either chitinous or calcareous, rarely siUceous 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 exliibits 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 w'de aperture — sometimes more than one — through which the pseudopodia are extruded, as in 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, fuie pores in addition to the principal opening. Wliether perforate or imperforate, the shell remains a single chamber in the simple forms, as in the Amoebsea 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, rem%ins 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 smgle-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 O.PIanorbulina 10 ll.Nummulires Fig. 93. — Shells of various genera of Foraminifera. In 3, 4, and 5, a shows the surface-view, and 6 a section ; 8a is a diagram of a coiled shell without supple- mental skeleton; 86, of a similar form with supplemental skeleton (,s.sk.) ; 10, of a form with overlapping whorls ; in lies half the shell is shown in hori- zontal section ; 6 is a vertical section. In all the figures a marks the aperture of the sheU ; 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 exliibit 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 ^vill be discussed further under the reproduction. It may be noted that if, in this order, a species were to forni 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 Amoebaea Reticulosa. It is very probable that many of the large marme " Proteomyxa " are allied to the true Fia. M.—Biloctdina depressa: transverse sections of (a) the megalospheric form, magnified 50 diameters, and (&) the microspheric form, magnified 90 diameters. After Schlumberger, from Lister. Toraminifera, as forms either primitively or secondarily without a test ; and Rhumbler unites the Foraminif era 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 fsecal matter in the form of masses of bromi granules, termed by Schaudinn the " stercome " (compare also Awerinzew, 281). Periodically a process of defeecation 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 dififerent forms, even in the same species, as wm 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! A Fig 95 —Pdystomdla crispa : decalcified specimens to show the structure of the two forms. A, The megalospheric type ; B, the microspheric type : b. 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 is Polystomella, which has been investigated by Lister (285) and THE SARCODINA 235 Schaudiim (131) ; their results have been confirmed in the case of PeneropUs 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 animal grows, and which also give ofE 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 amoebulse (Fig. 96). Each amoebula contains a nucleus and chromidia, and secretes a single-chambered shell, which is the initial chamber of a megalospheric individual. The amoebulee separate, and 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- bulse). The megalospheric 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 o£E 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 PeneropUs, however, the gametes have a single flageUum, and in AUogromia ovoidea the gametes are amoebulse (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 gamont, 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 amoebulaj (agametes), and fl. the flagellulce (gametes), the lite-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 ot 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.—3I. . . . ■•>>-., *n.\t , ' ~ 'T- ^ \ Fig. 96. — Stages in the reproduction of the microspheric form of Poly- stomella crispa. In a the protoi^lasm is streaming out of the shell ; in 6 and c it is becoming divided up into amcebulse ; in d the amcehulae, 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 one specimen attached to the walls of a glass vessel. ...Cr-'"""*^ ' o ■ — ^ eh M ■Oe,..*^i,";ce' Q . .0' P'' 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 sexuaUy, and is always an agaraont ; a megalospheric iorm is produced non-sexuaUy, and may be either a gamont or an agamont. Very little is known of the life-cycle of the non-marme genera, ihe only form of which the cycle is known with any approach to completeness is Chlamydophnjs stercorea, the only entozoic member of the order, which is found in the fffices of various vertebrates ; a second species, C. schaudinm, is distinguished by Schiissler (A.P.K., xxii., p. 366). The adult form has a chitinous single-chambered sheU, and its protoplasm contams a smgie nucleus and a ring of chromidia. It reproduces itself vegetatively by binary hssion, and also by multiple fission producing gametes. In the gamete-formation, 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 up into as many cells, the gametes, each of which becomes a biflagellate swarm-spore, and is set 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 germmates in the hind-gut, setting free an amcebula which forms a shell and becomes a young Chlamydophrys, 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 EuglypJia, are found in fresh water, and can 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 f^onges allied to Keratosa, such as Spongelliidce, horny sponges which load the spongin-fibres of the skeleton with foreign bodies of various kinds. Schulze established definitely their relationship to THE PROTOZOA the Rhizopoda by showing that the soft body was a plasmodium contaming numerous nuclei and chromidia, and forming a pseudopodial network, but with no cell-differentiation or tissue- lormation. 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 hyahne organic substance resembling spongin. In the interspaces between the tubes great numbers of foreign bodies (" xenophya," Haeckel) are deposited, such as sand-grams, sponoe- spicules, Radiolarian skeletons, and so forth. In one family (StannomidcB) the xenophya are held together by a system of threads, " ImeUffi," in the form of smooth, refringent filaments, approxi- mately cyhndrical, which pass from one foreign body to another, and are attached to them by trumpet-like expansions of their ends. The substance of the hnellse is doubly ref ractile. and aUied to spongin in its chemical nature. Schulze compares them to the capiUitium of the Mycetozoa (see p. 241, infra). The protoplasmic body withm 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-ref ractile granules, or " granellse," which consist chiefly of barium sulphate. Schulze terms the system of stercome-contaiiiing tubes the " stercomarium," and those that contain granellse the " granellarium." 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 whicli* 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 l.nelljB is Imown 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 " myxamceba." After a time the amoebula develops a flagellum, and becomes a flagellula or zoospore (" myxoflagellate"), which feeds and multipUes by fission. The flagellula (Fig. 98) retains its amoeboid form, and sometimes also the amoeboid method of locomotion, the flageUum appearing to act as a tactile organ. It captures bacteria and other organisms by means of its pseudopodia, nourishing itself m a holozoic, perhaps also in a saprophytic, manner. It also may become temporarily encysted. The flageUate 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 plasmodmm, the 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 amoebulse, with relatively few nuclei, It becomes a sheet or network of protoplasm, which may Fig. 97.--The hatching of a spore of Ftdigo 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 Fig. 98. — Flagellula of Stemonitis fusca, show- ing successive stages in the capture of a bacillus. In a it is captured by one of the pseudopodia at the hinder end ; in c it is enclosed in a diges- tive vacuole. Another bacillus is contained in an anterior vacuole. From Lister, magnified 800. 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 differentiated in any way. The plasmodia are often brightly coloured. From their mode of life, the plasmodia are naturally liable to desiccation, and when this occurs the plasmodium passes into the sclerotial condition, in which the proto- plasm breaks up into numerous cysts, each containing ten to twenty nuclei. When 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 protoplasmic body, and takes the form of a rounded capsule, attached to the substratum by a disc-like attachment known as the hypothallus. Between the sporangium Fig. 99. — Part of a plasmodium of Badhamia uiricularis expanded over a slide. From Lister, magnified 8 diameters. THE SAJRCODINA 241 proper and the llypothallus the body mcay be drawn out into a stalk. The first events in the reproductive process are tlie 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 completely formed the actual spore- formation begms. 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. o Jr!i! -^^'P""* given above may be taken as describing the typical series of events m the life-history, which is Hable to considerable Variations in particular 16 Fig. 100. — Badhamia ulricularis. a, Group of sporangia, magni- fied 12 ; &, a cluster of spores ; c, a single spore ; d, part of the capillitium containing lime- granules : h and d magnified 170. From Lister. Fiu. 101. — Trichia varia : part of a section through a sporan- gium after the spores arc formed ; threads of the capil- litium arc seen in longitudinal and transverse section. From Lister, magnified 650 dia- meters. 242 THE PROTOZOA types. In the subdivision termed the Sorophora or Acrasise there is no flagellula-stage m the life-history, and the amoebula) which are produced from the spores aggregate togetlier, but form only a psoudo-plasmodium, in which the constituent amoebulae remain distinct, without fusion of their protoplasmic bodies, each amcebula multiplying independently. The details of the reproductive process also vary greatly. In the division known as the Exosporose, represented by the genus Ceratiomyxa, no sporangium is formed, but the Plasmodium grows up into antler-like processes, sjwrophores, 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 earUer 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 reducmg divisions. According to Jalm'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 flageUulae and amoebulse, 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 amoebulas copulate in pairs as gametes. The zygotes thus formed are the foundation of the Plasmodia ; when one zygote meets another it fuses with it, the nuclei remaining separate, and by repeated fusions of this kmd the plasmodia are formed. When, on the other hand, a young plasmodium or a zygote meets an amosbula (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. Withm the spore the nucleus divides twice, forming four nuclei, and as soon as the spore germinates the contents divide into four amoabute, which adhere in the form of a tetrahedron. Each amosbula has eight chromosomes in its nucleus, and divides into two amoebulae, also with eight chromosomes. Each of the amoebulae develops a flagellum and swnns off Possibly in this genus the syngamy takes place between flagellulae. From the investigations of Jahn, it is clear that the swarm-spores ot Mycetozoa, like those of other Sarcodma, are the gametes ; then- nuclei have undercrone a process of reduction, and represent pronuclei, which after a certain number of divisions give rise by syngamy to synkarya, from whicli the nuclei of the vegetative phase, the plasmodium, takes origm. The Mycetozoa are classihed by Lister (297) as follows : Suborder I. : Etjplasmodida (Myxogastres, Myxomycetes sens, strict.).— Mycetozoa with a flageUula stage and a true plasmodium formed by plasto- gamic fusion of amcebulaj. This suborder comprises forms Mith the full liie- cycle described above. .... . ■ir^„„,r.w. Section 1 . ^/KZosporetB.— Spore-formation withm a sporangium. Examples . Badhamia, Fidigo {Mhalium), etc Section 2. ^/ctos^JorecE.— Spores formed on the exposed surface ot sporo phores. Example : Ceratiomyxa. THE SARCODINA 243 Suborder 11. : Sorophora (Acrasiae, Pscudoplasmodida). — With no flagellate stage in the life-liistory ; the amoebulas do not fuse completely to form a true plasmodiura ; the spores are formed in clusters (" sori "). Here belong various genera, for the most part found in dung, such as Dictyostelium and Copromi/xa. Acrasis occurs in bcer-ycast. 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 Doflem in the suborder Phytomyxinm, 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 msects have been assigned to Phytomyxinoi. The best known example of the group is the common Plasmodiophom brassicce, which attacks the roots of cabbages and other Crucifer£e, 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 flagellulse, 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 myxamoebse after loss of the flagel- lum. The youngest myxamoebae seen have two nuclei. They grow in the cell-contents with multiphcation 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 certam whether the myxamoebse can pass from one cell to another, and so spread the infection, or whether all the infected cells are derived from the multiphcation of the first cell infected. The second view, mamtained by Nawaschm, is supported by Prowazek, and also by Blomfield and Schwartz with regard to the aUied genus Sorosphcera. ' When the host-cell is exhausted, the reproductive phase begms, accordmg to Prowazek (127), by the nuclei of the plasmodium throwing out numerous chromidia, and becommg m consequence very indistinct. In Sorosphcera at this stage (Blomfield and Schwartz) the nuclei disappear altogether being entu-ely resolved mto chromidia from which secondary nuclei are formed bpore-formation, preceded by sexual processes, takes place in the manner described above (p. 149, Pig. 76). In Sorosphcera, Blomfield and Schwartz louncl that, after reconstitution of the generative nuclei, the Plasmodium divides up into unmucleate cells, each of which divides twice by karyokmesis • after these divisions the cells become arranged as a hoUow sphere, the " soro- were'obseTver ' ^ ov syngamic processes As stated above, certain parasites of insects are referred to this order Lier fnri Hp«^ ^^^/^e genera Sporomyxa, Leger (295), M2jcetosporidium, i^egev and Hesse, and Peltomyces, Leger {G.B.A.S., cxlix., p. 239) Zoomvxa ZtiewZr'' ^''^l' P"^"^^^^ «^ t^'^^^' i« also tobe rSeS ometv& drbtfnl P^'^'T " n?"'^ ^'^^^ considered trt 7bp nffln f the suggestion fSL.lf.v, of PeZtom2/ce5 are rather with the Cnidosporidia (p 409) through the genus Paramyxa recently found by him (761 ^ ^ ArcLl anSSww n- "^^"^^ ^^^i^rkable genera Chlamydomyxa, By Skcstwin t^^^^^^^^ Cienkowski, the affinities of which are still obscure underthe I T f.^^'^V''' independent order of the Sarcodina thev nn ,1 r ^^^^^""thulidea ; by Dolage and Herouard (6) and others ChfZ P ^"'^o'-^er, Filoplasmodida, of the Mycetozoa. It. ^™^f"uf f fresh-water genus occurring either free or encvsted S'to"S;VLT?ofo:^:t"" " chfomatophore: 12ZSe when ^cysted of S^nn'^^ consequently to assimilate and grow cu encystea. Un the other hand, when free it fornls a network of long, 244 THE PROTOZOA filamentous pseudopodia, by means of which it is able to digest food in the ordmary holozoic manner. The body is a plasjnodium containing, in addition to numerous nuclei, chromatophores, and peculiar "oat-shaped bodies," "spmdies," or " physodes," stated to consist of phloroglucin. The cyst- onvelopo 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 remam 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-protoplasni 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 medullary 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 stifE, 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 tlie pseudopodia are centred on it. Actinosphcerium (Fig. 3) can be derived from this type by multi- pUcation of the nucleus, originally single, until there may be some hundreds present in large specimens. The marine form Gampto- 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 Glathrulina (Fig. 19). In the interesting marine genus Wagnerella (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 stall?:, 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 Glathrulina. In Wagnerella 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. — 'Repvo&uction 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 " primary " cyst is secreted within the gelatmous " 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. Tlie zygote develops into a young ActinospJicerium with several nuclei, which emerges from the cyst and begins a vegetative life, but appears to divide frequently at the start into uninucleate, ActinophrysAike forms. In other genera, on the other hand, and especially in those of the Acanthocystis -ty^e {Acanthocystis, ClathruUna, 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 mdicate 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 rise to amcebula? (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 itseK 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 the 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 BhajMdiophrys, 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 Wagnerella-form. 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 centrosome. 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 displaced 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 nviclcus 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 biflagellate swarm- spore which is set free, while the parent-individual degenerates. The destniy 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 mto a number of daLKThter-nuclei, as in gemmation ; each daughter-nucleus grows, its karyo- some multiplies by Hssion, and it breaks up in its turn mto granddaughtcr- nuctei Continued multiplication of the nuclei in this manner proceeds until the body is filled with vesicular nuclei ; it then breaks up mto as many amcebulffi, which are sot free, leavuig a residual body with the central gram, which degenerates. FiQ. 102. — Wagnerella borealis, showing budding and fission. A, Specimen with a single bud (&) : e.g., central grain ; B, specimen with four buds (6) ; G, en- larged view of the head of a specimen containing two buds (b) in process of extrusion ; D, specimen in which the head has multiplied by fission to produce a Rhaphidiophrijs-like colony ; six individuals are seen, five of them eacli 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 aiUceous spicules, only formed during 248 THE PROTOZOA encystment. Examples: Actinophrys (Eig. 46), Actinos'phoerium (Fig. 3), Camptonema (Fig. 47), etc. Suborder II. : Chlamydopiiora.— 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 ehitinous. Examples : Acanthocystis (Figs. 18, 64, 68), Wagnerella (Figs. 48, 102), He'erophrys (Fig. 103). Suborder IV. : Desmothoraca.— Body invested by a continuous, lattice-like skeleton. Example : ClathruUna (Fig. 19). Fio. 103. — Heteroplirys fockei, Archer. c.,c., Contractile vacuoles ; s., radial ehiti- nous sj^ines surroundmg the envelope. A nucleus is present in the bodj-, but is not shown ; the bodies in the protoplasm represent zooxanthellse. 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. Nuclearia, 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 Fig. 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 Penard (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 ajnoebpid, .and a flagellum grows out; finally THE SARCODINA 249 tho animal becomes a pear-shaped flagellate swimming by means of its flagellum (Schcwiakof¥, 8G3 ; CauUery, 300). Ciliophrys thus recalls Pseudo- spom in its two phases (p. 218), and there can be little doubt that the two forms are closely allied. , -r j Dimorpha nutans, Gruber (Fig. 104), has radiating pseudopodia strengthened by axial rods, and m addition a pair of flagolla arising close together at one pole of the body. Both flagella and pseudopodia arise from a centrosome situated near tho flagellated pole ; tho 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). Those facts appear to indicate an origin for the Heliozoa from Flagellates such as those of the genus Midticilia (p. 270, Fig. 113), in which the body bears radiating flageUa planted evenly over the surface ; transformation of tho 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- FiG. 104. — Dimorpha 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 Ciliophrys and Pseudospora. Przesmycki has described a species, Endoplirys rotatorium, parasitic in Rotifers, which he considers as a connecting-link between Nuclearia and Vampyrella. The exact systematic position of such genera must be considered at present an open question. VI. Radiolabia. General Characters.— 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 250 THE PROTOZOA structure of the body, and especially the skeleton, may depart more or less widely from the radiate symmetry wliich 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 Fig. 105. — Acanthometra dastica, Haeckel. sp., Radiating spinea of the skeleton (twenty in number, but only twelve are seen in the figure) ; ps., pseudo podia ; c, calymma ; c.c, central capsule ; N., N., nuclei ; x, yellow cells ; 7ny., myo- phrisks. After Biitschli, Leuckart and Nitsche's " Zoologische 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 251 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 sole 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. — The 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 tliis 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 phceodivm, 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 fresh 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 " zooxantheUae " or " zoochloreUse," according to their colour. Absent in the Tripylaria, these yeUow cells are found, as a rule, in the calymma, but in Acantharia they occur in the intra- capsular protoplasm (Fig. 105, x). The nature of the yellow cells of Acantharia has been much disputed, 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 Sphserozoa in the first place, and then to Radiolaria generally. The difiiculty in the way of such an interpretation which arises from the co-existence, in Thalassicolla 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. — Lithocircus productus, Hertwig, showing a bilaterally symmetrical skeleton consisting of a simple siliceous ring pro- longed into spicular processes. sk., Skeleton ; c.c, central cap- sule ; pf., pore-area, surmounted by a conical structure (c), the so-called " pseudopodial cone " ; N., nucleus ; o., oil-globule. After Biitschli, Leuckart and Nitsche's " Zoologische Wand- tafeln." THE SARCODINA 253 stages of other Radiolarians have penetrated into the calymma, and live there symbioticaUy— 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 {Acanihometrida) 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 MoroS and Stiasny, the myonemes are formed in the interior of the central capsule, and are derived from nuclei. In a certam 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 radiaUy-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 Tripjdara 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 rap;d 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, Fio. 107. — Actinomma asteracanthion : semi-diagrammatic to show the mode of growth of the skeleton. S.^, S."^, S.'', 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 ; ps., pseudopodia. After Biitschli, Leuckart and Nitsche's " Zoologische Wandtafeln." B, G), 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 S ARC ODIN A 255 sinks to a considerable depth. The swarm-spores are Hberated 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, Hartmann (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 Forammifcra). 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 or monokarya, 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 more than a thousand chromosomes, is interpreted as bemg in reahty made up of as many mitotic figures as there are chromosomes, since each so-caUed " chromosome " is regarded as a single Fig. 108. — Swarm-spores of Collozoum inerme. A, Crystal-bearing swarm-spores, agametcs ; B, G, swarm-spores without crystals, gametes ; B, microspores (microgametes) ; G, macrospores (macro gametes). 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 Acanthometra pellucida a process of multiphcation is proceeding continually within the central capsule, until it is entirely filled up with cells, from which the swarm-spores arise. In this multiplication, termed by the authors " schizogony," trophic nuclei (" macro- nuclei ") and generative nuclei (" micro nuclei ") 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 FlageUata (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. : Peripylaria setj 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 cxtracajDsular region, siliceous. Legion 1 : CoUodaria. — Skeleton wantmg or simple in structure ; monozoic forms. Five families. Examples : Thalassicolla (Fig. 13), Tlmlassophysa. Legion 2: /Sp/icere^Zarm.— Skeleton complex, usually with lattice-work shell ; monozoic, generally small. Four families. 256 THE PROTOZOA nnir 3 ^ ^pWozoa se« Polycyttana.-Coloni^X forms consisting of numerous mdmduals embedded in a common jelly ; their central capsules are aistmct, but their extracapsular regions anastomose. The colonies reach a length of several centimetres. Two families. Example : CoUozoum. bUBORDER II : AcANTHAKiA.— Skeleton composed of strontium sulphate typically in the form of spicules radiating from the centre of the body within tne central capsule ; in addition lattice-work shells may be developed. Central capsule with pores evenly developed, or grouped in areas. A nuniber of families are recognized, grouped in different ways by different authorities. Example : Acanthomelra (Pig. 105). Fig. 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. StTBORDBR III. : MoNOPYLARiA SETT Nassellaria. — Central capsule monaxon in form with the pores aggregated at one pole into a pore-platc, 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. : Tbipylabia seu Phjsodaria. — 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 : Aulacanilia. 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 flageUum 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 CystoflageUata, 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.— T\\Q 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 aUied genera, are small as compared with the larger species of the Sarcodina and other classes. As a rule the FlageData are free- swimmmg 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 m this group. Li the process of multiplication by fission ot 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 largo, 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), diiierent forms being holozoic, holophytic, saprophytic, or parasitic ; and one and the same form may live in different ways during different periods of its hfe-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 of their hosts. 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 combmed in a suspicious manner with great numbers of the parasites (compare Bohne and Prowazek, Noc). It is worthy of note that in some cases an intestinal parasite may pass from the mtestme into the blood or lymph under pathological conditions of the host. This condition seems to have been noticed first by Danilewsky, 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, especiaUy in the oedemata and trans- udations, abundant flagellates of the genus Hexamitus ( =Octomitus, big. lib), of a species which in normal, healthy animals is found only m the intestme. A number of similar cases have been recorded by Plimmer (383 and i resi- dential Address to the Royal Microscopical Society, 1912), who found both Odomitus and Trichomonas in the blood of various batrachia and reptiles The conditions under which these intestinal parasites pass into the blood annears to be strictly comparable to those under which the Leydema-ioTm TSMamydopkrys paLs ii^^to the ascitic fluid (p. 237)^. Whether in sijch cases the migration of the parasite is the cause of the diseased state of the host or whether, as seems more likely, the abnormal condition of the host Tes the parasite an opportunity of spreading into fresh Pastures m t Remain for the present an open question; but, accordmg to Plimmer, the presence of intestinal flagellates in the blood-circulation is associated with dSnS and recognizable lesions of the intestiiial wall. In any case the tt that intestinSl flagellates can pass into the blood is a pomt which is probably of phylogenetic as well as of practical importance (p. rf/^). THE MASTIGOPHORA 259 Siructure. — The body -form is of three principal types : (1) Au 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), Lophomonas (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 such 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, enclosmg 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 flageUa 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, as tractella, anterior, and pulsella posterior in movement. The flagella vary in number and in arrange- ment m different species, and for the different types of the flagellar apparatus a number of technical terms are in use : monomastiqote with a single flageUum (Fig. 38) ; isomastigote, with two or four flagella of equal length (Fig. 43) ; paramastigote, with one long principal flageUum and a short accessory flageUum (Fig 15) heteromastigote, with one or more anterior flageUa directed forwards' and a " trailing flageUum " directed backwards (Figs 5 25) ■ polymastigote, with a tuft of flagella (Fig. 45) ; and holomastigoU with numerous flageUa scattered evenly over the body (Fia 113)' Of these various types of arrangement, the heteromastigote te 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 c.u- Fig \V(S.—Godonosiga botrytis. A. Young specimens attached singly to the stalk of a VorticeUa ; B, colony of six individuals on a common stalk ; G, 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 h! marginal flagellum of an undulating membrane m the other -as, for example, Trichomastix and Tnchomonas (Fig. 5), Prnii^nzpkia (Fie. 141), and Trypanoplasma (Fig. 36). Tone group of flagellates-hence known as the Choanoflagellata THE MASTIGOPHORA 261 or Craspedomonads (Fig. 110)— a peculiar structure occurs, knovm 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 Mastigammha, 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 expeUed at any point of the body-surface in primitive forms, or through the oesophagus and cytostome in those more highly organized. (b) In holophytic forms the organs of nutrition are those of the plant-cell (p. 188)— namely, chromatophores, or corpuscles contain- 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 a3sophagus. In EugUna 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 : , ^. . .i 1 There is a single nucleus with a single centriole, which fimctions at tlie 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 (6) the centriole, and the flageUum it gives off, are quite independent of the nucleus, as in Mastigella (Fig. 40). THE MASTIGOPHORA 263 2. There is a single nucleus with its centrosome, and in addition one or more blopharoplasts in relation to the flagellar apparatus. Then (a) at division the old blopharoplasts 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 Hsemo- flagellates or Bmucleata (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 be, 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 lewisi and Lophomonas hlaitarum (Janicld, 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 m the life- cycle, or under special conditions. Moreover, the longitudinal division prevalent in tliis 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 S3m.gamy has 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 Oopromonas 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 flagellum 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 ordmary free individual at once, the interlude of encystment being omitted. Pig. 111. — Life-cycle of Gopromonas subtilis. A, Ordinary adult form ; B, G, D, " vegetative " reprodii'ction by binary fission ; E — J, stages of reduction and syngamy : F, 0, H, reduction ; I, J, fusion of the two pronuclei ; the zygote (I) 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 syiigamy 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 Sarcodma 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 FlageUata. The life-cycle (Fig. 112) has been worked out in full detail in Mastigella 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 faUmg-off in the supply of nutriment leads to gamete-formation and syngamy. In the earUest stages of the sexual generation a differentiation of the individuals into macrogametocytes and microgametoeytes is to be observed, though externally they are similar to ordinary individuals and contmue their vegetative life durmg 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, wliich has its cytoplasm crammed with the small gametes, now becomes encysted. Within the cyst the gametes acquire flageUa and become motile. At this stage the original nucleus of the gametocyte breaks up and disappears rather suddenly. FmaUy the cyst-waU is ruptured and the flagellated gametes escape. The formation of the microgametes takes place in a mamier essentially similar to that already described for the macrogametes, but with a few ditterences m detail. The microgametoeytes become encysted at the very begmmng 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 u diameter, and have a flageUum 15 to 18 ^ in length ; the microgametes are 2-8 a ill diameter, and have no f^agellum. A macrogamete seeks out a microgamete and fuses with it, cytoplasm and nucleus. The zygote retains the flageUum of the macrogamete, and becomes a smaU, monad-like individual which multiphes by fission as such. After several generations the monads cease in Jv, '''' ^ grows up into an adult Mastigella. A development ^h^ nl,.«^« """"fi,-' ^"bed by Goldschmidt for Mastigina, but some of tHe phases escaped his observation. Comparing the sexual cycle of Mastigella (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 hfe-cycle of Mastigella considered as a whole, there are two forms of mdividuals, each capable of multiplying vegetatively for many 266 THE PROTOZOA generations — namely, the monad form, product of syngamy, and the adult, mastigamcsba-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, 2b, repro- duction by binaiy fission; 4—10, gamete - formation ; a (in each case) microsamete-formation, b. 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 prmcipal nucleus persists to the last: 4—6, extrusion of chromidia from the nucleus and formation of secondary nuclei ; 7, 8, formation of the gametes round the secondary nuclei ; 9, extrusion of the gametes ; IQa, the small, non-flage lated micio- gametes; 106, the larger, flageUated macrogametes ; 11, «0P'f^^t;°" °J * ° gametes 12, 12a, 12&, multiplication by bmary fission of the mon-^™ fygote ; 13, 14, growth of the monad-form, after a period of multiplication, into the adult mastigamceba-form. After Goldschmidt (41 ). cases 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 series are primitive types, such as StephanospJicera, 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 bifiagellate swarm-spores, the gametes, which are set free and copulate. The sjmgamy is perfectly isogamous. The zygote grows in size, and finally multiphes 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 bifiagellate microgametes, comparable to tlae gametes of Siephanosphcera. In the macrogonidia, on the other hand, multi- phcative 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 ot 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 califomica dis- tinct male, female, or parthenogenetic colonies occur (Chatton), as IS the case in some species of Volvox. auSr^'^'ntf^'T?^" Flagellata are classified ia different ways by different authors, and in the present state of our knowledge of the group no svstem can be regarded as m any way final. As in otherVoups of^rotozoa S 268 THE PROTOZOA are a certain number of well-defined orders and families characterized by the possession in common of certain foaturqs 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 ease of many forms, as to whether they represent truly specific adult forms, or merely developmental stages of some other species of the Flagellata or Sarcodina. PLnally 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 Flagellata, . 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 arc 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 alhed 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 kepharoplast, 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 TryjMnosomidcB, allied to the Cercomonadidce, and in the trypanoplasms, which belong to the family Bodonidce, 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 hero 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 anythmg more than a cataloguer's makeshift for disposing of a number of forms of dubious position and uncertain affinities. Order I. : Pantastomina. — Holozoic, with no definite mouth-openmg ; food-particles ingested at any point on the surface of the body. Suborder 1 : Rhizomastigina.— Body amoeboid ; food captured and mgested by means of pseudopodia. i . j.i • Several genera, only known as yet from fresh water, arc referred to this very interesting group ; such arc Mastigamoeba, F. E. Schulze, Mastigina, Frenzel (Fig. 38), and Mastigdla. Frenzel (Fig. 40), distinguished from oiie another by the nature of their amoeboid movement and the characters of their pseudopodia. In appearance the species resemble amcebss which possess a long and well- developed flageUum, or in Dimastigammba two, m Tnmastig- 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 mastigamoeba-phase ; m 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 f /P; ^f ^), ^ s ngle centriole which f imctions both as centrosome and blepharoplast ; in M^f S^^^ and Mastigamoeba the flagellum arises from the nucleus Type la) ; aella the origin of the flagellum is distmct from the nucleus (Type lb). Ihe fife cycle of Mastigdla is clescribed above (p. 265). In many J; ^'^P^^'f^lly in the formation of secondary gamete-nuclci from chromidu^ the d^^^^^ ment resembles more that of the Sarcodina than that of tlie Fl^'-gel'^'t?'' bv many authorities the affinities of the Rhizomastigma ^^e considered to be ShTS the flrst of these two classes. The ^^f^S^-'^.^^TSZ^ tliP f riip fl'iffpUatcs with the Proteomyxa and Mycetozoa ; and if the nagciium Ite lost fn't^^^^^^^ they would be classed in the Sarcodina without hesitation. Fia. 113.—A, Multtciha lacustris, after Lauterbom. /Z., Flagella, one of which IS curled up into a loop ; fs.. pseudopodium-like process ; N., one of the nuclei (the others are hidden by the ingested food-masses) ; C, ingested Ohlamydomonads ; c, chlorophyll-bodies, the remains of other Chlamvdo monads m process of digestion. B, MuUicilia palustris, after Penard, N The single central nucleus. 270 THE PROTOZOA Suborder 2: HolomastigirM. —With numerous flagella radiating from a sphorical or approximately spherical body. This suborder contains the single genus Multicilia, Cienkowski, to which several species, some fresh-water, some marme, 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, Penard (Pig. 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 of the aflBnities of this genus. Doflein (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 Multicilia, 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. Okder II. : Protomonadina. — 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 comprises 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 flageUum 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 BodonidoB {Proioa- zelcia). 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. Gercomonas, type of the family Cercomonadidce (Fig. 114), has a single flagellum ; the hmder end is frequently drawn out into a long tail-like process, and is capable of change of form. (Ecomonas {Oikomonas) differs in having the body rounded. Monas, type of the family Monadidce, has a principal flageUum and one or two accessory flagella. Cladomonas and Spongomonas (Figs. 41, 42) form arborescent colonies; the constituent monads have two flageUa of equal size, both dkected forwards. Alexeieff (327) considers that the Monadidce should be placed in the suborder Chrysomonadina (see below). Bodo (Fig. 115), type of the family Bodonidce, has two flageUa, one directed forwards, the other backwards as a trailmg flagellum; the species of this genus are free-swimming and do not form colonies ; they occur both free- livinc^ and parasitic, for the most part in the digestive tracts of various animals. Bodolacertoe, from the cloaca of Lacerta spp., has been studied by Prowazek (354) who has described a process of autogamy, but doubt has been cast upon his observations by Dobell (333). Note also the O3curro;io3 oE ^oio- iko forms in the developmsnb of GryptoMffl'^jia (p. 230, snpm). The flagellar THE MASTIGOPHORA 271 FiJ. 114. — Gercomonas crassicauda, Dujardin, showing amoeboid changes of form. After Stein. recently described by Wonyon (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 flageUum, and may remain single, but more usually form colonies often of cotisiderable extent (Pig. 110). The flagellum is used mainly for food- capture, in which the collar also pro- bably plays an important part ; but an individual may become detached from its 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 Plagellata, which are then divided as a whole into Choanoflagellata and Lissoflagellata, 'the second of these divisions being used to include aU other flagellates. Since, however. the choanoflagellates scarcely differ from ordinary monads except in the possession of the characteristic collar, a speciaHzation of the food-capturmg function related to a sedentary life, they are now generally ranked as a family of the Protomonadma, the Choanofiagdlidce. Order III. : Polymastigina.— FlageUa from three to eight in number, usually all more or less equal in size ; m other points of structure similar to the last-mentioned order. Two famihes, 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 flageUum ; in the latter case the trailing flageUum may or may not be united to the body by an undulating membrane. The species referred to this famUy are for the most part parasitic. Endoparasitic forms of common occur- rence, especially in the digestive tracts of vertebrates trp.-Hnrrflo^ n f^!^ Tnchomashx, with three anterior flageUa and a free men of the flnSr^, (Fig. 5), with the same number and arraige- Suliin. i ° i ' ''"^ S,™gthe trailing flageUum united to the bodybyfn undulating membrane. These two forms occur frequently in the same hnT and are perhaps to bo interpreted as two developmVtal ^iLes oUhe same Fig. 115.— a, Bode saltans, Ehren- bcrg. B, Bodo gracilis, Stein. After Stein. 272 THE PROTOZOA organism rather than as distinct generic types. Trichomonas hominia is cntozoic in the human intestine, T. vaginalis in the human vagina ; they appear to be harmless scavengers rather than parasites. The cncystment of Trichomonas has been the subject of some controversy. According to Alexeioff (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 (Aloxeieff, 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 ; 31. mesnili occurs in the human intestine. According to Alexeieff (324), Macrostoma is a synonjmi of Tetramitus. M ono- cercomonas, includmg a number of common intestinal parasites, has four anterior flagella of equal length, or two longer, two shorter (Alexeieff, 325). Gostia 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 ceUs torn away from the epidermis (see also Neresheimer). 2. Octomitidce.* — With six or eight flagella, arranged in pairs ; the body is bilateraUy 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 weU, and the entire nuclear structure is doubled, with right and left halves. Octomitus (synonym, Hefcamitus ; see Dobell, 236), with four pairs of flageUa (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, Fig. 117) is a common inhabitant of the human intestine. It becomes Suhe stmXrerindicated encysted, and is probably disseminated in this are paired, and the letters form. Withm the cyst it divides into two - • " - ' (Rodenwaldt). L. sanguinis, described by Gonder {A.P.K., 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 Protomonadma except in the compUcation of the flagellar apparatus, correlated probably with the entozoic habit. Hartmann and Chagas propose to merge the Polymastigma * Doflein terms this family the Pohjmastigidce, but the name is clearly in- admissible, since the genus Polymaslix belongs to the f ^ f ""1^',^"^ closely allied to Trichomonas, but has six anterior flageUa and no tiailing flagellum (compare Alexeieff, 325). Fig. 116. — Octomitus dujar- dini. U.^, Anterior blep- haroplast, from which the first and second flagella of that side of the body arise ; U.^, second blepharoplast, giving off the flagellum of the third pair ; N., left-hand nucleus ; ax., left axostyle ; bl.^, third blepharoplast, at the extremity of the axo- style, giving off one of the flagella of the fourth pair. indicate the member of each pair on the left side of ths body. After Dobell (236). THE MASTIGOPHORA 273 in the Protoinonadina, and then to divide the order into two suborders ; the fii'st, entitled the Monozoa, would include the Protoinonadina as constituted above, with the exception of the Trypanosomidce (" Binucleata "), and with the addition of tlie Tetrainitidce. The second suborder, Diplozoa, would in- clude only the Odomitidce. This arrangement certainly seems more natural than that which is usually adopted, so far as the Tetramitidoe and Octomitidce are concerned. Order IV. : Euglenoidina. — 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 compUcation of the individual is concerned, the liighest type of organization among Flagellata. The body may be metaboUo, or of definite contours, with thick cuticle. The free-living A B Fig. 117. — Lamblia inteslinalis. A, Ventral view ; B, side view. N. One of the two nuclei ; ax., axostyles ; f,.^, fl.^, f,?. flA, the four pairs of flageUa ; s., sucker- hke depressed area on the ventral surface ; x, bodies of unknown function After Wenyon (277). forms are either holozoic or saprophytic, if colourless, or holophytic if pro- vided with chromatophores, in which case they may be capable of nourishmg themselves by more than one method. The flagellum may be single or there may be a second flageUum, usually smaller than the prmcipal flagellum ^'S*^*'^^^ du-ected backwards as a trailing flageUum. The attachment ot the flagellimi is of the second type (p. 263), with blepharoplast distinct from the centrosome. Accordmg to Hartmaim and Chagas (62), in Peranema irichophorum the centrosome first divides to furnish a blepharoplast, and tue latter, havmg become completely independent of the nucleus, divides into two, a distal blepharoplast or basal granule of the flageUum, connected by a rhizoplast (centrodesmose) with the proximal blepharoplast or anchoring granule. The authors consider that this should bo regarded as a fourth typo 18 274 THE PJaOTOZOA of flagellar 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 Coj)romonas suhtilis the blepharoplast remains undivided, so that this species shows a flagellar attachment strictly of the second type. In Eagkna, according to Wager (213), the flageUum 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 Cupro- 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, Pig. 111). The order comprises three families. The first, Euglenidce, contams forms provided with chromatophores, holophytic, saprophytic, and parasitic (HasweU) in habit. Examples : Euglena (Fig. 4), Phacus (Fig. 118). The second family, Astasiidce, contams 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 subfamihes Meter onemince and Anisonemince are heteromastigote. Example : Anisonema (Fig. 25). Order V. : Chromomonadina. — Small forms, with- out oesophagus or vacuole-system, with delicate cuticle and one or two flagella ; their characteristic feature is the possession, usuaUy, 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 yeUowish-brown chroma- tophores ; body often amoeboid or metabolic ; colony- formation frequent; nutrition holozoic and holophytic. Fig. 118. — Phacus Three famihes. ^yL^.m^^les : Chrysamoeha, Chromulina, triqucter. as., CEso- Dinobryon, etc. According to Sclierffel, Chrysamosba phagus; c.v., con- is the amoeboid, non-flageUated phase of Chromulina ; tractile vacuole ; sL, compare also Lauterborn (345"5). To this suborder stigma ;iV^., nucleus, must be referred also the Coccolitlwphoridce, marine After Stein. flageUates which secrete the calcareous sheUs known as coccoliths {vide Lohmann). Suborder 2 : Gryptomonadina.—^md^W forms with one or two flagella, colour- less, or with chromatophores ranguag in colour from yeUowish-brown to olive- green or blue-green. Holophytic or saprophytic, not holozoic. Examples : Chilomonas, colomless ; Gryptanonas, some species of which are symbiotic in Sarcodina (p. 15). Doflein refers the SilicoflageUata to this order (p. 255). Order VI. : Phytomonadina seu Phytoflagellata.— Completely and exclusively holophytic, Avith cellulose envelope and without mouth-aperture. This order comprises the most plant-like flagellates, to aU intents and purposes uniceflidar algaj which retain throughout life their flageUar apparatus and their motihty. 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 ofl: from the body, and is perforated by pores tlurough which the fla^ - -hiol^ it 276 THE PROTOZOA sixteen, or thirty-two, up to many thousands. Examples are Gonium (Fig. 119), ^^tephanosphcera, Volvox, etc. In addition to the six orders of flagellates enumerated above, there remain some peculiar parasitic forms, the systematic position of which is extremely doubtful. Such arc the family Lophomonadidoe, represented by Lophomonas blattarum, a common parasite of the end-gut of the cockroach and otlier Orthoptera, and the Trichonijmphidae, including the genus Triclionympha 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 flagella arising at the anterior polo of the body from a double ring, or rather horseshoe, of blepharoplasts, situated at the edge of a fuimel- shaped or cup-like structure, the calyx, which is prolonged into an axostylo (Fig. 45). The nucleus lies within the calyx, which is surrounded iir its turn by a peculiar thickening or support, termed the " collar," consisting of free, radially-disposed rods crowded together to form an aureole-like figmre, 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 Trichonymphidce comprises a number of peculiar parasites fovmd in the digestive tract of various species of Termitidce ; such are the genera Joenia, Lophophora, Calonympha, Devescovina, etc., and finally the genus Trichonymplm, 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, Trichonymplm 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 beheved 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 aUied 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 TricJwnympha ; the recently-described genus Lopliophora (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 TrichonymphidcB have been placed with the Ciliata, while Hartmann considers that they should rank as an independent class of the Protozoa. Subclass II. : Dinoflagellata seu Peridikiales. The characteristic feature of this subclass is the possession of two flagella, which arise close together about the middle of the body. One flageUum (Fig. 120, e) runs longitudinally backwards as a trailmg flageUum ; 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, investmg the body. The two flageUa are usuaUy lodged in grooves in the cuirass, the longitudinal flageUum in a longitudinal groove or sulcus, the transverse flageUum in a circular groove, or annulus. THE MASTIGOPHORA 277 The transverse flagellum executes undulating movements whi(!ti were formerly mistaken for those of a ring of ciUa ; hence the name Cilioflagellata formerly appHed to this group. The cuirass, composed of callulose or an aUied substance, is in its typical form a perfectly rigid structure, and is often prohnged 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 afi&nities by the formation of numerous flagellated swarm-spores exhibiting the typical DinoflageUate structure. The pelagic species generally possess chroma- tophores, and frequently a red stigma, which in some geneva— Pouchetia (Fig. 31), Erythropsis~is modified into an eye-like organ. The deep-sea forms, on the other hand, are colourless. In many Dinoflagellates a peculiar system of vacuoles 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 bmary fission in the transverse plane of the body, in which each daughter-individual receives a half of the cuirass of the parent and regenerates the half that is wanting. Fission rapidly repeated may li.- 1 r. . lead to the formation of chains of indi-dduals. In f orma fon n^ ''^^' fission within the cuirass has been observed, leading to the tne sexual processes of these organisms. The Dinoflagellates are an exceedingly abundant and widespread group, Fig. 120. — Olenodi- nium cinctum, 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. Fig. m.—Ceralocorys horrida: cuirass. After Stein, from Lankester. 278 THE PROTOZOA highly differentiated as regards forms and species. The vast majority arc pelagic in habit, and constitute an important element of the plancton-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 (ProroccntraceiB). — Primitive forms in which the typical peculi- arities of Dinoflagcllate organization are not fully developed. The body-envelope consists of a bivalve shell without furrows. The two flagella emerge through an aperture between the two valves, and one fiagellum projects freely into the water, while the other twists round it at the base. Example : Prorocentrum. Order II. : Dinifera. — With the typical characters of the subclass, as described above. Families: (1) Oymnodinidce, without a well- developed cuirass — example : Gymnodinium ; the marine genus Oxyrrhis (Pig. 123) is referred to this family by Senn (358) ; it is holozoic in habit. (2) Peridinidce, Avith a weU-developed cuirass made up of definite plates — examples : Glenodiniiim (Fig. 120), Ceratium., Ceraiocorys (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) BlastodinidcB, a family created by Chatton (366, 367) for certain parasitic forms ; such are Blastodinium, an internal parasite of various copepods, and Apodinium myceioides, an ectoparasite of appendicularians (Fritillaria). 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. Fig. 122.- — Peridinium diver- gens : ventral view showing the vacuole-system. c.p., The coUccting-pusule 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 fundus (/.), from which both the trans- verse fiagellum (<.), lying in the annulus (a.), and the longitudinal fiagellum (I.), arise. After Schiitt, from Lankester. Subclass III. : Cystoflagellata seu Rhynchoflagellata . 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. 123. — Oxyrrhis marina, Duj. P., Peristome ; N., nucleus; f.v., food- vacuoles ; ex., excretory mass about to be ejected. After Blochmann, from Senn (slightly modified) ; magnification 1,000. THE MASTIGOPHORA 279 The best known form is the common Noctiluca miliaris 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. From this central mass of protoplasm strands extend in an irregular network through the whole body, which is limited by a thin pellicle. Tlio 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 " eilium " ; 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 Cmspedotella (Kofoid, 373), both remarkable for their superficial resem- blance to medusfe. 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 H^MOFLAGELLATES 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-suckmg 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 Haemoflagellates. 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 Hsemo flagellates 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 JoUos (390) as a distinct order of the Flagellata termed the Binucleata. The Hsemo 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 flageflum 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^MOFLAGELLATES 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 (Pigs. 36, 134), with two flagella arranged in a heteromastigote manner, and with the posterior trailing flagellum miited to the- body by an undulating membrane for the greater part of its length. A number of species are Icnown, 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 tract of leeches ; (2) species parasitic in the digestive tract of marine fishes ; (3) species parasitic in various invertebrates. 3. Grithidia (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 Grithidia arc 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 kmetonucleus, 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-ioim. On the other hand, as a developmental phase this form represents simply a non-flageUated, resting stage which may occur m the life-cycle of either Trypanosoma, Grithidia, 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 reaUy distinct, and it differs from Bodo only in the possession of a kineto- habitat" ^^''^^''^ '^'''''''^ described, free-living or intestinal in 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 Leptomonas, of which Leishmania may be regarded as the resting, non-flagolUed phase ; and tlie bodonid or heteromastigote type witli two flagella, seen in Trypanoplasma and Prowazelcia. 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 dehiutions most commonly accepted, but it is necessary to state that the application and significance of the names Crithidia, Leptomonas, and Herfeto- 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- domestic(B (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 of Herpetomonas as its normal adult form employ the older genus Leptomonas of Saville Kent* for forms with a single flagellum (Chatton.Roubaud, 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, G. fasciculata, from the intestine of Anopheles maculipennis, and defined as a small uniflagel- late form shaped like a grain of barley (Greek, Kpidrj). 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 SaviUe Kent, "Manual of Infusoria," vol. i., p. 243, for L. UltscMii, parasite of the nematode worm Trildbus gracilis; the genus Herpetomonas was founded on p. 245 of the same work for H. vmscoe- domesticce and H. lewisi ( = Trypanosoma lewisi). Leptomonas is therefore techni- cally the older genus. B A Fig. 124,. — H erpetomonas muscce-domesticw (Burnett). A, Motile individual with two flagella ; B, cyst : n., 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 Hsemo flagellates 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 Trypanosoma is T. rotatorium (synonyms, T. sanguinis, Undulina ranarum) of the frog [Rana esculenta). Trypanosomes are now known, however, to occur commonly as blood-parasites in all classes of vertebrates. Li 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- f^^- various reasons, it may often be extremely diflicult to decide whether a given animal is infected with trypano- somes or not ; and in recent years trypanosomes have been dis- covered m animals in which their presence was previously quite unsuspected-f or instance, in calves (Crawley, Carini, 423, Stockman • see also BuUetm of the Sleeping Sickness Bureau, No. 29, p. 320) and m sheep (Woodcock, 527, p. 713, footnote). Fig. 125. — Trypanosoma viega, from the blood of African frogs, fl.^. Marginal flagellum of the undulating membrane ; fl?, free flagellum ; m., myoneme-striations (it is doubtful whether the granular streaks or the clear interspaces correspond exactly to the actual myonemes) ; n, kinetonucleus ; N, space in which the trophonucleus lies, but, not being stained, it is not clearly defined in the preparation. After Minchin, magni- fied 2,000; compare Pigs. 11 and 12 at the same magnification. THE H^MOFLAGELLATES 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 aU cases, our laiowledge 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 " trypanosomiases." 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- biense, of sleepmg 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 shghtly different type is T. equinum, of " mal de caderas " in South America, with a very minute kinetonucleus ; but the recently- described T. Uppicum of " murrina " (Darling, 428) appears to be a typical member of the brucii-gvovc^. T. theileri, on the other hand, from cattle, is very distinct in size and appearance from the members of the &mcw'-group. Finally, T. cruzi, the cause of human trypanosomiasis in Brazil, stands apart from aU the others in pecuharities 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 Umited 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 in the hosts to which they are pathogenic. As contrasted \vith the natural, non-pathogenic forms, their most striking pecuharities are that they are not specific 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 usuaUy broader, and tapers more abruptly, or ends bluntly ; but in different forms, even of the same species, there may THE PROTOZOA be great variation, from long, slender to short, stumpy types and ated The prmcipal nucleus or trophonucleus is usually situatorl Snd TT^ 1 1°'^* ^''"^ kinetonucleus is almost fn^arlty behuad 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 arrangemen the blepharoplast is lodged within the kinetonucleus itseU, 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 usuaUy 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, m 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 HageUum 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 fui-like ridge or fold of the periplast of variable width (c/. Fig. 126). The flagellum may in some cases end with the undulating membrane at the anterior end of the body, but more usuaUy 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 smuous body, the undulating membrane, and the flageUum, are ahke 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 caimot 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 iield of the microscope in a straight line {mouvement en fleche), sometimes, on the other hand, pushing its way a lowly through the blood- corpuscles, mth the flageUum directed either forwards or backwards in movement ; and wriggling movements, when the animal writhes incessantly in serpentine contortions with httle or no displacement * The only Imown exceptions are furnished by certain forms of the recently- described T. rhodesiense {vide Stephens and Tantham), and by some of the smaU forms seen during the multiplication of T. lewisi (Fig. 127, L). It is needless to point out that the statement made above applies to the typical trypaaosomo-form as found in the vertebrate blood, and not to the developmental forms through which they pass in the invertebrate host (crithidial and other types). THE HiEMOFLAGELLATES AND ALLIED FORMS 287 from a given spot. Many trypanosomes, especially 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 Ziomann 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 Trypawplasma and Crithidia, that the extremity Fig. 126.— a, Trypanosoma tincw of the tench ; note the veiy broad undulatiae membrane in this species; B, C, T. percoe of the perch, slender and stout forms. After Minchm, 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 ff ""'n Y'^^'^l " ' " ^"'^ " posterior," and substitute for S!^. 1 •''"'^ aflageUar respectively, to denote the two poles of tlie body, iliere is as yet, however, no concrete evidence for regarding the fry^n^o'some.'""^^ morphologically posterior in any known specils of 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 Z Is^' rSnr«?l''' ^^"^ ^^'^y ^ is sometimes spoken of as aorsal, an unnecessary rehnement of terms. The free cdse of the ruembrane, with its marginal flagellum, can be shown by direct mSfurements IVhlT ^«"^^d«™bly m length that portion of the body to which itTs St! r^aS ^t^^^i^^""^, «dge is thrown into folds or pleats more o less marked. In preparations, trypanosomes are seen to lie, speaking generally 288 THE PROTOZOA n one of the throe ways ; a certain number show the body extended nearlv in a straight hnc, with the free edge of the membrane much pSed but as a rule the body is curved, and then either with one principal bend like a 0 or with several b- iko serpentme bends. In either case the lindulatii g membmne IS seen almost invariably to run on the convex side of each curve InC-vZ forms (Fig 125, A ) the membrane runs evenly along the outside of the prin^c S curve, and the myonemes parallel to it. In S-like forms (Fig. 125 ^) the membrane is often seen distinctly to be spiraUy 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 wmd. Wave-like undulations rmi 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 rmuiing in the opposite direction (Minchm and Woodcock, 42). Much confusion exists in the nomenclature of the parts of the trypanosome- body, more especiaUy with regard to the small body for which Woodcock's term " kinetonucleus " (" Geisselkern ") is here used— a confusion due to differences of cytological mterpretation. While it has never been doubted that the larger body [N.) is a true nucleus, various views have been held with regard to the smaller body (w.), 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. Laveran and Mesnil (464, 391), on the other hand, regarded n. as the " centrosome," the name by which it is generaUy known in France. Schaudmn (132) emphasized strongly its nuclear nature, and stated that n. was not a centrosome, but nevertheless used for it the term " blepharoplast," by which it is still generaUy known in Germany, although a true blepharoplast is a body of centrosomie nature. Moore and Breinl (484) reverted to the centrosomie view, and termed n. the " extranuclear centrosome," behoving that it arose by division of the intranuclear centrosome contained in the principal nucleus {N.). Hartmann and Prowazek (63), on the basis of their nuclear theory of the centrosome (see Chapter VI., p. 95), regarded n. as a body both of nuclear and centrosomie 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 of 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 in 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 trypaiiosomes — namely, the various modiflcations 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 restuig 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 bo regarded as representing THE HiEMOFLAGELLATES 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 flagoUum is lodged. In some species of the ftntcu-group, an axial filament, apparently a sup- porting structure of the nature of an axostyle, has been described (c/. Swel- lengrebel, 514). The system of fibrils, however, with which Prowazek decorates the trypanosome-body are probably artefacts (c/. Minchin, 479). Many trypanosomes contain granules in their cji^oplasm 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 eases 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 growing out at first in its sheath, from which it separates later (c/. 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 itseK 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 Cocc/ta described above (p. 106, Fig. 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 pomt, and his statements require independent confirmation before thev can be accepted unreservedly, since in objects of such minuteness, requiring delicate and elaborate technique, imagination may aU too readUy outrun perception. j <^ 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. Wlien the host is a terrestrial vertebrate the transmittmg agent is generally an insect, such as a mosquito or some bitmg 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 m all cases that have been investigated. Li 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- Verdum), and has been suspected, but not proved, to take place in ilTt Tt T' vertebrate to become infected by devouring animals containing living trypanosomes, 19 290 THE PROTOZOA whether it be the blood-suclcing 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 mth 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 trjrpanosomes 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 (c/. Kleine and Taute, 459). Brumpt (419), however, asserts that T. inopinatum is transmitted hereditarily from parent to off- spring of the leech Helohdella algira. According to Porter (554), " Griihidia " melophagia 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 Avith 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 mamtaining itself only in a particular species, or limited group of species, of vertebrate hosts, so it may be said, as a general rule, that in transmission by the cyclical method the parasites are specific in the same way to certain invertebrate hosts, in Avhich 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 abeady cited, of trypanosomes and their right hosts. Many pathogenic species of trypanosomes in Africa are transmitted by tsetse-flies— e.j/., T. gambiense and T. vivax by Glossina palpalis, T. hrucii by G. morsitans* etc. The recently- described T. cruzi of Brazil was discovered in its invertebrate host, a blood-sucking hemipterous insect, Gonorhinus megistus, before it Avas found m the blood of human beings. The trypanosomes of certam fresh-water fishes — namely, goldfish, perch, etc. — pass through their developmental cycle in the leech Hemiclepsis mar- gmata (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 Glossina palpalis (Kleine, 458 ; ICleine and Taute, 459), and stages m Its life-cycle have consequently been confused Avith those of T gamhense in the same fly. The trypanosomes of birds are prob- ably transmitted for the most part by mosquitoes, but the details of to Taute, G. morsitans can act as a true host for T. mmhiense mrl conversely, accordmg to Fischer, 0. palpalis can do the same forlr.CS ' 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 Icptoraonads have a trypaniform phase in their development (see p. 314, infra), so-called " Icpto- trypanosomes." In Drosophila conjusa, a non-biting, muscid fly, Chatton and Alilaire (compare also Chatton and ledger) found in the Malpighian tubules a trypaniform type of flagellate which they consider as a " eutrypanosome," as a species of Trypanosoma distinct from the Lepiomonas 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 Lepiomonas {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 Lepiomonas indicates that, like the " leptotrypanosomes," they are merely a phase in the cycle of the Lepiomonas. 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 durmg 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 flrst appear in the blood, their most striking pecuHarity is the extraordinary diversity in type which they exihibit. Besides " ordinary " individuals of the normal dimensions of the " adult " form, there are others smafler 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 bemg those Fig. 127.— Various forma of multiplication in Trypanosoma lewisi from the blood of the rat. A, Trypanosome of the ordinary type ; B, small form resulting from division ; G, stage in equal binary Bssion ; the nuclei have divided and two liagella arc present, but division of the body is beginning, and is indicated by a hghter streak down the middle of the body ; D, final stage of binary hssion, which IS complete except for a bridge of protoplasm, much drawn out connecting the hinder ends of the two sister-trypanosomes ; E, form with hinder end drawn out {longocaudense type), the result of binary fission as seen in the last figure ; F unequal binary fission of a large trypanosome ; Wlfvi.?,?"! rr'""'' *yP*^ ''^^^ Pf^i-ci^t and three daughter- mith .1 t\ \ ''•^ ^^"^ daughter-mdividuals, can bo distin- ffiatlb^ri nf^r'f i"r '^''^T/ ^^"'^ °^^i-'^^^d by the possession of L i. •''■^"^^ ''^'^g*'^' ^hUe the daughter-individuals, formed 8imiarro'vrbn7wrv^n° ^fs^"^ ^^^'^'^g '-^ i^-^g^^^^' ^ --^ f«™ similar to B, but with the kinetonucleus in front of the trophonucleus ■ J S^l™?-^ ?"i° "V^ '°7^" 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 arc those which have resulted from a recent act of reproduction. The multiplication of T. lewisi in the rat's blood takes various forms ,n^'r.^^\ 1^ ^ trypanosome divides by equal binary fission (C, JJ), but tins is comparatively rare. More usually the fission is markedly unequal, and of a multiple type. Small daughter-forms are split off from large parent -mdividuals, and usually many at a time ; the nucleus of the parent- lorna divides several times, and subsequently the body divides into as many portions as there are nuclei, thus producing rosette like forms (Fig. 127, F, G, H) in which the original parent can usually be distinguished by its long flagoUum from the small daughter-individuals with their flagella 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 distmguished (Fig. 127. K). A curious type of trypanosome found during the' multiplication -period of T . lewisi is a form with the posterior end prolonged to a great length, so that it abnost 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, D) ; 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 tweKth or thirteenth day the trypanosomes, now usually present in vast numbers in the blood, are of uniform size and appearance, exhibiting, apart from 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 diflferent cases, but is usually one or two months. The infect'on of the rat is sometimes spoken of as " acute " when the trypanosomes are multiplying, and as " chronic " when multiphcation has ceased, not, however, 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, sometime? it takes place with great rapidity. In either case the rat gets rid of its infection entirely sooner or later, without havuig suffered, apparently, any marked inconvenience from it,* and is then immune against a fresh infection with this species of trypanosome. * Instances are 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 HiEMOFLAGELLATES AND ALLIED EORMS 295 A typo of development in the vertebrate host contrasting in many points with that described in tlie 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 fii-st type of multiplication proceeds in the capillaries of the lung (Fig. 128, b—e). An adult trypanosome loses its flagellum, and in some cases its kinotonucleus also ; its body then becomes rounded off into an oval mass ; the trophonucleus, 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 Fig. 128. — Phases of T. (Schizotrypamim) 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 ; i, similar phase, the flagellum in process of formation. Stages b—e are found m the lung, the others in the peripheral blood. After Chagas (425). which Chagas regards as sexual ; those produced lay trypanosomes which retamed their kmetonucleus have both trophic and kinetic nuclei and a rudunent of a flagellum (male forms) ; those derived from trypanosomes which lost both flagellum and kinetonucleus have only a trophonucleus (female lorms) ; in the latter ease the single nucleus divides into two unequal parts, ot which the smaller becomes the new kinetonucleus, and a flagellum is formed subsequently. In either case the merozoites penetrate into blood- f rom even the most swarming infection with T. lewisi (for the action of the " ren- forces strams see p. 28). Those who study habitually the lethal species of trypanosomes often display a natural bias, not in tlie least justified, to assume that a similar virulence is an inseparable attribute of all other species of these parasites, it that were so, it would be necessary to consider practically every Rpecimen of pike, bream, perch, or tench, in the Norfolk Broads, for instance to be in a cliscascd condition. THE PROTOZOA corpuscles, and so pass into the general circulation Wi+i,,-., +i On account of its power of multiplication by schizogony Chaeas has marlp obscr^cdtn ^1%??^ '^f Z ty% oStl^lS oDscrveci in the lung-capillanes is not essentially diflPerent however frnm tlipt t'^^o cS.t^'^^"',' ^"^^p* ^n^^v^^^^ dirorphiTrand^r^^^^^^^ Tlfp ?nV^. i / 1 ^' u ■'''v P''"'^^^^^^ of schizogony occur in other trypanosomes The intracellular multiplication in the tissues, however, recalls strongly Siat an impKni w'"'"' f f,' 't""^- ^'^^^^otnjpanum thufform and a tis^uf ,.nr^U '7 ^ ^l^''^^ b bod-trypanosome, such as T. lewisi, trvnanolZ;?.^ ' f""'^' ^-^''^ "P""^"' ie^•s;^ma7^m, in which the free trypanosome-phase no longer exists, apparently non seltf.roni?"'' ^-^^ multiplication of ScMzotrypanum cruzi in the tissues as X^h ?n S« ]f ^ 1 "^^'^^a^e thc 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 groundslor S interpretation are, first, that in human blood the adult trypanosomes exhibit a dimorphism rare y found in guinea-pigs infected artificially, in which also schizogony in the lung is seldom observed ; secondly, that the invertebrate liost, Conorhinus, is always rendered infective if fed directly on infected human blood, but very rarely becomes infective if fed on guinea-pigs, even wiien these ainmals show an intense infection. He suggests that the greater resistance of tho human organism to the parasite stimulates the production hoSr"* trypanosome may cease to produce in a less resistant In the more familiar pathogenic species, such as T. hrucii, T. gamhicnse etc. the development m 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, oven when fatal to their host. T. hrucii, 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 tho 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 flagollum, diminish in size, and become small, rounded " latent bodies," 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 active trypanosomes are developed again. Laveran (462), however, denies that there is a non-flageUatcd 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 BLEMOFLAGELLATES 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 Macha,do 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 flageUum ; 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 smaU 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 karyosomes, followed by budding off of small nuclei from the originally single nucleus. Thus the body of the rounded-off trypanosome becomes fUled, 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 flageUum ; 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 trypanosomes, 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. granulosum of the eel (Fig. 129) ; or, finally, the trypanosomes may be practically uniform m size and structure, as in T. lewisi after the multiplication-period, T. vivax, etc. A satisfactory explanation of the polymorphism has not been found m all cases ; the various forms may be in some instances stages of growth related to multiplication, as in T. lewisi during the multipUcation- period ; in other cases the polymorphism— for example, of T. gamUense—ma,j be sexual cliflerentiation 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 nnTw f JS^' ^^'^ vertebrate host, as in T. noctuoe (Minchin different cases I^^^erent explanations must probably be sought in 2. The cycle in the invertebrate host always takes place entirely or mainly m the digestive tract, though the extent to which this region is invaded varies greatly. Li the development of T. lewisi m 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, 603). l^mally, ni the many species of pathogenic trypanosomes which are transmitted by tsetse-flies of various species, two types of develop- mental cycle can be distinguished : in the one, the parasite invades Fia. 129. — Trypanosoma granidosum 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 in 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.EMOFLAGELLATES 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 tjrpical 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 Avithin 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 Avith 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 shoAvn 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, intracellular stages : A, a trypanosome curled on itself ; B, similar form in which the body has become rounded ; C, 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-hajmatoxylin ; in such preparations the trypanosomes always appear appreciably smaller than in films stained with the Romanowsky-stain (see Minchin, 479) ; con- sequently these figures, though drawn to the same magnification as Figs. 11, 127, etc., are on a slightly smaller scale ; compare the trypanosome drami in 0 -with those in Figs. 11, ^, and 127, A. remain so for at least three months, without bemg reinfected. From the rectal stock trypaniform 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 HiEMOFLAGELLATES AND ALLIED FORMS 301 brane running the length of the body is established. The trypani- form individuals thus formed are of small size and broad, stumpy form ; they represent tlie 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 ; G, D, division of crithidial form ; E, clump of crithidial forms detached from wall of rectum, hanging together by their flagella, one of them beginning to divide ; F, G, 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. leivisi 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 Swellengrcbel 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- pmus spinulosus). Experiments have shown that this insect is also capable of transmitting the trypanosome from rat to rat, but only, to judge from the 302 THE PROTOZOA wSh f ? T ^"«tanccs, in striking contrast with the ease with rcSecf th?T''''°" is effected by the rat-flea. The rat-louse ^1^^ S exceptSr'nn'rl f ^^^oh the trypanosome establishritseH omy exceptionally, and by no means as the host to which it is best adantorl and other forms have been seen in the louse, but the in raceCa; phase has not been observed, and it is probable that ikost of tlie forms de scribed from this host are degenerating forms maintaining a feeble a™ pre carious existence under adverse conditions, and destined to die or and dis appear sooner or later. "ii>_uu__anu ais- The developmental cycle of ScUzotrypanum cruzi in the bug Conorhinus megtstus has been described by Chagas,* and is briefly as follows 13?^ Fig. 132. — Phases of ScJdzotrypanum cruzi in the bug Gonorhinus megistus. a, b, and c, Forms 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 ; i, tiypaniform type from the salivarj? 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 difl'ering in minor details ; in both cases an early multiplicative phase in the stomach is followed by a crithidial i^hase, also multiplicative and constituting the principal stock of the parasite, in the hinder part of the digestive tract ; to this succeeds a propagative trypanif orm 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, 503) 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 try^aniform 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-iovm. ; 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 agam. In the development of T. raice in the leech Pontobdella muricata (Robertson, 500, 502), the ingested trypanosomes miiltiply in the crop in a similar manner by unequal binary fission, budding off smaU 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. vittatce, from the blood of the Ceylon tortoise, Emyda vittata, m the leech GlossipJwnia sp., is of a similar type, but takes place almost entirely in the crop (Robertson, 501). The development of T. gambiense in the tsetse-fiy, Olossina 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 development'al cycle iiaving been missed by so many investigators, until it was first dis- covered by Kleme. 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 m which the development contrasts with the usual type seen in other trypano- somes Finally an invasion of the saUvary glands takes place, though in what way It IS brought about is not clear ; short, stumpy trypaniform individuals TohoZill ^{ which represent the ripe, propagative phase destined to be inoculated into the vertebrate host. These ripe forms first make their appearance, according to Kleine, in the intestine. THE PROTOZOA mnHo'^rj ^^r'^^ °^ trypanosomes transmitted by tsetse-flies, a peculiar r So^l ^^r^^r'^r '^I'-^^^y «t'^ted, iA the proboseis ^termed ?^lo? i"^' ""^f, discovered it, a culture d'attente. The rrvpanosomes rZtZo^T' RoubTnfr'^ change very rapidly into t^roZnad lff„ 1 T ' ^•^"'^^"d) forms, with the kinetonuclcus far forward ajid attach themselves by the tip of the flagellum to the wall of the prol'^c s-'tX la tins situation they multiply in the salivary fluid by binary fissioT untii great numbers arc present. In some eases this culture in the probosc s aprars to bo the sole form of developmental cycle in the fly, as for ex'mnloTn T. cazalboui (Roubaud, 506, Bouffard), T. vivax (Bruee%Ti, m ) StvS IS termed by Roubaud evolution par fixation directe. In other spedes l^-^'^^P^^on T. pecaudi) the parasite multiplies first in the digestive tract Lt^rZ/nrRn K " f^9a<\s forward into the proboscis-ewZwiton par fixation indirecte of Roubaud ; m this case, however, the possibihty does not seem to be excluded that the forms seen in the digestive tract may have belon-ed to the deve opmental cycle of a distinct trypanosome. Development of this Kind has only been observed in tsetse-flies. According to Bouffard, T. cazalboui can be transmitted mechanicaUv bv t^tomoxys, but goes through its developmental cycle only in the proboscis of Mossma palpalis ; Stomoxys may therefore cause epidemics of the disease { souma ), but endemic areas are always in regions where G. palpalis occurs. The tsetse-fly is not infective until six days after first feeding on an intected animal, and it then remains infective permanently, or at least for the greater part of its existence. Hence the proboseis- 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 Glossina palpalis. The cysts result from the encystment of a crithidial form, and are very similar to the ej'sts of Herpetomonas, described by Prowazek (Fig. 124), from the hmd-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 (c/. Cystotrypanosoma, 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 &rMcu"-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, wliich 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 Hemidepsis, or rounded Leishmania-iorms which later become crithidial, as in T. raice and T. vittaoet ; 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 Sbhaudinn (132) for T. noctuce is dealt mth in a subsequent chajDter (p. 390). THE H^MOFLAGELLATES AND ALLIED FORMS 30S parasite, keeping up the infection of the invertebrate host. In hunger-periods the flagellates may persist as simple, rounded, Leishmatiia-likQ 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, j)urely 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 fertihzation described by Schaudinn (132) in " T. noctuce " is the well-known conjugation of Halteridium, which can be observed without difiSculty ; 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 (497) for T. lewisi 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 habihty to error in the mterpretation of observations is greatly increased, first by the fact that trypanosomes divide longitudinally and often unequaUy, secondly by the phenomena of agglomeration (p. 128), which occur readily under un- lavourable conditions. Consequently the adhesion together of two trypano- somes may be due to quite other causes than sexual affinity. In some cases tbe aUeged occurrence of syngamy has been based merely on the fact that non-llageUated 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, m view of the paucity of data, that so many investigators, foUowing Schaudmn's lead, should persist in ascribing all form- aitterentiation m trypanosomes to sex, and should be miable, apparently, to conceive of any other cause of polymorphism in parasites which have to adapt tbemselves, in the course of theh 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. borne investigators have upheld the mifashionable view that the syngamv of toypanosomes occurs m the vertebrate host ; so Bradford and PhmnTer, and ?^LS.^ Ottolenghi, who has described in T. hruaii, T. eguinum, TwnT ' ^""^ T'P"''^'''^' foUowing process of sexual conjugation ?o onfSer hv f^""^"?' ''^^ "^^^ appearance attach themselves mo?e lonl? 7 ' One, regarded as the microgamete, is Td tin t^ 'T'^r^*^"'"' trophonucleus or a larger nucleus of this kind lari^and^; f ' i^^crogamete, is much mSrXr n J h?^'^ '^T T'^'"' ^^'"^ kinetonucleus and two or Su-Slv thJoo « P'*"?' ' °^ degeneration. The macrogamete also has K thf two , ^'"^ °' ^^'^g^"^^ ^'^'"^ unduladng membranes, budded off Zrf r ^'''"^'i "^''^'f ^ ^ ^'^^^ll is body of the mT rn L Prmcipal nucleus of the microgamete, passes over into the the micro.^^^^^^^ ? ^ ^^^h its principal nucleus. Subsequently ..no microgamete appears to degenerate, and the fertilized macrogamete to 20 306 THE PROTOZOA divide up into trypanosomes of the ordinary type. Tliose who consider that sjaigamy can only occur in the invertebrate host will doubtless regard the process described by Ottolcnghi as phenbmena 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. gamhiense, Moore and Broinl (484) have described a process of fusion between the kinctonucleus 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- ceptibiUty of the invertebrate host. In the case of T. lewisi, only about 20 per cent., approximately, of the fleas fed experimentally on infected rats become infective in their turn, and m 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 establishmg 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 7ioctuce 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 determming whether a fish was infected was to feed a newly-hatched Hemiclepsis 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 ex|)erimental 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 wliich, when liberated, possess a certain motihty 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 exlaibit movements due to molecular or other causes is higlily 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- cha3tes be considered as a legitimate argument in favour of any such con- clusion. , , , 1 • 1 . There remains for consideration the development which trypanosomes undergo in artificial cultures, in which they exliibit 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 critludial * It is doubtful whether the forms of which Schaudinn made this statement were really trypanosomes or spirochsetes. THE H.EMOFLAGELLATES AND ALLIED FORMS 307 or trypanomonad typo of flagellate. Until the cultural development of a trypanosomo has been compared in detail with its natural development in the invertebrate host, it is impossible to estimate precisely the bearing of tl:e cultural series of stages from the jDoint 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 Schizoirypanum cruzi the same three principal phases — namely, rounded, crithidial, and trypaniform — that occur in the natural cycle, and in the same order of sequence. At present, therefore, it would be miprofitablo 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 McNeal (489), Bouet, Franga (438, 443), Eosenbusch, 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 am"mal is infected with trypanosomes or not. Lebedew has described what he beheves 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 Fig. m.—Endotrypanum schaudinni from the blood of Choloevus didaciylus. f hr"^ °* intracorpuscular parasite; F, tiypanosome 1 500 dfamet'Ss" ^^""^ ^^^""^^ Brimont, magnified about 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 the foundation by Chagas (425) of the genus ScMzotrypanum, 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 {Chol^Bpus chdaclylus), 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 Schizoirypanum. 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 iiitra- corpuscular habitat is probably commoner in trypanosomes than has usually been supposed. It has been described quite recently by Buchanan in T. hrucii. Attempts to subdivide the genus Trypanosoma as a whole have been based on the possibihty that the trypanosome-type of structure may have had two distinct phylogenetic origins, one through Leptomonas and Grithidia 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 Hke Trypanoplasma by loss of the free anterior flageUum, in which case the flageUum of a trypanosome is to be regarded as posterior ; on the other hand, if, in a form Hke Leptomonas, the kuietonucleus and with it the origin of the flageUum, be shifted backwards to the neighbourhood of the trophonucleus, and if at the same time the flageUum runs forwards along the body connected to it by an un- dulating membrane, a Criihidia-likG form results, from which, by stiU further displacement backwards of the kinetonucleus and flageUum to near the posterior end of the body, a trypanosome- form is produced in which the single flageUum 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 flageUum morphologicaUy anterior ; and Trypanosoma, ni a restricted sense, with heteromastigote ancestry and flageUum morphologicaUy posterior. The genus Trypa^iomorpha mcluded only one species, T. noctuce of Athene noctua ; aU other species of trvpanosomes 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 • Hmmatomonas (Mitrophanow) for the trypanosomes of fresh- water fishes beheved to have a heteromastigote ancestry ; Trypano- zoon for the trypanosomes of mammals, such as T. lewisi, T. bruai, etc regarded as having a cercomonad ancestry and an anterior fl l\}nrn ■ and Trypanosoma sens, strict, for the trypanosomes of frofs and'reTt™ noctu., on the other hand, he regarded in agreement with Schaudinn (see p. 390, infra), merely as a develop- THE HiEMOFLAGELLATES AND ALLIED FORMS 309 mental stage of Hcemoproteits. Althougli, however, it is quite possible that some trypanosomes may have a heteromastigote ancestry, all the developmental facts hitherto discovered indicate a cercomonad ancestry witli 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 tlieir biflagellate, heteromastigote type of structure throughout their development in all active phases, and never pass through a trypaniform or critliidial 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 must 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 ") grqbbeni, formd in the gastro vascular system of Siphonophora (Keysselitz, 453) ; T. (" Bodo ") helicis, from the receptaciilumseminis of Helix pomatia and other snails (Friedrich) ; T. dendrocceli, from the digestive tract of Dendrocoelum lacteum (Fantham and Porter, P.Z.S., 1910, p. 670) ; T. vaginalis, from the female genital organs of leeches (Hesse, G.R.A.S., 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 aU 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 in vestigated, but in all probabihty 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) aYeSiLool '''"^ digestive tract of tsetse-fiies was in reality 310 THE PROTOZOA former host. T. hdicis, according to Friedrich, passes from one siiail to another mechanically in the spermatophores during coitus Ihe lollowmg account refers mainly to the blood-inhabiting species • The body of a trypanoplasm is relatively broader and shorter' less smuous 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, Trypanoplasma abramidis from the blood of the bream ; B and 0, T. Jceyssditzi from the blood of the tench : B, small ordinary form j G, large form. After Minchin, magnified 2,000. other solid particles. The principal structural feature is the possession of two fiagella, which arise close together at the anterior extremity from a pair of blepharoplasts or diplosome, or from a single basal granule (Martin). One flagellum i)rojects freely for- wards ; the other turns more or less abruj)tly 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. gryllotalpce the un- THE HiEMOFLAGELLATES AND ALLIED EORMS 311 dulating incmbrcane only extends along two-thirds of the length of the body, after Avhich 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 JoUos, 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 Idnetonucleus, 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, G). 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 mvariably biflagellate. Resting forms witliout a locomotor apparatus may occur. In T. helicis, Friedrich describes winter 312 THE PROTOZOA forms Avith a single nuclous, wliich is in some cases the tropho- nucleiis, m others the kinetonucleus. . ASd^rrrMaSh^^^^^^ ^r^^'^ °* --^^^ ^^^^^^^^^ conflicting. Accorcluig to Mart n, divjsion ol T. congeri is initiated by tlic division of tho oTo^IuSirSJxt^^^ flagella followed by splitti/g oieZ^Z^^ S • « 1 trophonucleus divides amitoticaUy, the karyosome becommg fii-st drawn out into a band, after which the nucleus as a whole is constricted mto two. Lastly the kinetonucleus becomes elongated and divides simply by a transverse constriction into two pieces. JoS, however following Rosenbusch's statements for trypanosomes, affirms that the dhn,Jon tLt°thoZ?/ ^"^T- °" °«ier hand Ses that the knietonuclcus of Trypanoplasma is a nucleus at all. This author also describes a series of chromatinic blocks at the base of the undulating memteane otl. tntestmahs, similar to those seen in Trichomonas (compare Fig 5) Keyssehtz (454) has described syngamy in the development of T. " borreli " m the leech Pisctcola, but the description and figures are miconvhicing, and the matter requires reinvestigation. No other investigators have fouSd 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 alreadv pointed out, the application of the name Griihidia, as the denomination of a genus is involved in considerable confusion and perplexity — partly because the distinctive morphological characters shade ofE 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 tryjianosomes 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, tmtil the contrary has been clearly established. At the same time the possibility must always be taken into accomit that a blood-sucking invertebrate may harbour flagellate parasites peculiar to itself in addition to those bl. Tia. 135. ~ Orithidia minuta, Leger, from the gut of Tahanus tergestinus. A, Or- dinary motile indi- vidual; B, O, young forms, with flagel- lum short or rudi- mentary. After Leger. THE HiEMOFLAGELLATES AND ALLIED FORMS 313 which it takes up in vertebrate blood, and tliat 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-sucldng insect has been proved satisfactorily to harbour flagellate parasites not derived from vertebrate blood. After deducting doubtful species of GritUdia, 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 0. campanulata, recorded from the digestive tract of Ghirononms plumosus (Leger, A.P.K., ii. 1903, p. 180), from that of the larva of Ptychoptera (Leger and Dnboscq) and of caddis-worms (Mackinnon, 547) ; G. gerridis, from Gerris spp. (Patton, 550 ; Porter, 555) ; and possibly others. The life-cycle of G. 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 flageUa. 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 (Hbrpetomonas). 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 iafection 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- biacese, and in this case an alternation of hosts occurs. The para- sites are taken up from the plants by bugs (Hemiptera) which suck then- juices, and by the agency of the bugs the flageUates are moculated into other plants again (Lafont ; Bouet and Roubaud, 530 ; Fran9a, 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 considerably. 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 flageUum, 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 ; Fig. 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 Avhich may Fig. 136. — Leptomonas jacidum, Leger, from the intestine of Nepa cinerea. A, B, Monad forms ; 0, division of a monad form ; D, monad form with short flageUum ; E, F, G, gregarine-like forms : E, in division, F, attached to an epithelial cell by the rudimentary flagella, which resemble the rostra of gre- garine sporozoitcs. 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 Herpetomonas should be restricted, but which on another view represents merely an early stage in binary fission, with a daughter- flageUum precociously formed ; (2) smaller flagel- lates with a single flageUum (Fig. 136, B ; Fig. 137, d), the type for which the name Leptomonas is employed by those Avho regard the true Herpetomonas as typically biflagellate, while by those who hold the contrary view the two genera are ranked as synonjnns ; (3) cri- thidialforms (Fig. 137, g) ; (4) trypaniform flagellates (Fig. 137, j, h), with the kinetonucleus at the posterior end, and the flageUum running the whole length of the body with a more or less distinct undulating membrane — the " leptotrypanosomes " of Chatton. In THE HiEMOFLAGELLATES AND ALLIED FORMS 315 addition to these four types of active flagellates, there may occur also non-flagellated individuals or with the flagellum rudimentary— namely, (5) long " gregariniform " individuals (Fig. 136, E—G; Fig. 137, q) and (6) oval or rounded ie*57imama-forms. The latter may become encysted and function as the propagative stages. If the four active forms are all distinct species, one and the same host may have intestinal flagellates belonging to four different genera ; if they are all phases in the development of one species, it becomes a subtle point for discussion which of the four forms is to be regarded as the " adult " generic type. Fig. 137.— Flagellates from the digestive tract and Malpighian tubules of Dro- sophila confusa. a, h, c, Trypanosoma drosophilce, three forms, from the Malpighian tubides; d—q, various forms of Leptomonas drosophilce from the intestine : d, e, f, leptomonad forms ; g, crithidial form ; h, i, transitional forms from the preceding to j, k, the leptotiypanosome-forms ; m, n small cnthidial (" barley-grain ") forms ; o, p. forms transitional from the preceding to q, gregarmiform mdividuals attached to the epithelium by a rudimentary flagellum, the middle one of the three in process of division. After Chatton and Leger (533). Not in every case, however, does such complexity of form occur in the same host. The development of a typical leptomonad, such as L. (H.) jaculum of Nepa cmerea, as described by Porter (556), is of a comparatively simple type, hJie that of Grithidia gerridis described above. Non- flagellated Leish manza-like mdividuals give rise to flagellates of the true leptomonad type wh>ch multiply by fission ; these in their turn pass into a non-flagellated condition 111 the hind-gut, there becoming encysted and being cast out lith the feces to infect new hosts. Prowazek (557) has described in H. musccB-dojnes. hcce an extraordinary complication of male and female types -av^ogaZ parthenogenesis of female forms, and " etheogenesis " of male formsTS of these statements can be accepted as even probable until the weighty si'per struc ure of theoretical nterpretation is supported by a more f bstantial sp" : irhis so°X'''Jtf " ""^"^-"^ ^^^^^^ ^^^^^^^^-^ by Prowazek especially his so-called etheogenesis," represent stages in the development of Plu'DunCv belonging to the group Flagellata ; compare 316 THE PROTOZOA V. The Genus LEisHMANrA. This genus was founded by Ross to include two human parasites • the so-called Leishman-Donovan bodies, cause of tlie disease known in India as "Kala-azar" ; and Wright's bodies {L. tropica) cause of boils known by various local names, but termed compre- hensively " Oriental sore." To these a third specics-namely L. infantum— has been added by Nicolle (570), causing a disease in children ni Tunis, Algeria, and Italy, and foimd also in dogs, which are regarded by Nicolle as the primary host of the parasite and the source of the infection in human beings. In all cases the type of parasite found in the vertebrate host is very uniform (Fig. 138)— small bodies, usually rounded or oval, contained within ceUs and Fia. 138. — Leishmania donovani in cells. A, A macrophage ; B and C, endothelial cells containing the parasites (p.) ; n., nucleus of the infected cell. After Christophers. multiplying by fission (Fig. 139). Each parasite possesses two distinct nuclear bodies, which the development shows to be a tropho- nucleus and a kuietonucleus respectively. The cells which harbour the parasite are mainly, if not exclusively, of two classes — namely, leucocytes and endothelial cells ; the latter become greatly hyper- trophied, forming the so-called " macrophages " (Fig. 138, A), which may contain 150 to 200 parasites at a time. L. donovani was believed originally to occur also in red blood-corpuscles, and was first described as a species of the genus Piroplasma (p. 379). The balance of evidence, however, is against their occurrence in the hsematids. If set free by the disintegration of their host-cell, they THE H^EMOFLAGELLATES AND ALLIED EORMS 317 are probably taken up by leucoeytes, and in them they may be carried into the general circulation. Although the diseases caused by these parasites are termed comprehensively " leishmanioses," they are not all of one type. L. donovani produces a systemic disease, very deadly m its effects, and the parasite is found in immense numbers in the spleen, bone- marrow, liver, etc. L. infantum is similar in its effects. L. tropica, on the other hand, produces a purely local infection, manifested in the form of one or more boils on the skin, each of which, accord- ing to Wenyon (84), represents either a single infection by the insect, as yet not known with certainty, which transmits the disease, or a secondary infection by a house-fly or by the m- dividual hunself from another boil on the skin. The infection by L. tropica has an incubation-period of about two months. The disease lasts from twelve to eighteen months, and one attack, after it is healed spontaneously, confers absolute immunity for the rest of the patient's life. Corresponding with these difierences in A B C D E Fig. 139. — Leishmania donovani. A, Three parasites in the ordinary condition, each showing a larger trophonucleus and a smaller kinetonucleus ; B, G, D, stages of binary fission ; E, multiple fission into three parts. After Chris- tophers. the effects produced by the parasites, there are also slight structural differences to be made out in them. L. donovani (Fig. 138) is very uniform in shape, being rounded or ovoid ; L. tropica (Fig. 140), on the other hand, shows more variety of form, with every transition from elongated, narrow forms with one end pointed to the typical oval body (Row, Wenyon). No other stage than that described above is known from the -human body ; but it was first discovered by Rogers (576) for L. donovani, and subsequently confirmed by other investigators for this and other species, that in artificial cultures the parasite develops into a typical leptomonad form (Fig. 140). The LeisTi- mania-ioims m the cultures grow considerably in size, and at the same time multiply by fission. The relatively large rounded forms become pear-shaped, and a flagellum is developed at the blunt end of the body ; finally the organism assumes the typical elongated form of a leptomonad, with a long flagellum arising close beside the kme- tonucleus, which is situated near the anterior end of the body. Leishman and Statham have described a further stage in the THE PROTOZOA ott tiom the Icarge Jeptomonad forms. There can be little doubt but that the cultural development observed in mrSLToortl^' -P^---^t« tl^o natural deX^J ntXdf tie parasite goes through in some invertebrate host. As regards {\Z r ril vam, arguments have been brought forward incrimi^atSfthc bef^ af ^^^^^ Fia. 140.— Leishmania tropica. A, Parasites from the sore, showing different forms ; B and G, development in cidtures : B, parasites growing and multi- plying prior to the formation of the flagellum ; 0, adult flagellated leptomonad forms, with a couple probably the result of binary fission ; D, adult leptomonad form ; E, similar form with the kinetonuclcus dividing ; F, G, stout forms, two stages of division ; note the flagella arising direct from the kinetonuclei! which are connected by a centrodesmose, indicating that the centriole is con- tained within the kinetonucleus (compare p. 87). A — G, after Row, from preparations stained by the Romanowsky method, magnified 2,000 ; D — G, after Wenyon (84), from preparations stained with iron-hajmatoxylin after wet fixation. transmitting agent, and Patten (573) has found that the parasite goes through the same stages of development in the digestive tract of the bug (Cimex rotun- datus) as in the artificial cultures ; but Donovan believes the true intermediate host in Madras to be another species of bug, Conorhinus rubrifasciatus, and Wenyon (84) considers that the development in the bug obtained by Patton is, like the development in artificial cultures, only an imitation of the develop- ment in the true host, and not a proof of transmission by the bug. Basile THE HJ5M0FLAGELLATES AND ALLIED FORMS 319 claims to liavo transmitted L. infanium by fleas. In the case of L. tropica, VVenyon points out that the sores occur almost invariably on parts of the pci-so-n not covered or protected by clothing, a fact which is strongly agamst. the infection being eflected by fleas, bugs, or ticks, and indicates that the invertebrate host is some biting fly, probably either a species of mosquito or a sand-fly {Phlebotomus}. Experimental evidence of transmission, however, is as yet lacking. . t . ■, It is clear from the development that the species of Leiskmania are non- flageUatcd phases of a true leptomonad, and it has been proposed by Rogers to abolish the genus Leishmania, and to place the parasites in question m the genus Herpetomonas. The life-cycle of a Leishmania is, however, so different from that of a typical Herpetomonas {Leptomonas), which is parasitic solely in the digestive tract of an insect, that the genus Leishmania may well be aUowed to stand. So long as the development is only known from artificial cultures, the significance of the " spirillar " forms of Leishman caimot be determined. Assuming that they are not merely degenerative forms, they may possibly represent the i^ropagative stage in which the invertebrate host inoculates the parasite back into the vertebrate ; the fact that L. donovani causes a sys- temic disease rather suggests that the initial phase in the vertebrate may be a flagellated form which is carried all over the body in the circulation, and from which the typical Leishmania-'pha,se is developed. Another possible explanation of the spiriUar forms is that they may be gametes, perhaps of male sex ; but there is no evidence in support of this interpretation either from observation or analogy, since sexual phenomena in leptomonads have not been observed. Marzinowsky claims, however, to have observed copula- tion of male and female gametes in cultures of L. tropica. Darlmg has described midcr the name Histoplasma capsulatum an organism causing a disease in human beings, and believed to be aUied to Leishmania. It is stated to develop flagellated phases in lung-smears. For Toxoplasma, referred by some to a position near Leishmania, see p. 387. " Leucocytozoon " jnroplasmoides, foimd in epizootic lymphangitis of horses in Senegal (Thiroux and Teppaz), is possibly a Leishmania, but only a single mass of chromatin appears to be present in the body, and no flagellated forms were obtained in cultures ; possibly, therefore, its proper systematic position is near Toxo- plasma. VI. The Genus Prowazekia. This genus was created by Hartmann and Chagas (62) for P. cruzi, a species discovered in a culture from human faeces on an agar-plate in Brazil. Two other species have also been described from human faeces — namely, P. wein- hergi, Mathis and Leger (Fig. 141, A and B), and P. asiatica, Whitmore. It would appear, therefore, that several species (or possibly a single species) of this genus occur in various parts of the world in human faeces. Martini considers P. cruzi to be a cause of human diarrhoea and intestinal catarrh in Chma. Nagler has described a species P. parva (Fig. 141, C), which is free-living, and Dunkerly has found a Prowazekia in the gut of the house-fly. The form which Walker has described under the name " Trypanoplasma ranee " very possibly should be referred to Prowazekia; it was obtained from cultures of tho intestinal contents of the frog. In its structure, Prowazekia resembles the genus Bodo in the heteromastigote arrangement of the flagella, and in its nuclear apparatus it resembles Trypano- plasma, with trophonucleus and kinetonucleus. It difl'ers structurally from Trypanoplasma only in the fact that the backwardly-directed flagellum is free from the body, not united to it by an undulating membrane ; it bears, in fact, the same structural relation to this genus that Trichomastix has to Trichomonas. Alexeieff (388) denies that the genus is distinct from Bodo, and considers that the name Prowazekia should be cancelled ; he identifies P. cruzi with B edax Jvlebs, and P. parva with B. saltans, Ehrenberg. * 320 THE PROTOZOA Afjimiies and Phylogeny of the HcBmo/lagellales. —Two opposite views have been held with regard to the origin of the Flagellates para- sitic m blood and their aUies : First, that they have a double origin along two distinct lines of descent, some bemg derived from hetero- mastigote, the others from cercomonad ancestors (Woodcock Doflein, Senn) ; secondly, that the HiemoflageUates are closely allied to certain forms hitherto classed as Hfemosporidia (p. 388), and form with them a homogeneous group or order of the FlageUata,' the so-called Binucleata (Hartmann). The question of the Hsemosporidia will be discussed below (p. 389) . It is sufficient here to deal with the Htemo flagellates alone. From the general survey of the " forms " or " genera " dealt with in this chapter, it is very evident that Trypanosoma, Grithidia, Leptomonas {Herpetomonas), and Leishmania, are very closely EiG. 141. — A and B, Prowazekia weinbergi, Mathis and Leger (473), magnified 2,400 ; G, Prowazekia parva, Nagler (585), magnified about 2,250. related to one another. Structurally the first three types shade off insensibly into one another, the dividing Ime between Trypano- soma and Grithidia, or between Grithidia and Leptomonas, being quite arbitrary, and far less definite in reahty than it appears when reduced to words ; while Leishmania is a resting non-flagellated phase of any of the three. Developmentally the four types, or any two of them, may occur as phases in the life-history of a single species, so that the selection of a given form as the " adult " in- dividual, by means of which the generic name is to be determmed, is also, in many cases, quite arbitrary. Phylogenetically an evolu- tionary series can be recognized begmning -with Leptomonas, and passing through Grithidia to Trypanosoma, of Avliich the central feature is the gradual development of an undulating membrane, which finally runs the whole length of a more or less sinuous and THE HzEMOFLAGELLATES AND ALLIED I^ORMS 321 serpentine body, probably as an adaptation to life and movement in a brotli-like medium, containing numerous suspended bodies, such as occurs in the gut of an insect, especially a blood-sucking insect, or in the blood-fluid of a vertebrate. LeisJimania, on the other hand, represents an offshoot from the main stem in which the resting, non-flagellated phase has become the most promuaent stage in at least one part of the hfe-cycle. On the other hand, the Hsemo flagellates of the biflagellate type, Trypanoplasma and Prowazekia, stand sharply apart from the uniflageUate genera. The orientation of the body, and of the undu- lating membrane, when present, in particular, is entu'ely difi^erent in the two types. The development in the invertebrate host of Trypano- plasma and Trypanosoma, respectively, are quite distinct in type, neither form passing through any stages which suggest the slightest afiinity with the other. The only feature common to the two types is the possession of a Idnetonucleus in addition to the principal nucleus, and it is questionable to what extent this structure can be relied upon to indicate afi&nity. The large kinetonucleus of Trypanoplasma is very different in appearance from that of the uniflagellate genera ; and, according to Alexeieff (324), it is a struc- ture of quite a different order from the cytological point of view. Finally it should be remarked that it is only in the biflagellate genera that parasitism in the gut of vertebrates is known to occur. With regard to the origin of the forms parasitic in blood, two theories have been put forward. Leger (545) and Brumpt (389) have upheld the view that they were origmally parasites of the digestive tracts of invertebrates, as many allied forms still are ; that in many cases their invertebrate hosts acquired the blood- sucking habit, whereby the intestinal flageUates became accus- tomed and adapted to life in blood ; and that, finally, forms so adapted passed from the invertebrate host into the blood of the vertebrate itself. Minchin (476), on the other hand, suggested that possibly the ancestral forms may have been parasites of the digestive tract of vertebrate hosts, and may have passed from the digestive tract into the blood, whence they were taken up by blood- suckmg invertebrates and transmitted to fresh hosts, acquiring finally the power of being parasitic upon, and establisliing themselves in, the invertebrate host. It must be admitted that all evidence which has accumulated of recent years is in favour of the view of Leger and Brumpt, so far as the uniflagellate forms are concerned. The types denoted by the generic names Leptomo7ias, Crilhidia, and Trypanosoma, form a perfect evolutionary series, beginnmg with monogenetic parasites of invertebrates and cuhninating in digenetic blood- parasites. From the same stem other forms of parasitism are seen 21 322 IBB PKOTOZOA to arise in other directions, as in the digenetic flagellate parasites of Euphorbiaceae. The biflagellate genus Trypanoplasma , on the other hand, com- prises species which, like those of Prowazekia, appear to have been primarily parasites of the vertebrate digestive tract, and which in some cases have established themselves in the blood and have acquired an alternation of hosts (they can hardly be said to have an alternation of generations), having become parasitic in an inter- mediate host, always, so far as is known, a leech, in which they pass through a simple type of development, consisting of little more than simple multiplication by fission. Their structure indicates affinities with heteromastigote types such as Bodo and Trichomonas, common intestinal parasites, rather than with miiflagellate forms. The suggestion is, therefore, that the flagellates parasitic in the blood of vertebrates have two distinct lines of ancestry : the one from heteromastigote forms such as Bodo and Trichomonas, origin- ally parasitic in the gut of the vertebrate and culminating in the genus Trypanoplasma ; the other derived from uniflagellate cer- comonad ancestors originally parasitic in the digestive tracts of invertebrates, and culminating in the genus Trypanosoma (compare also Senn, 358). It must be emphasized strongly, however, that any such conclusions are of a tentative nature, and can have no finality, but are liable to modification with every increase of know- ledge concerning these organisms. Bibliography. — ^For references see p. 488. CHAPTER XIV THE SPOROZOA : I. THE GREGARINES AND COCCIDIA Under the common denomination Sporozoa are grouped together a great number of parasitic organisms extremely varied in form, structure, habitat, and life-history, but of which the most general though not invariable characteristic is that the propagation of the parasite from one host to another is effected by means of spores, in the primary sense of the word (see p. 165, footnote) — that is to say, resistant seed-like bodies within Avhich one or more parasitic germs are protected by a firm envelope or capsule, whereby they are enabled to resist the vicissitudes of the outer world until they pass, in one way or another, into the body of a suitable host ; when this end is attained, the spore germinates— that is to say, the contained organisms are set free and a fresh infection is started. It is very obvious that propagation by means of resistant spores is a character very inadequate for diagnosing an extensive group of Protozoa. In the first place, many organisms, parasitic or free-hvmg, which are not included in the class Sporozoa, are propagated by means of resistant spores. In the second place, many forms included in the Sporozoa do not produce resistant spores^ being propagated by methods which render any such phase un- necessary. The class therefore ceases to be amenable to strict verbal definitions, and it is not surprismg that the limits assigned to it have varied at different times, and are even now debated. The class Sporozoa was origmally founded by Leuckart to comprise two closely allied orders— the Gregarines and the Coccidia. To this nucleus other groups were added, in particular the various forms termed vaguely " psorosperms "* — a word corned originaUy by Johannes MiiUer to denote the spores of the Myxosporidia, but soon extended to other parasitic organisms. Ihus 'Sporozoa" and " psorosperms " became practically synonymous terms, and the class to which these names were applied became a most heterogeneous assemblage of organisms * From the Greek xj^d^pa, mange, and o-Tr^p^a, a seed, on acponnf nf 323 324 THE PROTOZOA having nothing hi common except the parasitic habit and the adaptations arising from it, more espcciaUy the propagation by spores. The modern tendency is rather to spHt up this vast assemblage into smaller groups, and to abohsh the Sporozoa as a primary subdivision of the Protozoa. It is practically certaua, at least, that the two main subclasses into which it is always divided are per- fectly distinct in their origm. The class Sporozoa is retahied here solely m 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 wliole has been surveyed. The life-cycle of a Sporozoon may be started conveniently from the mmute 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 amoebula, a minute amoeboid organism ; or it may be of definite form, a little rod-like or sickle-shaped animalcule (" falciform body," " Sichel- keim ") which is capable of twisting or bending movements, but retains its body-form, and progresses by ghding forwards ; for this second type of sporozoite the term " gregarmula " 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 Avithin cells (cytozoic), or in tissues of the body amongst the cells (histo- zoic), or in some cavity of the body in which it either Ues free or is attached to the wall (coelozoic). Whatever their 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 i^hases 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. MultipUcation of this kmd, which is non-sexual, is known as schizogony ; the trophozoites which multiply in this manner are termed schizonts ; and the minute THE GREGARINES AND COCCIDTA 325 daughter-individuals, products of schizogony, are termed mero- zoifes, to distinguish tliom from sporozoitcs which tlioy may resemble closely. Sooner or later, however, the propagativc 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 sporozoites. 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 Aggregatci (p. 353), Wliether 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 groAV 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 (Metchnikofi) ; and the germs or sporozoites produced are gregarinulse, hence Rhabdogenise (Delage and Herouard). In the second subclass the trophic and reproductive phases usually overlap — that is to say, the stiU-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 Amoebogenise (Delage and Herouard). Of the three contrasted characters by which the two subclasses arc 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 Telosporiclia, as mentioned above, includes the three orders Gregarinoidea, Coccidiidea, and Hsemosporidia. 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 hsemoccele. The full-groAvn 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 famihes. 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 cephdlont (" 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 Rhabdogcnise, as instituted by Delage and Herouard, included the Sarcosporidia, which, however, arc almost certainly true Amcebogenife. 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 prolomerite and detito- merite (Figs. 7, 142) ; but in the curious genus Tceniocyslis (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- plasra. 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 arc 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 Y'u «r "^"^^lous-like body observed by Siedlecki in Lankesteria ascidice, and by Wenyon (84) in L. culicis, occurring at the point of contact of the two associated sporonts in the cyst, is perhaps a body of similar nature. The nucleus of CallyntroMamys phronimce is remarkable for being surrounded by a halo composed of radiating processes, each a thin tubular evagination Fig. in A, 142. — Examples of gregarines the " cephalont " condition. . Actinocephalus oligacanthus ; B, Stylorhynchus longicoUis. ep., Epimerite ; 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 smootli. C'liromidia arc stated to occur in the cytopiasm of some grcgarincs (compare Kuschakewitsch). According to Comes, they arc 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 growi,h 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 vesiculis 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). Accordmg 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 hfe-cycle. In tlie first suborder, known as the Eugregarinse, 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 Schizogregarinte, 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 Eugregarinse 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 eugregarme, 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-ceUs. 2. In the early cytozoic phase the trophozoite may be con- tauied completely withua 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 tlie cell attacked (Fig. 143, B) ; later, hoAvever, the cell atrophies, dies, and shrivels up (Fig. 143, G). {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 Oiona intestinalis. A, Young intracellular stages in the intestinal epithelium ; B, older intracellular stage ; 0, 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- nne ; n., nucleus of normal cell ; n.', nucleus of infected cell. After Siedlecld, 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 ceU {e.g.', Pyxinia), or may penetrate so far that the nuclear region of the parasite is within the ceU {e.g.,8tylor7iynchus), or, finally, may become completely nitracellular {e.g., Stenophora). Ultimately, in all cases the chief mass of the body of the gregarine projects from, or grows out of, the host-ceU mto 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 cpimerite 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 Pterocephalus. Ultimately the epimerite breaks oif, and the body of the sporont drops into the cavity of the digestive tract. In some cases (Pyxinia) 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, M^hich, 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, occui-s 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 Stylorhynchus 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, b) 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. ScheUack (630) has discussed the question in detail, and is of opinion that in septate eugregarines solitary encystment either does Fig. 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 m 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 m the right upper quadrant complete zygotes (sporoblasts) are seen • c, 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 St B° cki^^ ' ^""^ ^^^^ ™ cross-section. Modified after Calkins and not occur, or leads to nothing if it does, but that amongst the Acephalina and schizogregarmes it can take place ; a clear case has bin described by Lver m Ltthocyshs schneiden, parasite of EcUnocardium ; and m Monocvftis Srifd: MaTlL^r*^^ '^^''^^ sporo-form^tion is deSerby Lognetti de Martns. In some species cysts containing three sporonts have nuclei With regard to the dilfcrences in the sizo of the spores the noT bihty has to be taken into account that in some eases thoy may be deveCd parthenogenetically-that is to say, the gametids may each bLomrrsporo blast directly, without copulation wth another. ut^comc a sporo- 332 THE PROTOZOA Tho first division of the nucleus of the sporont has given rise to considerable pSZh"' '''' ""'f:"' f mu'ch. study, 'in the r^^^Z^ spoiont-nuclous ,s a body of relatively huge size, but the first spindle formed m tlie sporont is, like all tho subsequent mitoses, a minute structure Some authors have believed that the sporont contains two nuclei, comparable to tnoso ot Intusoria— namely, a very largo macronucleus of purely vegetative nature, which takes no part in the subsequent development ; and a minute micronucleus of generative nature, from which tho first and subsequent I: D Fig. 145. — Stages in the formation of a generative nucleus (" micronucleus ") from the primary nucleus of Pterocephalus (Nina) gracilis. A, Primary nucleus showing the first appearance of the micronucleus in a clear space ; J3, 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 ; G, further stage in the formation of the micronucleus ; D, micronucleus com- plete with the first centrosome ; the remainder of the prima ry nucleus in j)roeess of absorption. After Leger and Duboscq (621) ; A magnified 800, B, 0, D, 1,000, diameters. mitoses arise. Recent researches, however — more especially those of Sehnitzler on Oregarina ovata, Schellack (629) on Echinomera hispida, Leger and Duboscq (621) on Pterocephalus, Robinson on Kalpidorhynchus, Duke on Metamera, and especially Mulsow (123) on Monocystis rostrata — leave no doubt but that tho 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 tho nucleus. THE GREGARINES AND COCCIDIA 333 The generative chromatin may organize itself into a definite secondary nucleus (" micronuclous ") during the break-up of the sporont-nucleus, as in Ptero- cepJuilus (Fig. 145) ; or the fu-st spmdle arises withm the sporont-nucleus before it breaks up, as in 0. ovala (Fig. 146) ; or a number of distinct chromo- somes are formed in the sporont-nucleus durmg the process of its disintegration, which pass to the exterior of the nucleus and form the equatorial pla,te of a spmdle of which the achromatiuic elements appear to arise chiefly outside the nucleus, as in Monocijstis rostrata. In either case the first spindle consists only of the generative chi'omatm ; 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 clu:omidia, 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 B Fig. 146.-TWO stages in the formation of the first division-spindle of Greqarina ovata. showing its ongm irom a very small part of the primary nucleuf 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 primai-v nudeu! degenerates. After Schnitzler ; magnification 850 diameters ^ d™n begins as a gram or a pair of grains placed at the apex of a "cone ?bw t"! ' '"l^rr^^' ^ntrosomes appear to be hnf M ^ "'T^^'' chromosomes in the equatorial plate is usually four but m Monocyshs rostrata the number appears to be eight, and in pTSahi's and the aUxcd genus Mchinomera there ire five chromosomes fouVS oXarv rMeTazortfer^"'' chromosome. Unlike the unpaired elmoS ot Metazoa that of the gregarmes is present in both sexes : it gives rise during the reconstitution of the daughter nucleus, to the karyosome and the kTvvo ThTsiLt^:of^^^ the-imdear sphxdle at tL s'L'equi^'ni^Ss: furtheTcS^^^^^^^ ""^'""^ Chromosome is far from clear; and requires .u^.Z^l^.l^ the nuclei of the preceding stage grows out from the ant h ' -grounded by a small quantity of protoplasm, and thus a great number of small cells are budded off over the 334 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 residua] protoplasm, which may contain nuclei, but which takes no further direct share in the development. The cells that are produced are known as the " prunary EiG. 147. — Stages of nuclear division in the cyst of Pterocephalus [Nina] gracilis. A, Resting nucleus with a centrosome at one pole ; B, division of the centro- some ; 0, 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 ; G, 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. sporo blasts," 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 midergoes 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 maJe and female remain in the undifferentiated condition of the gametid. For an account of the gametes of gregarines, see above (Fig. 79, p. 174). Keduction has been described in several cases in the formation of the gametids. In the genus Gregarina 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 iii the gametids of Gre- garina ovata. In Monocystis rostrata, 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 oS of the gametids. In this case the ordinary number of clu-omosomes is eight, as seen in aU 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 foiir. 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 dividmg 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 sporo- 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 schizogregarmes. The spores of gregarines differ enoi-mously in different species in form and appearance, and often have the sporocyst prolonged into tails, spines, or processes of various kmds. Various mechanisms may be developed for liberating the spores from the cyst; for instance, in the genus Gregarina [Glepsydrina) 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 Mo?iocysiis 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 feeces. In all cases, so far as is known, the new host is infected by the casual or contammative method, and in its digestive tract the spores germinate and liberate the sporozoites. In the case of CystoUa mincUnii, 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 camiot be described in general terms ; a few examples must suffice. 1. Selenidium cauUeryi (Fig. 148) : Tlie sporozoite penetrates into a cell of the intestinal epithelium, and grows to a large size, remaining luiinucleate. When fuU -grown, the intracellular parasite gives rise by a process of multiple fission to a great number of motile merozoites which penetrate into epithehal ceUs, 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 coll, 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-hke organ to the epithelium, or massive m form, and quite free. When fuU-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, mto 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 limder end of the body, apparently from chromidia given off from the principal nucleus, which loses its clii'omatin. Round the secondary nuclei protoplasm aggregates, and fuiaUy 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 Porospom giganiea of the lobster, the largest gregarine known, the fuU-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 sporogonj- 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 Eripliia 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 jjrocess of non sexual multiijlication. Tlio 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 Cephaloidophora the sporogony, of the same cycle. 4. In the peculiar genus Ophryocystis (Fig. 151), parasitic in the Malpighian ^'^I'-^^^'l^i^m ^<^avlleryi. A, Full-grown intraceUular schizont x 850 • B. stage m the multiplication of the nuclei of the schizont v 1 2nn • n I t' ^iltZf.Tl^'v^^^f^^i^''''^^^^^^ Gurculionidce, etc.), and formerly 22 338 THE PROTOZOA reproduce their like for many generations, but which finally produce mcro- zoites which grow up into paucinucleate sghizonts, 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 small cell is a gamete, which is Fig. 149. — General diagram of the life-cycle of Schizocystis gregarionides, after Leger (617, ii.). A, Sporozoite escaping from the spore ; B, C, D, E, growth of the sporozoite into the multinucleate schizont, 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, which may either grow into schizonts again {0^, 0^), or may grow into sporonts (G^) ; 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 sporczoites; the two binucleate envelope-ceUs form a protective envelope to the spore dunng its development, and die off when it is ripe (Leger, 617, i.). (For Scliandinnella see p. 355.) THE GREGARINES AND COCCIDIA 339 The Gregarinoidea are classified as follows : Stihorder I. — Eugregarince {wifJtout Schizogony). Tribe 1 : AcepJialina. — Witliout an epimerite and non-septate ; typically, tliough by no means invariably, " coelomic " parasites. Example : Monocystis, with several species parasitic in the vesiculse 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 : CepMUna. — With an epimerite in the early stages, at least, of the trophic phase ; m 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 ^ famihes, genera, and species ; see Minchin f^q. 150. _ " Gymno- (589). The type-genus Gregarina {Clepsy- spore " of Porospora drina) comprises many common species, such Tn^XrTf '*sp?r? as G. ovata of the earwig, G. blattarum of zoites arranged radi- the cockroach, G. polymorpha of the meal- ^^^^ * central -r,r^„w, /■XT'- n n.\ 1 ■■ residual mass which worm (J^ig. 7, p. 9), etc. Other well-known contains a chromatinic species are — Pterocephalus [Nina] nobilis, from -^^^^ Leger and the centipede {Scolopendra spp.) ; Stylo- rhyncJmslongicollis (Fig. 142), from the cellar-beetle, Blaps mortisaga, and many others. The family Doliocystidce contains species parasitic in marme AnneHds. Suborder II .—Schizogregarince (with Schizogony). Various methods of classifymg the Schizogregarines have been proposed. Leger 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, m which the schizont is a free trophozoite, as in Ophryo- cyslts and Schizocystis ; the aberrant genus Siedleckia 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 • nZ'l the families of the Schizogregarin^ appear to be more closely aUied to particular famihes of Eugregarina. than to one another Leger (617, ii.) points out that the family Schizocystidce shows close affinities with the eugregarine family ActinoceplZ^ 340 THE PROTOZOA Pfeflfer asserts that the young intracellular stages of the mealworm- gregarine multiply by fission. Porospora, with its remarkable schizogony, is apparently a septate cephaline gregarine of tlie Fig. 151. — Diagram of the life-cycle of Ophryocystis, after Leger (617, i.). A, The spore setting free sporozoites ; B, the sporozoite attached by its rostrum to the epithelium of the Malpighian tubule ; G, 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 {0, 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 sporoblast ; P. the sporoblast has as- sumed the form of the spore, and its nuclei have divided into four; lUtimately eight nuclei and as many sporozoites are formed. ordinary type. A character such as the possession of the power of multiplication 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 schizogregarines amongst these two divisions. At present the following families of schizogregarines are recog- nized : Ophryocystidce, Scldzocystidce, Selenidiidce, Merogregarinidce, and Porosporidce. For the family Aggregatidce see p. 353. Oeder II. — COCOIDIA. 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 kmgdom. 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 (TurbeUaria) and Nemertuies. A parasite of the gregarine Gystohia 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 famihar parasite of the rabbit now generally known as C. cuniculi (or G. 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 mfection 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 mto cells of the intestinal epithehum, but they may under- 342 THE PROTOZOA Fig. 152. — Life-cycle of Ooccidium scJmhergi. A — E, Schizogony ; F — I, gamctog- ony ; K, L, syngamy ; L — 0, sporogony. A, Sporozoitc 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 pone- [Conlinued at fool 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, Iddneys, 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 ultunately 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 itseK, and the host recuperates its health, but without acquirhig immunity agauast remfection. As a typical coccidian life-cycle may be taken that of Goccidium schubergi (Eig. 152), from the common centipede, Lithdbius forficaius, described by Schaudinn (99) in a classical memoir. The complete life-history may be divided into eight phases, which are described Fig. 52 continued : trating the cell ; the other two cells contain parasites (p. ) in different stages of growth (schizonts) : n., nucleus of the host-cell ; G, 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 yo\mg sporonts, the one male ( S ), with fine granules, the other female ( ? ), with coarse plastinoid granules in its cytoplasm : GS , full-grown male sporont ; G ? , full-grown female sporont : h, its karyosome : H S , male sporont with nuclei divided up ; the remains of the karyosome are seen at the centre of the body ; H ? , female sporont which has expelled the karyosome : h?, fragments of the karyosome in the host-cell ; I S , ripe male gametes round the residual mass of the body of the sporont ; J ? , female gamete ripe for fertilization, throwing out on one side a cone of reception towards the male gametes (S ) swarming round it; J, fertilized zygote which has surrounded itself by an oocyst (ooc.) • msido the body the female pronucleus (?) has taken the form of a spindle, at one pole of which is seen the chromatin of the male pronucleus {S^); outside the oocyst is seen a clump of degenerating male gametes {S^);K the fertihzation-spindle complete, with male and female chromatin spread over it ; synkaryon dividing ; M, the synkaiyon has divided into four ; N four sporoblasts arc formed, each of which has surrounded itself with a sporocyst lying in a mass of residual protoplasm (cystal residuum) ; 0, ripe oocyst containing four spores, each enclosing two sporozoites and a small quantity ot residual protoplasm (sporal residuum). After Schaudinn (99). 3i4 THE PROTOZOA in the sequel, together with a brief summary of the chief variations which each phase may exhibit in other coccidia. 1. The sporozoites, liberated in the digestive tract, are small gregarmii^ which move by gliding movements, and penetrate into epithehal cells by means of their pointed anterior end (Fig. 152, A, B). 2. In the cytoplasm of the cell they grow into the large rounded schizonts, distinguished by the absence of reserve food-materials in their cytoplasm, and by the large vesicular nucleus with a karyosome (Fig. 152, B). In a few rare instances— namely, Coccidium mitrarium, Lav. et Mcsn Cryptosporidium muris Tyzzer, and the recently-described Selenococcidium intermedium (see p. 351, mfra), the trophozoite is free as in gregarines. In Barroussia spiralis, from Cerebratulus sp., the schizont during its early phases of growth IS vermiform and spiraUy twisted, but becomes rounded o/ when iuU grown Awerinzew, 47). In the intranuclear parasite of the mole, Cydo- spora caryolytica, the schizonts are stated by Schaudinn (147) to be sexuaUy differentiated, as also the merozoites to which they give rise. In the case oiAdelea omto however, a sexual differentiation of the schizonts aUeged by Siedlecki (i^opI"°, P^f se and in its schizogony ; the trophozoites Jo PenetrTJinln ^^^^''^^^"Itiply J^sb as in Schizocystis. The parasite only hZd tt '""^^ '^^^'^ '^"^.'^'f "P°^ reproductive phases. On the other nana, tJio sporogony, so far as it is known, and especially the sexual processes rceidia'n^ ^^^^^^occidiul link^ the gregLrCcs Tnd below. convmcmg manner, as will be discussed further CTa55i^m authors confirms in any way the peculiar account ot the life-hi3tory of //. stepanowi given by Hahn, whose work is criticized by xteichonow. 374 THE PROTOZOA I Fig. 159. — Life-cycle of TIcBmogrcffarina stepanowi. The figures to the right of tlie dotted line represent the phases in the blood of the tortoise ; those to the left, the phases in the leech. [Continued at foot of y 375. THE H^MOSPORIDIA 375 destined to grow into gametocytes (Fig. 159, J—L). In H. stepanowi the microscliizont sporulates in the bone-marrow or in the circu- lating blood, and produces six micromerozoites. In H. nicorim 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 (&) develop mto sporonts (gametocytes). (5) The sporonts (Fig. 159, ilfS are sexually differentiated. They represent the end of the development in the tortoise, and can only develop further in a leech. H. stepanoivi develops in Placob- della catenigera {=^Hcementena costata), H. nicorim in Ozohranchus sJiipleyi. (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—E). (7) The zygote forms an oocyst with a thm 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. Hovr 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 hsemogregarine, 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, 0, early schizogony, in which a large number of mero- zoites are produced ; D, merozoite penetrating a blood-corpusole ; E — H, later schizogony, in which few merozoites are produced ; in F the recurved vennicule 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 scliizogonous generation which produces the gametocytes ; L^, L^, sexually-differentiated merozoites of the final generation, which grow up into male (M^) or female {M^) gametocytes respectively ; these arc the stages which develop in the leech when taken up by it. N, Association of male and female gametocytes in the gut of the leech ; 0, fonnation of four male gametes by the male gametocyto ; P, one of the male gametes has penetrated into the body of tlie 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- cyto attached to it, which is seen also in the ney 'c four stages ; R, 8, T, succes- sive divisions of the synkaryon ; U, ripe cyit containing eight sporozoites, residual protoplasm, and the remains of the male gametes. After Ecichcnow (78), modified in arrangement. 376 THE PROTOZOA ?n S ^P^- I",<^heso cases the invertebrate host appears to be always arthropod. The only life-cycle of such forms which has S described completely is that of the parasite of the leucocytes of rats, which has been described by Miller under the name Hepatozoon perniciosum This parasite appears to be identical with that named by Balfour (694) Leucocylo- zoon muris and by Adie L. ratti; its correct name, therefore, is HceLgregarina (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 Puniaub ( Adie). liliartouin (Balfour), North America (Miller), Brazil (Carini), and various other parts of the world (see Franca and Pinto, A.I.B.G.P., iii., p. 207) The hfe-eycle of H. muris, according to Miller, is in the main as follows- Ihe 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-lour 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 hver ; m 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 hsemogregarine which, strictly speaking, is a para- site of the rat's liver, and not of the blood at aU ; 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 stomach. In the stomach of the mite the hsemogregarines 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 mierogamete. 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 surroimding 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 propagative phase, and in order that 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 hsemogregarines, 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. gerbilli of Gerbillus indicus (Christophers, 699) ; H. bcdfouri (jaculi) of the jerboa (Balfom-, 693) ; and the three species recently described by Welsh and others (Journ. Path. JBact., xiv.) from marsupials, one of which (H. peramelis) is remarkab e for having been found only in the free, extra- corpuscular condition. The rchizogony 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 mcrozoites (compare //. canis, below). In both H gerbilh and // jactdi free vermicules occur, and are set free readily m vitro / those ot H qerhilli are recurved when contained in the blood-corpuscle, btagcs of the development of H. gerhilli were found in a louse, H(smaiopinus stephensi; first free vermicules in the stomach and intestine, later large cysts m the body-cavity containing a great number of spores, each of which encloses six to eight sporozoites. It seems impossible that the parasites encysted m the body-cavity of the louse should get back into the gerbille m any other way than that of being eaten by the gerbiUe. Christophers found that, though the sporozoites were liberated in the intestinal juice of the gerbiUe, they soon died in it, but that in the blood- plasma of the gerbiUe they became extremely active ; this observation may perhaps be interpreted as indicating that the spores germinate in the intestme, and the sporozoites, when liberated, pass at once through the wall of the mtestine into the blood- ckculation. The crithidial forms seen by Balfour in Pulex deopatrce can have no connec- tion whatever with the hsemogregarine 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, 7O0), 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 prodTXction of 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, Bhipicephalus 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 apphes to the male sex alone. 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 epithehal cells of the gut or in the cells of the liver, lung, or bone-marrow ; 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 hamogregarines 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 haemogrcgarines of terrestrial reptiles is practically ^"^^ THE PROTOZOA Z^kZ^,''' '^^.^"^f^' the transmission appears to be effected by ticks • ioofnn if^T ^^'''^"'''"^ Ixodes ricinus (Schaudinn, ii.,V 339' 718) andVf;,r"'^ by //^/aZomma c.gy,nium (Laveran 'and ^Pe'tit; t*^® liicmogregarmes of snakes (Flu, 707). frnnrf^?."?*" "^^^^P^^idi^- '.' of f^ogs and newts appear to stand rather apart from the true ha^mogregarines ; beyond the fact that they multiply bv Sr^Toorr " red blood-eorpuscles, but little is knowia of^S Zelop"^ ment. Accordmg to Hmtze, Lankesterella ranarum has no invertebrate host s'lreTstsX'^ "'^^ whIS H ffrms t: S the frT coccidian parasite. The cysts were believed to pass out method^Tr?= 1 and infect other frogs by the direct contaiinative be W to fiV ' ^^Tlfx: described by Hintze really thIfTj ,1 ^'i' i'f ^e^^ereZZa ; from other observations it is possible f ^^"^ h^mogregarines at aU, but stages in the life-cycle of a trypanosome (compare BiUet, 696). According to Franca (709). " Dacty- losorna ' splendens of the frog produces iei.W^a-like LrozoUes, S clistmct kinetonuclei (compare also Seitz). Until further researches have been undertaken, the position of the drepanidia must remain uncertain. ^eresheimer (720) has described the penetration of the red blood- corpuscles by Lankesterella sp., a process in which remarkable phenomena are extnbited. When a Lankesterella, in approaching a blood-corpuscle, is within a distance from the corpuscle about equal to the length of the parasite, the eage 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 hajmogregarines, 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. canis, H. muris, and H. gerhilli, 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 " Hcemogregarinidce " 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 strikmg symptoms being an enormous destruction of blood-corpuscles and a red coloration of the urine by hsemoglobin (haemoglobinuria). From this peculiarity are derived popular names, such as " redwater," etc., applied to diseases caused by piroplasms. THE H.MOSPORIDIA 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, Piroplasrna), but of which the correct name is probably Babesia hovis (or higemina). The typical form of this parasite is a pear-shaped body withm the blood-corpuscle. It multiphes by binary fission, and is often double in consequence — whence the specific name higemina. Many other species are now known, parasites of domestic animals in various parts of the world, and of recentyears a number of species have been made known from wild animals, but our knowledge of piro plasms in a natural state is not very extensive. No species is Imown with certamty to be parasitic upon human beings, but a disease known as " spotted fever of the Rocky Mountains " has been stated to be caused by Piroplasrna hominis, and it is possible that the organisms Fig. 160. — Piroplasrna higeminum {Babesia hovis) in the blood-corpuscles of the ox. a, b, Youngest forms; c — /, binary fission; g — j, 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 flavigenum, 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 " Piroplasmidoe " may serve at the same time to indicate the structural varieties exhibited by these parasites (compare Tranga, 736). (1) Piroplasrna, Patton {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. cahalli of " bihary fever "), dogs, monkeys, rats, and various wild animals. (2) Theileria, Bettencourt, Fran§a and Borges. — Bacilliform or rod-shaped parasites arranged in a characteristic figure of a THE PROTOZOA cross.* T parva is the parasite of " East Coast fever " of cattle in Ainca^ Other species have been described from the fallow-deer and irom 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. Une species, N. quadrigemina, from the gondi, Otenodactylus (JSlicolle, 746). (4) NuttaUia, Franga.— Parasites oval or pear-shaped (not rod- shaped) ; multiplication-forms Hke a cross. N. equi, of equine piroplasmosis ;N. herpestidis, of a mongoose {Herpestes icJmeumon). (5) Smiiliia, Fran§a.— Pear-shaped forms, occupyhig the whole diameter of the corpuscle, not in pairs ; quadruple multiplication m the figure of a cross. ;S'. 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 foregouig classification, the form of the para- site varies considerably ui 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 rmg-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 (schieonts) 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 evei-y other respect — namely, Theileria parva, the true parasite of " East Coast fever " of cattle, and Babesia {Piroplasma) nmtans, 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 miitans the cross-forms are produced by quadruj)le 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 inocidation of blood from an infected to a healthy ox docs 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. Sec especially Gonder (739). THE H^MOSPORIDIA 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 Breinl 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 Eomanowsky 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 baciUary 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 kuietonucleus ; 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 flageUa ; but the published figures of these structures do not in the least favour any such interpretation. Eanoshita suggests that the " flageUa " figured by him may represent formation of microgam'etes. 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 flageUum becomes retracted when preserved ; if so, it is a structure of a very different kind to a true flageUum, such as that of a trypanosome, and its relations to the progression of the parasite also differ. Breinl and Hindle (730) have figured bifiagellate organisms from the blood of dogs dying from piroplasmosis. The flagellates in question were of transi- tory appearance, and were onlyfomid 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 mtestmal 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 THE PROTOZOA individuals, would seem to indicate the existence of some form of fZZ^J'm ""^f^^' - tissues or inte^alX ot the body. When the parasite or parasites have destroved thp corpuscle an which they are lodged, they are set free t the bt^d plasma and penetrate other red corpuscles. originally single nucleus diviSepeatedlv aT t r^o^ ^^''^'y- dial masses arc formed which finaS v H .^"^ multmucleate plasmo- zoites, " agamonts," as I ere a?e nuclei ^^^^ ""'^"^^ ^'^'o- up of the lymphocyte. The first schifn<.nnA,;^ „ I- *° ^'"''^k- several times, but at last a generation of "1 S^^^f may be repeated are distinguished from the £^1^0^. ^! ' is produced, which nuclear structure The SmontT^ni.- V ^'^'^^^^t^ristic differences in the The forms found m the red corpuscles in the perinheral Wnnrl nr. -.i. gamonts or gametoeytes, incapable of developin^. Se ' except in the tick ; this explains a peculiarity of this parasitfna^^^^^^^^ Xtn"" '^"^^ ^ ^^^1*^^ animal 'doeT^n'ot";"odtet' . J-?'' P°^^*?°" ^c/iromafocws, founded by Dionisi for A vesveru ginis, parasitic in the blood of bats of the genus Vesverunn L Ju^Z.7!f ^' It occurs under a number of diiicrent torn^lTollT^Tt^^^^^ others, more common, within the corpuscles. The free forms are rounded o^ 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 bv a nmpp«« olv,- Aeeording to Gonder (737), the parasite har/do^ublTuruslntu^^^^^^^^ but this IS not confirmed by Yakimoff and Co. (753), who regard the paraSe as a true Piroplasma. Neumann (745) states that in the balmite (Pf^^ vesverhUonis) the parasites midergo a transformation into flageUated organisms and considers Achromahcus alhed to trypanosomes. It is not improbabS that stages ot AchrormiUcus, both in the vertebrate and invertebrate hosts! have been confused with stages of the trypanosome found in the blood of the same vertebrate hosts. .3''/r'ff ""l^lJ'^'^'^^J'Pi'^Vlasnm^m^^^ (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 gemniation 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 continuaUy-diminishing remains of the body of the original parent-individual. The comiectinc^ 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-mdividuals, as in Babesia mutans. THE H^MOSPORIDIA 383 Tho 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 tho parasite occurs, and the side of the corpuscle becomes greatly distorted Gradually the parasite sinks more deeply into the corpuscle, and fmally disappears within it when the movements of the corpuscle cease and it resumes its rounded shape " (NuttaU and Graham-Smith, 748, vi., p. 235 ; compare the penetra- tion of blood-corpuscles by Lanhesterella 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. FiQ. 161. — Diagrams showing the mode of division of Piroplasma canis 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, G, growth of the buds at the expense of the main body ; H, I, J, final stages of the division of the body. After NuttaU 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, Ehipicephalus decoloratus. See especially Theiler (752). The transmission of piroplasms was first discovered by the American investigators Smith and Kilborne, who in a classical THE PROTOZOA Z2Z slw f '1" P'^^"*^ cattle-fever iBahesia boms or 6^5r6m^7^a) was transmitted from sick to healthy oxen bv tfl; f .f Planation in the habits and life-h/story of hTl;. ^ l^",,^^'^^^^^^^ have typically three stages in their L- history- 1) the minute six-legged larva hatched from the egc, 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 remoculated 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 ot 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 m- vertebrate host of any species of piroplasm appears' to be a tick. In P. boms (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 estabUshed, 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 cams, and Koch (743) for P. bovis, whose observations supplement each other, since Koch studied chiefly the' earher stages, while Cliristophers' 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 pubhshed by Kleine (742) and by Nuttall and Graham-Smith (750). Accounts differ chiefly as to the events at the begmning of the development. So far as it is possible to make a connected story out of the pubhshed 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 set 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. UsuaUy 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 HieMOSPORIDlA 385 staining moi'e deeply, on which the pseudopodia are centred ; and a smaller, paler body placed more oxceutrically. 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 Fm. 162.— Stages in the deTOlopment of Piroplasma in the tick. A~0 Amcebnirl orms (gametes?): ^ pear-shaped, with the pseudopodia g^ven off at tl^ The star-hke forms would appear to represent the gametes ; they congregate 25 386 THE PROTOZOA m clusters, and according to Koch they fuse in pairs (Fig. 162, D—Q)- cytological details of the syngamy, if such it be, are lacking (but compare Theilena, 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 thu:d day, according to Koch (Fig. 162, J). Whether these bodies have arisen by division of the zygote, or represent simj)ly 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 ookinetes (Fig. 162, K — M). They are motile and gregarme-like, and in some cases have an organ resembling an epimerite, regarded by Christoijhers as a boring organ, at the anterior extremity. Their size is about four times that of the piroplaams 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. canis, the globular bodies are foimd in the tissue-ceUs of the body. This globular stage, termed " zygote " by Christophers, very probably corresponds to the oocyst of the hsemamcebse. 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 Theilena 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 oif, 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 limax-amodba, ; 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 gregariniform, which penetrates into the salivary glands, and there goes through a multiplicative process, very similar to that of Haiteridium in the lung of the pigeon {cf. Fig. 157), producing a swarm of sporozoites which are inocu ated into the vertebrate host by the tick. Thus in Theilena also there is no flagellated stage at any part of the life-cycle— a fact which does not, however, prevent Gonder from seeing " blepharoplasts," 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- amoeba; 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. canis in the dog's blood described by Breml 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 Hcemosporidia. — 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 H£emosporidia ; 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 {Cienodactylus gondii) ; other species have smce been described— namely, T. cuniculi, Carini, from the rabbit, T canis MeUo, 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 frequentlv fatal ihe parasite is a crescent-shaped body, with one end thicker than the other" and contamuig a single nucleus ; they multiply by bmary or multiple fission' NicoUe and Manceaux regarded them as aUied to Leishmania, but [their resemblance to this genus appears to be purely superficial, since in Toxovlasma no kmetonucleus IS present, and in cultures no flageUated stage is developed Mleipsisoma thomsoni is the name given by Franca (441) to a nara^if a he blood of moles discovered by ThoLon (624). It oUurs as a^ amSboid mtracorpuscular parasite with a single nucleus situated at the margin of the body, which contams no melanin-pigment. Multiplication takes place ex clusively m the Imig, and is by binary or multiple fission, according to Franca the young forms are either vermiform, with the nucleus drawn out or oval' with a compact nucleus ; they penetrate into the corpuscles ^d grow t£' Franga considers this form to be allied to Toxoplasma ^ 1 w "^f^^ ^"i^?""'^ S^^*^^ ^^^"5^ (440) to certam bodies in th« red blood-corpuscles of batrachia, first described by Todd. The e?rl est i!;! in the corpusc e is a small globule of chromatin! Franca believe^ttf S parasite when it penetrates the corpuscle is reduced to 11^^ alone an J that It gradually forms a cytoplasmic body which becomersubst^S f that of the corpuscle. As the cytoplasmic body is SorS crvstals^^^^^^^ in It, one large crystal or as manyls three smaller ones. SlKt^l IS seen ^vl th a si ghtly hypertrophid nucleus pushed to one si^^^^^^ consisting chiefly of substance which stains intensely bhi^S the p± owsky stain, m which are the crystals and the nuclLs of the pariifeTr" bodies,eaeh.^;ti:^l?^??-;^~^^ 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 youngest forms show sometimes a grain near the nucleus, possibly a kinetonuclcus. With the bodies desciibcd by Neumann may bo compared those observed by Mathis and Leger (473, pp. 417-419, Plato XIII., Pigs. 12-16) in a fish, Glarias macrocephalus ; possibly they have some connection with the trypano- some found in the same host. Immanoplasma scyllii, 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 Romanowsky stain, and its nucleus appears usually as if separate from the rest of the body of the parasite, lymg 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-caUed " KurlofE-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 spirochsete, found free in the blood. The author proposes for this parasite the name Lymphocytozoon cobayce. 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 Hsemosporidia are closely aUied 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 Gregarmoidea and the Coccidiomorpha, the latter comprising two subdivisions, Coc- cidia and Hsemosporidia ; while Mesnil placed the Hsemosporidia, 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 utiHzed in this manner now that some heemogregarines have been shown to form sporocysts. 2. Hartmann and others {e.g., Aweruizew) mamtam that the H^mosporidia should be removed altogether from the Sporozoa, and should be classed, together with the HsemoflageUates, as an order of the Flagellata, for which the name Binucleata is pro- posed smce 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- pounSy Hartmami and his school, is not merely one of a general relation- shTbetwSn Hcemosporidia and FlageUata. This wjder pomt of view will be Lcus^rwEhe affi^^^^^ of the Telosporidia as a whole are considered The ques ion at present under discussion is whether the Ha=mosporidia, more THE H^MOSPORIDIA 389 than the other Telosporiclia, are allied specially to the Hneraoflagellates, more so than to other FIamosporidia. 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 mcluded in the Hsemosporidia, the piroplasms exhibit the least indications of flagellate affinity. From a general consideration of the life-cycles of the typical Hsemosporidia, such as the h8emamoeba3 and hsemogregarines, 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 haemamoebse, lialteridia, 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 haemogregarincs, gamete-formation is of the type of that seen in Adeleidce, 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 (c/. Sambon and Seligmann). In the face of such profound homologies v/ith Coccidia, what are the argu- ments from the developmental cycle in favour of a contrary opinion ? The case for the alleged Hsemoflagellate affinities of the Hsemosporidia 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 noctua) harbours the full number of kno\ra 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 spirochsete. 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 spirochsete (6), are supposed to represent different phases of one and the same life-cycle. The halteridium {Hcemoproteus noctuce) was stated by Schaudiim to be the THE H^MOSPORIDIA S91 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. Male, 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 Ml 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, Culev 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 trj^anosomes 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 occiu-red 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 tlu'ough 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 been, for the last seven years, a veritable riddle of the sphmx, have met with general scepticism except from a few devoted partisans, who have been striving continually to find corroborative evidence for Schaudinn's 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 imder observation in hanging drops under the micro- scope, trjrpanosomes 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 columbce, 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 hfe-cycle, and the invertebrate host is not a gnat, but a biting fly of an altogether different kuid. To meet this objection, Mayer proposes to restrict the name Hcemoproteus to forms which develop after the manner of H. colwmhce, and to revive the name Halteridium (in itahcs 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 largest 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 forms which have been confused with the true haltorida ; on such an assumption so emment an investigator as Schaudinn can be acquitted of having made what would appear at first sight to be a gross error of observation, and Mayer's observations are easily explained. Mayer seems, in fact, to have figured 392 THE PROTOZOA Buch forms on his Plate XXIT., Pigs. 2-4-small intracorpnscular forms, more or less Letshmanm-hko, jvUhont pigment, and wiih, apparently, distinct tro- phomiclcns and kinetonuclcus. ^ It is not necessary to deal Avitli Scliandinn's statements concerning Lenco- cytozoon further than has been done above (p. 370). It is now as certain as anythmg can ever be ni such matters that Leucocyiozoon has nothing whatever to do with cither trypanosomes or spirochaetes. The six forms of biood- 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 Icucocytozoon (5), and a spirochete (6). Of these five, it is probable that only the proteosoma, the trypanosome, and possibly the spirocha;te, can develop in, and be transmitted by, a gnat ; the halteridium and the Icucocytozoon 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 spirochjetes 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 Haemoflagellate affinities than the argu- ments deduced from the development of Hsemosporidia are those based on the development of Hsemoflagellates. 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 hcemamoebse. 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 Schaudinn for the non-sexual multiplication of Trichosphoerium sieboldi, a marine Rhizopod (p. 181). 2. Cyiological Data. — The theory of the Hsemoflagellatc affinities of the Hsemosporidia has led to the most laborious and painstaking efforts to discover in the body of each and every Hsemosporidian parasite, in at least some of its phases, a second nucleus, the homologue of the kinetonuclcus ; and any little granule, however minute, that can be coloured like chromatin is pro- claimed triumphantly to be the inevitable kinetonuclcus, or any streak of similar staining properties to be a flageUum. Consider first by itself the case of a ceU 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-caUed " ehromatin-stains " ; the body in question may therefore be chromatin or some other substance. If it be oliromatin, 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 Hmiting mem- brane, a little way from the main mass of the nucleus ; or it may be a true kinetonuclcus. If it be not chromatin, it may be a centrosome or blepharo- plast ; or a grain of mctachromatinic substance, such as volutin ; 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 kinetonuclcus and nothing else. What are the criteria by whicli a grain that stains hke chromatin can be identified as a kinetonuclcus, to the exclusion of other possible interpretations of its nature ? In the first place, according to modem 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 H.-EMOSPORIDIA 393 micloiis when present is a permanent ccU-elcment which hkc the pr.ncipa nucleus, divided when the cell divides, and is propagated by fission cqiial y with the ceU itself. Thirdly, and this is the most important criterion ol alJ, 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. , ., ^ if The smaller ehromatinic body of Leishmama may be cited as an example ot a body which fulfils these conditions, and which can be identified unhes^ita- tin^rly as a true kinetonucleus, homologous in every way with that of a try- panosome. But with the alleged kinetonuclei of Hsemosporidia the matter stands quite otherwise. It is not possible to discuss fuUy here every separate instance, but a few typical examples of such bodies may be dealt with briefly. , . . , In female halteridia and leucocytozoa (Pig. 158), a large gram 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 flageUum, it is far simpler to regard it as a karyosome which, Hke that of the merozoites of Adelea (Fig. 153, F), is excentrie, or possibly extranuclear in position ; assuming, that is, that the body in question is a true eliromatiuic nuclear element. In the merozoites of Proteosoma, Hartmann (675) has discovered a flagellum- like 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 Sehaudinn (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 than with Hfemoflagellates. 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 Sehaudinn, and recently Hartmann, have maintained that the microgametes of halteridia and other Hsemosporidia have the structure of a trypanosome. Inasmuch as Sehaudinn 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 Biimcleata, a conclusion which, indeed, seems to foUow 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 imtil 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 Sehaudinn 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 Hfcmoflagellates and Hsemosporidia, and are supposed to indicate the course of evolution whereby scrum- parasites of the first type became * On the other hand, the parasite of Oriental Sore was first described by Wright under the name Ildcosoma tropicum. and referred to the Microsporidia. 394 THE PROTOZOA coU-parasites of the second (compare Leger and Duboacq, 646). However enticing such a view may seem when only tlie 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 bo imagined more different than the develop- ment of Leishnama, with its typical leptomonad forms (Fig. 140), and that of Piroplasma (Pig. 162), with no flagellated stages at all in its life-cycle. It becomes evident at onco 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 agamst the theory that the Hsemosporidia are to be removed from the vicinity of the Coccidia,and classified with the trypanosomes and aUied 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 Hsemosporidia, 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 Htemo- flagellates themselves the possession of the bmucleato 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 hsemamoebse, halteridia, and true leucocytozoa, descended from a Coccidium-like ancestor ; the other represented by the haemogregarines, from an ancestral form similar to Adelea or Orcheobius. Leger (644) has classified the hsemogregarines 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 hsemamoebse 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 Leishniania and Babesia directly from Orithidia as a common ancestor, do not seem to have taken the development of Babesia (PirojJasvia) into consideration at all ; they neither refer to it in their text nor cite any of the relevant memoirs in their bibliography. THE HiEMOSPORIDIA 395 parasitism, and consequent temporary loss of the locomotor apparatus. It may well be, therefore, that some forms now generally included amongst the Hsemosporidia {e.g., possibly the drepanidia) may prove, when better known, to be stages of Hsemo- flac^ellates, and to have in reahty nothing to do with the true HiBmosporidia. Affinities of the Telosporidia.—From the foregoing discussion, the conclusion has been drawn that the Coccidia and the typical H^mosporidia 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 Avas 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 Amceha (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 Neosporidia are considered at the end of the next chapter. The question here is of the Telo- sporidia alone. For this group opmion 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 flageUate origin of the Telosporidia is the existence of flagel- 396 THE PROTOZOA lated stages in the life-liistory. In the first place, the micro- gametes are very often fiagollated, as has been stated frequently m 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 Sarcodma 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 flageUated swarm - spores and of gametes representing a modification of such swarm- spores is not sufficient of itself to prove a flagellate ancestrj'- for the Telosporidia. Far more cogent arguments for the flagellate afiinities of the Telosporidia maybe 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 HiEMOSPORIDIA 397 form probably used its flagellum for the purpose of attaching itseK to "le opitliium of the digestive traet, as leptomonads do now (oornpare Figs. 126, 137) ; cand from this primitive type of attach- ie7the epLerite of the gregarines may have been derived by secretion of chitm round the attaehmg flagellum ]ust as the px^tTve tuft of fixing ciha, the " scapula - of the prmutive VoXeUids appears to become converted into the chitmous stalks of such forms as (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 ot the " bmucleate " type of structure. Bibliography.— FoT references see p. 496. CHAPTER XVI THE SPOROZOA : III. THE NEOSPORIDIA A TYPICAL member of the subclass Neosporiclia is a parasite of which the life-cycle is initiated by the hberation from the spore of one or more amoebulae within the body of the host, in the digestive tract in all known cases. For this initial amcebula-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 m most cases migrates thence into some organ or tissue of the host, where it lives and multiplies actively, bemg 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 amoebula (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-formmg 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 NEOSPORIDIA 399 the sporozoites are always, apparently, amoebulse, and never gregarinuljB.* ^ . , The Neosporidia are divisible into two sections, known re- spectively as the Cnidosporidia and the Haplosporidia. The Cnidosporidia are distinguished by the possession m the spore of peculiar structures termed polar capsules, which are lackmg m the Haplosporidia. , i j -4.1, 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, Actmomyxidia, 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, resembhng 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 species, 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- tdtidw of the Haplosporidia (p. 424). But the position of all the forms in this order 13 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 PS. other classes of tissue. A few species arc known to attack the nervous system— for instance, Lentospora {Myxoholus) cerehralis cause of " Drehkrankheit " in Salmonidce (Plchn), and Myxoholus neuroUus of trout (Schuberg and Schroder). In the tissue attacked the parasite may be concentrated at one spot, so as to form a dis- tnact 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 mstance in the lumen of the digestive tract, but frequently in the gall-bladder or urmary 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- thehum ; 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-hke 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 Ueberkuhni, 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- -cot Fig. 164. — Ghloromyxum leydigi, parasite of the gall-bladder of the dogfish, skate, etc. ; trophozoite (Plasmodium) in an active state. ecL, Ectoplasm ; end., endoplasm ; y., yellow globules in the endo- plasm ; sp., spores, each with four polar capsules. After Thelohan, from Mmohin, magnified 525. THE NEOSPORIDIA 401 stuffs by diflfusion, 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 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 lieberkuhni, 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 ahnost 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 nothmg 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. Do*fletn'r"'s?p£^^f;, ' r™' *° ^V'' "^^taken altogether the significance of nnrifo V,- ^*emm-pseudopodium," and applies the term to the anterior pseudo- podia, which appear to be rather tactile in function in such oases. ^ 26 Fig. 165. — Leptotheca agUis: young plasmodial trophozoites in which the spore-formation has not begun. A, Individual moving forward by means of the " Stemm-pseudopo- dium " {st. ps.) ; B, individual in which only the anterior pseudopodia are developed. After Doflein. 402 THE PROTOZOA THE NEOSPORIDIA " 403 An example of the Disporca is Ceratomyxa drepanopseitce, of which the spore-formation is described by Awcrinzow (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 tho 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, tho 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 certam amount of chromatin is extruded from tho 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 (Pig. 166, O), and the smaller divides again into two (Pig. 166, H) ; the result is an aggregate of three -cells. :^ a larger, which gives rise ultimately to the two parietal cells which form thb valves of the sporocyst, and may be termed the " sporocyst- mother-cell " ; and two smaller cells, one of which is the mother-cell of the two capsulogenous cells, the other the future sporozoite or amcebula. The sporocyst-mother-cell may become temporarily separate from the capsule- mother-cell and the amcebula. The three cells of the previous stage build up the spore in the following manner : The sporozoite grows in size, and its nucleus divides into two The capsule-mother-cell divides into two capsulogenous cells, each of which forms a polar capsule in its interior. The amcebula and the two capsulogenous ceUs are placed close together and arranged in a definite manner (Pig 166 /) The sporocyst-mother-cell divides into two parietal cells (Fig 166 j) which place themselves on either side of the ceU-complex composed of the associated am(Bbula and capsulogenous cells (Pig. 166, K), and each secretes one valve of tfie sporocyst enveloping the whole complex. Thus the trophozoite consists finally of an amoeboid body containing two Fig. lee.—Spore-formation in Ceratomyxa drepanopsettce. A, Trophozoite sketched m outhne showing in the Plasmodium two generative nuclei (^ )! each sur- rounded by a ceU-body, and two vegetative nuclei (v.). which lie n ?he toplasm of the body (left blank) ; note that the generative cells are of different sizes; B, 0. the two generative cells have dilided each into two so tW there are now two macrogametes (?) and two microgametes ( ••*•*. ; • I \-rryz psp. Fig. 174. — Glugea anomala, Moniez : part of a section of a cyst, c, Envelope (autocyst) ; bn, vegetative nuclei ; sp., spores ; psp, sporont lying in a space in the protoplasm. After Stempell. protoplasm contaming 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- bourmg cells, the effect of which is to produce a sort of host-plasmodium as It were, containing gigantic liost-nucloi of irregular form (Fig. 171), amongst winch the sporonts and spores of the parasite are scattered. Mrazek also interprets the supposed vegetative nuclei of Myxocyslis as hypertrophied host-nuclei (see below). This interpretation of the composition of the Plas- modium greatly diminishes, or even abolishes, the prmcipal distinction between Ulugea and the other genera of Microsporidia. In opposition to this view, 27 418 THE PROTOZOA Stempcll (78G) brings forward a number of arguments, the most cogent of which is the existence of the autocyst separating the plasmodium of the parasite, containing the nuclei of disputed nature, from the tissues of the host. The most recent investigations of Awerinzew and Termor confirm com- pletely Stempell's interpretation of the cysts of Olugea anomala ; compare also Weissonberg. These authors find nuclei of various sizes in the protoplasm of the cyst, larger or smaller. The larger nuclei are found in the outer, non- vacuolated protoplasmic layer of the Glugea ; they grow in length and become sausage-shaped, and are ultimately segmented into smaller iiucloi, 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 in toto from nuclei, -without visible participation of the 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 fine 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., he 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 de- scribed in the foregomg paragraphs have been utilized by Perez (779) to sub- divide the Microsporidia into two suborders, as given below. Stempell (786), 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. SuBOEDEK I. : ScHizOGENEA (seu Oligosporea).— The principal trophic phase is a iminucleate meront which multiphes by fission, and from which the sporont fhially arises. Several genera, characterized by the number .of spores produced by the sporont : One spore, Nosema ; two spores, Perezia ; four spores, G-urleya ; eight spores, Thdohania ; sixteen spores, Duboscqia (see below) ; n spores, Pleistophora ; but Stempellia (Leger and Hesse, 775), for S. mutabilis, parasite of the fat-body of Ephemerid larvse, produces spores to the number of eight, foui", two, or one indifferently ; Ociosporea, the species of which are parasitic in Muscidce, produces eight spores in one species, one in another. These anomahes indicate that the classification by the number of spores produced by the sporont is purely artificial (Chatton and Krempf). Telomyxa glugei- formis (Leger and Hesse), also from the fat-body of Ephemerid larva;, pro- duces eight, sixteen, or n spores, and stands apart from all other known Microsporidia in possessmg two polar capsules in the spore. SuBOKDEB II. : Blastogenea (seu Polysporea).— The principal trophic phase is a multinucleate plasmodium producing sporonts by internal cleavage ; example : Olugea. To this section, also, the peculiar form Ilyxocysti^ has been referred, which was discovered by Mrazek in the body-cavjty of Ohgo- chfBtes. Myxocystis occurs in the form of large masses floating freely m the body-cavity, each mass remarkable for an envelope composed of a fur of vertical filaments, not unhke 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 lymphocj^te 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 Mygcocystis is an intracellular parasite reterabie, apparently, to the order Schizogenea, and characterized chiefly by the peculiar form of its spores. Duboscqia legeri, Perez (780), from the body-cavity of Termes lucifugus, is perhaps an organism of similar nature; it is descnoea THE NEOSPORIDIA 419 as a floating plasraodium 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 Imown to be parasitic in invertebrate hosts of any kind. In their hosts the Sarcosporidia are tissue-parasites, occurruig prmcipally in the striped muscles, but occasionally in unstriped. In a few cases 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 milhmetres, while in the roebuck [Gervus capreolus) cysts of 50 milhmetres 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 contamed m chambers separated off from one another by partitions The whole organism is enclosed by a distinct envelope, often ex- hibitmg vertical striations, and the partitions between the chambers oontammg the spores are contmuations 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 khid 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 Avhen set free : for example, in S. 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 S. muris, 8. bertrami (of the horse), and S. 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 S. 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 mdicate 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 contaminative 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, indicatuig 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 tlie 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-stainmg, 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 aS*. muris and ;S'. bertrami as having the nucleus near the blunt end, while the opposite extremity appears hyaline and homogeneous for a certain distance. Betegh, again, describes a nucleus at the blimt 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 metaclu-omatinic 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 pomted 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 cupsule. 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 b. nleyi are binucleate ; compare those of Qastrocystis (Fig. 179, p. 428). It is not THE NEOSPORIDIA 421 clear which part of the spore contains the amoBbula which is liberated from it, as presently to be described. j ^ In addition to spores having the complicated structure described tor those of S. tenella, there appear to be also spores of much simpler structure, as, for example, in 8. miiris. 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 sporoblast, as a simple ceU not difler- entiated as a spore, serves for spreading the infection in the same host. The occurrence of the simpler type of spore in 8. 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 8. 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 8. rmiris 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 8. 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- — Spores sporidia occasionally observed in the human subject ^rcocystis ^tenella. are those of some domestic animal undergoing a condition • B after modified or abortive development in a host that is staining with iron- not their usual one. Erdmann also infected mice hematoxylin - N. with 8. tenella in a simiiar manner. It is remarkable nucleus ; c, striated' that parasites generally so harmless should be so body (po'lar'capsule ?). little specific to particular hosts, and the results of After Laveran 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 occurreiice 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 being 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 metachromatini c 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 mctachro- 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 m the polar capsule, and is set free by its discharge, like the poison in the nematocysts of the Coslontera. 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 lininf'- S^'^^^'i- 446 THE PROTOZOA plate. At tho upper end of the body fine continuations of the longitudinal myonemos can bo traced to the adoral zone, ending in the baisal rims of tho membranelliB (Schroder, 867). Stentor may be taken as a type showing the contractile system highly developed in functional efficiency, but more or less primitive in arrangement. Canals lodging tlie myonemes are not present universally, even in highly contractile forms ; they are absent, according to Lebedew (93), in Trachelo- cerca, but they are figured by Maier (73) in Prorodon teres (Fig. 186, B). In their general form the myonemes are simple fibrillsc, often beaded when contracted. In the more specialized forms the contractile system acquires a more com- plicated arrangement. In Campanella, Schroder (864) describes five systems of myonemes : (1) Annular myonemes of the basal part of the body; (2) longi- tudinal myonemes of the outer body- wall, doubtless representing the primitive system (Fig. 186, K) ; (3) annular myonemes forming the sphincter-like muscle of the margin of the peristome ; (4) a spiral myoneme rmming under the adoral spiral, and continued down the wall of the vestibule ; (5) a series of retractor-myonemes of the peristomial disc. In Epistylis plicatilis, on the other hand, Schroder (865) found only three systems : The longitudinal myomenes (2), the annular peristomial myonemes (3), and the vestibular myoneme (4). To these systems found in the Vorticellids with non-con- tractile stalks must be added, in the genera Vorticella, Carchesium, etc., the powerful stalk-muscle (" spasmoneme '") formed by the union of the longi- tudinal myonemes (Schroder, 866). In Vorticella monilata fine connections run from the hinder ciliary ring upwards and downwards to the longitudinal myonemes when the cilia are developed, but disappear when these cilia dis- appear. In Licnophora, according to Stevens, the fibril that runs under the adoral spiral is continued down to the disc or cup of attachment and ramifies in its waUa. In the aberrant form Pycnothrix monocystoides, Schubotz describes a re- markable development of the myonemes in the form of a dense plexus of fibrils at the inner limit of the ectoplasm. The fibrils are connected with the basal granules of the ciha, and run in two directions, forming a deeper layer of circular myonemes and a more superficial layer of longitudinal myonemes. The question has been much discussed whether the contractile system, often so highly developed, is accompanied by any conducting elements of nervous nature. That many ciliates react with extreme rapidity to stimuli has been noted above, and that their movements are co-ordinated is suffi- ciently apparent. Neresheimer (856) describes in Stentor filaments believed to be of nervous nature, neuronemes which take origin from the foot and run about halfway up the body, at which point each neuroneme either ends in a bulbous swelling or becomes thinner and disappears. The neuronemes are situated externally to the myonemes, and run parallel to them. By experiments with various drugs, Neresheimer tried to prove the existence in Stentor of true nervous elements, as compared with Paramecium and other forms in which neuronemes were not foimd, and concluded that the elements described by him were truly nervous in natmre. Schroder (867) casts doubt on the existence of neuronemes and criticizes Neresheimer' s technique. Lebedew (93), however, describes fibrils, possibly nervous in nature, runnmg parallel to the myonemes in Trachelocerca. For the present the existence of nervous elements in Ciliata must remam doubtful. But of the sensory function of the cilia there can be hardly any doubt, and the fact that their basal granules are always in close proximity to the myonemes is extremely significant. Such a direct contact between the sensory and contractile mechanisms may render conducting elements ot nervous nature unnecessary, except for purposes of co-ordination of move- ments. In some ciha, as akeady stated, the motile function is lost, and only the sensory function remains. The genus Mycterothriy: (TricJiorhync ms) is characterized by a rostrum bearing a number of stifl:", tactile cilia (J^aure- Fremiet, 839). In some cases, however, sensory organs occur whic i apF^^r not to be derived from cilia, as, for example, the tentacle-like or club-siiapea THE INFUSORIA 447 1 organs, probably tactile, between the membranolla3 of the adoral spiral of Tiyitinnidce (Schwoyer). The natiu-e and mechanism of tlic peculiar trichocysts remains to be ex- plained. The trichocyst in the imexploded state is a spindle-shaped body with a fine, hair-like process at its outer end which reaches to the pellicle (Fig. 18G, D, T.). The exploded trichocyst tapers gradually to a sharp point at its proximal end ; distally it shows a cap-like swelling (Fig. 187, D—O). According to Khainsky (170-5) the trichocyst consists of two portions : a distal or outer part which stains deeply, and a proximal or inner part which stains a lighter colour (Fig. 187, A—C). The unexploded trichocyst consists entirely or almost entirely of the darker substance ; in the process of explosion the dark substance is con- verted into the light, so that in the exploded trichocyst only a small portion of the dark substance remains to form the distal cap. The notion, recently upheld by JVIitrophanow (855), that the tricho- cyst consists of viscid fluid con- tained in a cavity in the ectoplasm, whence it is expelled by a sudden contraction of the ectoplasm, and stiffens to a solid thread under the action of the watery medium, cannot be maintained (Schuberg, 44) ; nor does there seem to be any ground for comparing it to a Ccelenterate nematocyst or to a polar capsule of a Cnidosporidian spore. According to Mitrophanow, the substance of the trichocysts appears first near the nucleus in the endoplasm as small grains which pass out into the ectoplasm. Tricho- cysts do not occur in any Peritricha, but in one species, Epistylis umbel- laria, large oval nematocysts occur, arranged in pairs— a phenomenon unique amongst the Cihata. The contractile vacuoles open to the exterior as a general rule, but in the Peritricha, as already stated, they open into the vestibule ; in this order there is usually a reservoix- vacuole into which one or two con- tractile vacuoles empty themselves, and which in its turn voids its contents into the vestibule. In Campanella, however, there is no reservoir- vacuole, and the single contractile vacuole opens by two canals into the vestibule (Schroder, 8G4). In Opalina there are no contractile vacuoles and m some species {e.g., 0. ranarum) no excretory organs are to be found • but m other species the endoplasm contains an axial series of more or less irregular vacuoles, opening one into the other and to the exterior by a pore at the posterior end of the body. These vacuoles are sometimes in close relation with the nuclei, often enveloping them to form a perinuclear space (Metcalf 90^). in Fycnothnx monocystoides the endoplasm is traversed by a branched V ^''f ^^^^^^ ^ s^"gle efferent duct opening at the surface of the body near the posterior end by I pore ; the duct is ciliated and IS homologized by Schubotz with the cytopyge, which in %cLw is E F Fig. 187. — Trichocysts of Infusoria. A — E, Stages in the explosion of the trichocysts of Paramecium caudatum, showing the manner in which the tricho- cyst grows in length, with conversion of a darkly-staining substance into a lighter material ; the fully-exploded trichocysts are seen in D and E. After Khainsky (170-5). F, G. Exploded trichocysts of Frontonia leucas. After Schuberg, magnified 1,500 diameters. 448 THE PROTOZOA also ciliated These excretory systems of Opalina and Pycnothrix differ in bemg endoplasmic from the ordinary contractile vacuoles, which are always formed m the ectoplasm. The endoplasra of the Ciliata may contain enclosures of various kinds • food -vacuoles ; metaplastic bodies in the form of excretory grains, crystals' pigment-grams, etc. ; zoochlorellae, and occasionally parasites of one kind or another, etc. Special attention has been drawn by Fauro-Fremiet (38-5 and 835) to the bodies termed by him spheroplasts, and considered by him to be homologous with the mitochondria (p. 41). The bodies in question are small spherules, which multiply by fission when the cell-body divides ; they are permanent cell-organs to the same extent as the nuclear apparatus, of which, however, they are entirely independent. As pointed out above, the form of the macronucleus and the number of nuclei_ vary greatly in different species. The cases will be considered below in which the micronucleus appears to be wanting (Opalina), or is contained in the macronucleus in the ordinary condition of the body (Trachdocerca, Ichthyophthirius). As a rule the macronucleus has a finely granular appear- ance, with the chromatin distributed evenly over the nuclear framework ; but in a few eases it has a distinctly vesicular structure, with a large karyo- some, as in Loxodes (Joseph, Kasanzeff), Chilodon (Nagler, 96), etc. The macronucleus divides by binary fission of a simple and direct type (Fig. 54). The micronucleus, on the other hand, divides by mitosis (Fig. 61). In Trachelocerca, a form which may possess one or many nuclei (but no separate mieronuclei), Lebedew (93) describes a peculiar mode of multiplication of the nuclei, which divide by multiple fission to form a morula-like body consisting of a mass of small nuclei which separate from one another (Fig. 66). In Lovodes, another form in which the number of nuclei varies greatly in different specimens, the macronuclei do not divide, but only the mieronuclei do so, and the macronuclei arise by growth and modification of the micro- nuclei (Kasanzeff). In many cases in which the macronucleus is of the elongated monihform type, or in which the body in the ordinary state contains two or more macronuclei, they come together to form a single compact macronucleus prior to division ; but in other similar cases this does not occur, and when the body divides the nuclei are distributed irregularly between the two daughter-individuals, as in Trachdocerca, Opalina, etc. The distributed form of nucleus is especially characteristic of the astomatous parasitic forms, and in the opinion of Pierantoni [A.P.K., xvi., p. 99) is correlated with nutri- tion by the osmotic method. The micronucleus is less variable in form or number, as a general rule, than the macronucleus, but is not infrequently multiple, especiaU y when there is more than one macronucleus ; but in Trachdius ovum a single large macro- nucleus is combined with thirteen mieronuclei (Hamburger, 841). The conjugation of the Ciliata conforms, as a general rule, in its main outlines to the scheme sketched out above (Fig. 77), but some important variations must be noted. In the first place, the conjugation is often pre- ceded by active division of the animals, so that the eonjugants* are much smaller than the ordinary individuals of the species. When the two eonju- gants come together, the micronucleus of each usually divides into four, but sometimes into eight, as in both eonjugants of Euplotes and the microconju- gant of Peritricha ; in either case, however, only one micronucleus persists, and furnishes the two pronuclei. The Peritricha exhibit in their conjugation certain peculiarities which are clearly of a secondary nature and correlated with their sedentary habit. Certain individuals divide two or three times successively to produce four or eight microconjugants (" microgametes ") which acquire a rmg of locomotor * It is preferable not to speak of two conjugating Infusoria as gametes, since it is very doubtful if they correspond to the gametes in the other classes of Protozoa. It is on the whole more probable that the eonjugants correspond rather wtli gamonts or gametocytes, which originally produced a number of gametes, reduced now to two, represented in each conjuganfc by the two pronuclei. THE INFUSORIA 449 oilia and swim off. Each microconjugant attaches itself to a macroconjugant — that is to say, to an ordinary sedentary individual ; each of the conjugants has a single microniicleus and macronucleus, but as soon as they become associated" the changes preparatory to syngaray begin. In the microconjugant the micronucleus divides tlireo times to produce eight mieronuclei. In Carchesium the first of these divisions is an equating division ; the second reduces the number of chromosomes from sixteen to eight ; and the third division is again an equatmg division (Popoff, 125). Meanwhile the macro- nucleus is in process of degeneration, and is breakmg up into fragments. Of the eight mieronuclei, seven degenerate, one persists and divides into two pronuclei. In the macroconjugant, meanwhile, similar events are taking place, but the micronucleus only divides twice, first by a reducing, then by an equating division, to produce four mieronuclei, of which three degenerate, while the fourth persists and divides into the two pronuclei. Of the two pronuclei now present in each eonjugant, one degenerates in each case ; the persistent pronucleus of the microconjugant -passes over into the macroconjugant and copulates with its persistent pronucleus. The frag- ments of the macronucleus also pass over into the macroconjugant, but are there absorbed slowly. The body of the microconjugant then falls off and dies ; only the macroconjugant is fertihzed. Variations of minor importance are seen in the behaviour of the synkaryon of the exconjugant after fusion of the pronuclei has taken place. For example, in Paramecium hursaria the synkaryon divides to form four nuclei, two of which become macronuclei, whereupon the exconjugant divides into two ordinary individuals (Hamburger, 842) ; in Licnophora the synkaryon divides into eight, which become a micronucleus and a macronuclear chain of seven segments (Stevens, 872) ; in Carchesium the synkaryon divides also into eight to furnish a micronucleus and seven separate macronuclei, but the micro- nucleus then divides six times, with subsequent divisions of the body and sorting out of the macronuclei, imtil seven individuals, each with a single micronucleus and macronucleus, are produced (Popoff, 125) ; in A7ioplophrya the synkaryon divides into four nuclei, two of which degenerate, the remaining two becoming a micronucleus and a macronucleus respectively. The method of nuclear reconstruction may vary even in the same species, as shown by Prandtl (126) in the case of Didinium. The most important deviations from the usual scheme of conjugation are seen in those forms in which there is no separate micronucleus in the ordinary condition. The cases of Opalina and IcUhyopUhirius, parasitic forms and therefore open to the charge of degeneration, are dealt with below. In Trachdocerca pJioenicopterus, a free-living species, conjugation has been described by Lebedew (93) between individuals containing many nuclei all similar m appearance, each with a large karyosome. Prior to conjugation the chromatin passes out of the karyosome into the nuclear cavity of each nucleus (Fig. 188, A, B), which then divides into four. The chromatin forms a compact mass at one pole of each nucleus. During conjugation these masses ^ P^^^ ^^'^ nuclei, and lie free in the ejrtoplasm between them (Fig. 188, G~0) ; each such mass is now to be regarded as a micro- nucleus and lies in a clear area, finally becoming a vesicular nucleus with a distmct alveolar structure ; the old nuclei can now be considered as macro- nuclei. All the nuclei now collect in a mass near the middle of the body Ihe macronuclei ultimately degenerate ; the mieronuclei multiply by fission but ultimately, accordmg to Lebedew, they aU degenerate with the exception ot one in each eonjugant ; the persistent micronucleus divides into two pro- nuclei which conjugate in the usual way ; unfortunately, the author's observa- tions contain so many gaps that this statement cannot be considered estab- lished so decisively as could be desired. The exconjugants contain each a single synkaryon which divides by successive divisions into a number of nuclei not differentiated into mieronuclei and macronuclei. tnnnlrT oi Trachdocerca, as it is described, furnishes an important clue to understandmg the ongm of the heterokaryote condition of Infusoria from 29 450 THE PROTOZOA that found in other Protozoa. In this case, during the ordinary vegetative condition, the generative chromatin representing the micronuclcus of other Infusoria, and tlic vegolativo cliromatiu representing the macronucleus, are contained in one and the same nucleus, and become separate only when syngamy is about to take place. Tlio first sigii of the separation is the forma- tion of chromidia from the karyosome within the nucleus, resulting in the formation of a secondary nucleus which becomes separate and which behaves exactly as an ordinary micronuclcus ; thus indicating a clear homology between the micronuclei of Infusoria and the secondary generative nuclei of Sarcodina. The production of numerous micronuclei in the conjugation of Trachelocerca N' Fig. 188. — Formation of micronuclei in Trachdocerca phwnicopterus. A, B, A nucleus has divided into two. and from the karyosome (Jc.) of each daughter- nucleus masses of chromatin are being given o£E into the nuclear cavity ; G, D, the two nuclei of the preceding stages have divided again, to form a group of four, and the chromatin-masses (n) have acquired a compact struc- ture and are passing out of the nuclei to form the micronuclei ; in G crystals are seen in the cavities of the old nuclei, probably a sign of degeneration ; E, F, two groups of nuclei, both from the same specimen ; the micronuclei given off from the old nuclei become surrounded by a vacuole (n' in F), and then acquire an alveolar stracture {n' in E) ; G, portion of a preparation of the body of a conjugant, the wavy contour on the right being the surface of the body which is in contact with the other conjugant ; numerous micronuclei (n) are seen, and also macronuclei, some of which still appear normal (A ), others degenerating (N'). After Lebedew (93). is noteworthy, and would appear to favour the theory (see p. 154) that primi- tively numerous gametes (swarm-spores) were produced in the conjugation °^Smpl'es of a complicated life cycle are to be found in Cihata chiefly, perhaps solely, among parasitic forms. As an example may bo taken Icli- thyopMhirius multifiliis, a parasite of the skin of various species of fresh-;^a^r fishes. In aquaria, where, owing to the limited space, the parasites, if present find their way to the fish very easily, and where, consequently, a fif^ becomes infected with vast numbers of the ciliates, the parasites are "/\;^"yj^^^^^ the host, and cause its death, according to Buschkiel, m f J^out ourteen dajs In Nature, on the other hand, " ichthyophthiriasis ' is seldom observed, prob- THE INFUSORIA 451 ably owing to the fact that under natural coiiditions only a very small propor- tion of the young parasites succeed in establishing themselves on a fish, and consequently the infectious produced are so slight that they are overlooked, and the fish is miharmed. The life-c3'cle of Ichthyo'phtliirms is as follows : The youngest parasites hatched out from a cyst are very small, and have a macronucleus and a micro- nucleus. They seek out a fish and bore into its epidermic', attaching them- selves by one end of the club-shaped body and rotating actively, with the result that epithelial ccUs are displaced, and either cast off into the water or form a ring-like wall round the parasite. In this way the infusorian works its way gradually into the deeper layers of the epidermis, which closes over it, so that the parasite lies finally in a closed hollow space in the epidermis. In this position it grows in size, and at a certain point the micronucleus disappears, passing into the macronucleus to form a nucleolus like body within it. The parasite appears to the naked eye as a little white spot on the skin, occurring on any part of the body-surface or on the gills. It retains its cilia, and can be seen rotating within the cavity in which it lies. The full-grown Ichthyophthirius may reach 1 milhmetre in diameter, but is usually less, about 0-75 millimetre. When full-grown the cihate breaks out of the cavity in the epidermis and sinks to the bottom, attaching itself to the ground or to water- weeds, and becomes encysted. Within the cyst it multi- phes by bmary fission repeated eight times, producing 256 small eiliates ; sometimes this multiplication takes place without encystment. Durmg this process of multiplication the micronucleus reappears, being extruded from the macronucleus of each individual when not less than four are present in the cyst ; but the exact period at which the micronucloi appear varies in dilierent cases. In addition to the micronucleus, one or two other extrusions from the macronucleus take place (Buschkiel) ; but whether these represent other micronuclei or expeUed vegetative chromatin is not clear ; in any case they degenerate and disappear. When the micronucleus makes its appearance. It divides by mitosis at each division of the cell-body, as in ordinary Oihata, macronucleus divides in the usual way by direct division. When the full number of tmy eiliates is formed, each with a macro- nucleus and micronucleus, sexual phenomena occur, but the events that ^ke place are deseribed differently by different investigators. According To fZl'^'T f ifidividual the micronucleus divides twice, and T produced degenerate ; the fourth then divides ?Sto?other ??Wn"^^^^^^^^ according to this account, similar to tfiatot other Cihata, and the orgamsm appears to be ready for coniugation n ^.P'wf' ' ^ Nejesheimer was unable to observe e JnjugatJSS place either m the cyst or after the organisms have become free • he observed however, sometimes two micronuclei,%ometimes one, both i^free f ormsTnd m those attached to the fish, and from this it was inferred Sat the two nro nuclei fuse autogamously, leavmg the possibibty open SwevL tharS" that thfbmodl iT M-f own parasite leaves the fish to the time -^rSSi^^rF— ^^^^^ as Tn^orlr ATomak^'S^ sometimes classified group, like ottrsTounded .^'''^''^ ' ^'^^^ doubt this of foSis i^ SthrchaicrrS!!'^' characters, is a heterogeneous collection wnicn tne characters thoy possess in common are due to convergent 452 THE PROTOZOA adaptation to their mode of life (c/. L6ger and Duboscq, 848). The best- known genera are Anoplophrya, a typical ciliato with micronucleus and macronuc eus and with a rudimentary cytostomo, constituting with Hopli- tophrya, Herpetophrya Discophrya, etc., the group Anoplophryinm ; Chromidina and Opahnopsis parasitic in Cephalopods, are probably allied to the fore- going (c/. Dobell, 833). The species of Opalina, constituting the group OpaUmnce, are parasitic in frogs and various cold-blooded vertebrates • their nuclei vary in number in different species from two to an indefinitely large number, but are all similar and without differentiation into inicronuclei and macronuclci at any period of the life-cycle. Cepcdc has monographed the section Anoplophryina;, and has described a number of new genera and species, distributed amongst eleven famihes. The Astomata are internal parasites of their hosts, especially of the digestive tract. Protophrya ovicola occurs in the brood-sac of the mollusc Littorina rudis, and is parasitic upon its eggs, causing their disintegration (Kofoid). The remarkable form Pycnothrix monocystoides, from the gut of Hymx capensis, described by Schubotz, deserves special mention. It reaches a length of 3-2 millimetres, and contains parasitic nematodes. The animal itself has a great superficial resemblance to a nematode or to a monocystid gregarine ; it has a very thick and distinct ectoplasm, covered by an even coat of short cilia, and with two longitudinal grooves which Schubotz regards as equivalent to the peristomial grooves of other Cihata. Each groove contains a series of pouch-like depressions, which open doAvn into the endoplasm, and are provided with special tracts of myonemes. Schubotz regards these pouches as a scries of cytostomes, but no food-particles or vacuoles are found in the endoplasm ; the interpretation, therefore, of these openings as cytostomes can only be taken in a phylogenetic sense ; actually they appear to represent perforations of the tough ectoplasm which may facilitate absorption of food by the osmotic method. For the cilia, myonemes and excretory system of this form see above (pp. 443, 446 447) ; the micronucleus and macronucleus are each single and of the ordinary type. Pycnothrix stands at present quite isolated. The species of the genus Opalina differ in certain peculiarities of structure and life-history from all other ciliates. The life-history of the common species of Opalina parasitic in the rectum of the frog has been studied by Metcalf (853) and Neresheimer (857), whoei.- accounts agree as regards the general life-cycle, but differ in some cytological details. Opalina ranarum multiplies in a vegetative manner during the summer and autumn months, but in the spring a special propagative cycle occurs in relation to change of hosts and is followed by sexual processes. The vegetative reproduction increases the numbers of the parasite in the host ; it consists of two processes, multiplication of the nuclei and division of the body, which go on independently. The animal contains a great many nuclei, and when it reaches a certain size the body divides either longi- tudinally or transversely to produce two daughter-individuals, each of which grows again to the full size. The multiplication of the nuclei is effected by a simple mitosis, similar to that of the micronucleus of other Infusoria, and without eentrosomes. In the spring the parasites divide rapidly and repeatedly, without groA^dng to full size between the divisions, so that they become continually smaller in size. A few individuals, however, do not undergo this process of rapid fission, but remain of the ordinary type, forming a stock which persists and carries' on the infection in the frog, while those which divide up are destined to pass out of it. At the beginning of the process of rapid division, the nuclei extrude ehromidia, some of which are absorbed, while from the remainder secondary nuclei are formed (Neresheimer). Finally the old nuclei are absorbed. The secondary nuclei also multiply by mitosis ; and, according to Metcaif, in the later mitoses preceding encj^stment the number of chromo- somes is reduced to one-half the ordinary number (in 0. intestinalis from eight to four, in 0. caudaia from six to three). The result of the repeated division THE INFUSORIA 453 is to produce small individuals containing, as a rule, from three to six secondary nuclei. Such individuals become encysted (infection-cysts), and pass out of the frog in the fteces. The animal at first fills the cyst completely and shows no cilia, but after a time the body shrinks within the cyst, and the animal is then seen to have a ciliary covering. The faeces of the frogs are readily devoured by tadpoles, which thus become infected with cysts. In the gut of the tadpole the Opalina emerges from its cyst. It at once divides up into uninucleate individuals, the gametes, elongated club-shaped forms with a sparse coat of cilia over the flattened body. Under unfavourable circumstances the gametes undergo agglomera- tion in rosettes, adhering by their pointed ends (Neresheimer). Under normal circumstances they copulate in pairs as isogametes, according to Neresheimer, in 0. ranarum ; but Met calf describes smaller uninucleate micro- gametes and larger macrogametes with one or two nuclei, in other species ; the male pronucleus then fuses with one of the two nuclei of the macrogamete. The zygote of 0. ranarum, with the two pronuclei still separate, rounds itself off and becomes encysted (copulation-eyst) ; within the cyst the two pronuclei, which have passed into a spindle-stage, undergo fusion. The zygote emerges from the cyst with a synkaryon, and it becomes an adult Opalina. Neresheimer considers that the hfe-cycle of Opalina proves that its affinities are with Plagellata rather than with Infusoria. In deciding this question, it must be considered, in the first place, whether in such a form the life-cycle, or the structural features of the body, are most likely to indicate affinity — that is to say, least likely to exhibit secondary peculiarities due to adaptation. Opalina is a parasitic form, and its life-cycle shows very obviously a direct daptation, of a type very common in parasitic Protozoa, to its mode of life ; multiplicative reproduction increasing its numbers within the host, and prop- agatiye reproduction, combined with sexual phenomena, leading to the infection of new hosts. On the other hand, its minute structure is that typical of Ciliata, a character hardly likely to be due to the influence of parasitism, as Popofl" (125) has well pointed out. The chief difference between Opalina and other Ciliata, which requires special consideration, is the fact that the animal contains but one kind of nucleus. This, however, is a character known in other genera of Ciliata a\so~e.g., Trachelocerca, Ichthyophthirius. There can be but little doubt that the heterokaryote " condition of the Infusoria, with distinct generative and vegetative nuclei, must have been derived phylogeneticaUy from a condi- tion m which, as m other Protozoa, the two kmds of chromatin were contained m one and the same nucleus ; and to find this condition stiU retained in some infusoria would not be remarkable. In such forms it is to be expected that prior to gamete-formation the vegetative chromatm, equivalent to the macronucleus, would be expeUed, and the pronuclei would be formed from generative chromatin. There is nothing, therefore, to be said against the view of Popoff, that Opahna show^ the most primitive type of gamete-formation laiown it present amongst the Ci lata. Its nuclei contain generative and vegetative chromatin IZtT ' '^"V VP''^.^ ^^^g^^^y ""'^l^i f«^-'^ed which are pure y generative, out o chromidia expelled from the primary nuclei. The forma oi generative nuclei micrcnuclei prior to the svnuamie Droeoa^ • l.iif l,„m sTtffinrtra-r^rSo-^^^^^^^^^^^^ 454 THE PROTOZOA ac- Tho quostion of tho exact systematic position of Opxlina cannot be decided h lTnnf . '"'n' 1'^^' '^'^"^^^^^ P^'-^^^i*^'^ C'l'^^t'^ ' but at present Ciliata! '"''^ ^«'«°^"^g this genus from the .dffT °Jc^'^ Oiliata-A typical ciliatc, such as Paramecium, with its even coat of fine cilia its hetcrokaryote nuclear apparatus, and its peculiar Snf°i/^^!f^/"*'\P^''*^''^^ karyogamy, stands apart and apparently isolated from the typical members of other classes of the Protozoa Never- theless, even within the limits of the class Ciliata, examples are to be found in which the hetcrokaryote condition is not developed, or only appears prior to syngamy in the form of a separation of generative from vegetative chromatin (Trachelocerca, Opalina), and in which the S3mgamy takes the form of total karyogamy between minute gametes, swarm-spores (Opalina). Such cases, while they minimize the gap between Ciliata and other Protozoa, do not bring the ciliates nearer to any particular class, since a similar type of syngamy and of preparations for it may occur either in Sarcodma or Mastigophora. As the most distmctive feature of the Ciliata there remains that wliich is implied in the name — that is to say, tho posses- sion of cilia. As has been pointed out above, how- ever, a cilium is similar to a flagellum in every essential point of structure and function. There can be no doubt that the ciliary covering represents a large number of flagella specialized in respect to size, number, arrange- ment, and co-ordination. It has been mentioned abov3 that some flagel- lates, such as the Triclio- nympJiida and allied forms, are regarded by some authorities as transi- tional from the FJagellata to the Ciliata. It is per- haps improbable, how- ever, that the transition from the one group to the other should have been through endoparasitic forms ; and it is on the whole more likely that free- living forms, such as the holoniastigote genus MuUicilia, arc the nearest representatives of the earlier ancestral forms of the Ciliata. Two interesting forms have been described which combine in some respects the characters of both Flagollata and Ciliata. Maupasia paradoxa (Fig. 189, B) is described by its discoverer, Schewiakoff (863), as having the body metabolic, with cilia in the anterior part of the bodyi and tho remainder covered with long flagella. At the hinder end of the body is a longer flagellum implanted close beside the aperture of the efl'ercnt duct of the contractile vacuole. The mouth-opening, on the ventral side of CM Fig o, fl., flagellum ; micronucleus ; 189. — A, Monomastix ciliatus mouth N, macronucleus ; n C.V., contractile vacuole ; a., anus, near which opens the efferent canal of the contractile vacuole. After Roux, magnified 1,000. B, Maupasia paradoxa : as., cesoiAagus ; other letters as in A. After SchemaliofE, magnified 1,300. THE INFUSORIA 455 the body, leads into a short oesophagus. The nucleus is smgle, without a micronuclous. Sclicwiakolf makes Mawpasia the typo of a distinct order of the Oiliata— the Mastigotricha. Monomastix ciliaius (Fig. 189, A), described by Roux (862), and referred by him also to the Mastigotricha, has an oven coat of ciba all over the body, and possesses two macronucloi, near each of which is a micronuclous ; its most remarkable featiu-e is the possession of a long flagollum implanted at the anterior end of the body close to the mouth. Prom these various considerations, it seems highly probable that the Ciliata are descended from flagellate ancestors ; but it is not possible at present to indicate with any approach to exactness the line of descent. Subclass II. — Acinetabia (Suctoria, Tentaculifera). The Acinetaria are distinguished from the Ciliata by the posses- sion of the following characters in combination : The adult organism is of sedentary habit, and has no ciha, though the youngest stage in the life-history is typically a free-swimming ciliated organism ; there is no mouth, but both the capture and ingestion of food is effected by means of special organs peculiar to this subclass, and known as tentacles. An aciuetan may be attached to various objects, and is frequently epizoic. Some species attach themselves indifferently to a living or a lifeless object ; others are constantly epizoic, and occur always attached to some particular animal, frequently to a particular organ of it. Very few species, however, are truly parasitic in the adult condition ; on the other liand, many species are parasitic in the early larval stages of their life-history, and frequently so within the bodies of Ciliata (Fig. 192). The marine genus Ophryodendron, however, is a true ectoparasite of liydroids, accordmg to Martin, and contains nematocysts derived from its hosts. In this case the parasitism is correlated with a peculiar dimorphism of " proboscidi- form " and " vermiform " individuals, the former possessing a tuft of tentacles on a proboscis-like process, the latter being without tentacles altogether. The vermiform individuals are budded from the proboscidiform, and either form can produce ciliated buds, which develop into proboscidiform individuals agam ; but the vermiform type does not grow into the proboscidiform. In Den- drosomides paguri, however, Collin (881) finds that similar vermi- form individuals become transformed into the tentacle-bearing form. In Bhabdophrya trimorpha, ectozoic on a Copepod {Gletodes longicaudatus), there are three forms of individuals— namely, in addition to tentaculated and vermiform specimens, peculiar " unci- form " individuals, which are also Avithout tentacles (Chatton and Colhn, 876). The form of the body varies greatly, but may be said to be typically vase-like, with or without a stalk or peduncle. In sessile forms the body is attached by a broad base to the substratum. In 456 THE PROTOZOA stalked forms the body is raised up from the point of attachment on a straight, non-contractile stalk of secreted substance, similar to that of many Vortioellids, and the animal as a whole may resemble in its general contours an Episiylis or other VorticeUid (Pigs. 10, 190). Collin (877) finds that the stalk consists of a sheath, a cor- tical layer thickest at the base, and a medullary substance stratified longitudinally to the longitudinal axis. The base of the stalk rests on a cushion of secreted substance— the portion which is first formed, and which is produced by a special organ of the larva comparable to the scopula (p. 441) of the Vorticellids. The body is often protected by a secreted house or theca, con- tinuous with the stalk in the pedunculate forms. In Astrophrya arenaria the house is built up of foreign particles of various sizes (Awerinzew). As in the attached ciliates, colonies may be formed of considerable size and extent, and of various forms. The non- pedunculate genus Dendrosoma produces spreading colonies, which bear a considerable resemblance to a polyi)-colony. The characteristic tentacles arc stiff j)rotoplasmic jDrocesses con- sisting of a parietal layer of ectoplasm in the form of a tube en- closing a canal containing fluid. The apex of the tentacle usually terminates in a sucker-like laaob ; suctorial tentacles (" Saugten- THE INFUSORIA 457 takel ") of this type arc always present. In the genus Evhelola there are present in addition prehensile tentacles ( Greiltentakel ), which end in a fine point. The exterior of the tentacle is clothed by a delicate peUicle, continuous with that of the body, and formnig in the suctorial tentacles a sheath or tube, from the end of which the sucker protrudes. The tentacles are slowly retractile. When expanded they appear homogeneous ; but in the process of retrac- tion they exhibit a spiral marking, due apparently to creases and folds in the pelHcle, and not to be interpreted as indicating the presence of myonemes. The tentacles are used for the capture of prey, which consists chiefly of cihates. As soon as the sucker-like extremity of a tentacle touches a ciliate it is held fast ; the substance of the prey is then slowly absorbed by the tentacle, and passes as a stream of granules down the axis of the tentacle. During this process the ciliate re- mains alive, Avith cilia movin gand contractile vacuoles pulsating, until about haK its substance is absorbed (Fihpjev). In the genus Rhyn- cheta there is but a single tentacle of great Fig. l9l.~Urnula epistylidis, epizoic on Dendro- . TT^^. 7 /T?- soma radians. ^, jB, Individuals with one or two length ; m Urnula (Fig. tentacles respectively ; 0, formation of a bud 191), one or two. Other (g) ; D, the same seen in transverse section genera bear usually passing through the bud and the macronucleus ° •'of the parent ; free-swimmmg larva ; J< , en- many tentacles, which larged view of the single tentacle, showing the may be distributed spii-al striation. After Hickson and Wads- 1 ji 1 1 worth (886). evenly over the body- surface, or, more commonly, occur in special regions of the body or are distributed in tufts and patches. In Dendrocometes the tentacles occur m bunches borne on branches or arm-like processes of the body-waU. Ishikawa describes in the larger prehensile tentacles of EpJielota huetschliana a system of filaments, consisting of fine threads running parallel to one another in pairs and continued into the body as far as its base. The filaments stain deeply with iron-hsematoxylin. According to Collin (877), each such pair of filaments is in reality the optical section of a fine tube. A suctorial tentacle, according to Collin, represents a deep invagination of the ectoplasm, opening at its innermost end into the endoplasm like the cytopharynx of a ciliate. The prehensile tentacles, on the other hand, are special formations of a different kind, simple cvaginations of the body-wall, pseudopodial in nature, and containing from one to three axial filaments, the number increasing with the age of the tentacle. THE PROTOZOA ii^^IiT,^'! ''^^^''^f — foJ^ osampio, a Paramecium— is captured by the tentacle tb piotoplasm streams down the tentacle to fonn a mass i.i t^ic cndonksm carr^^ l V l' 'T'"'' '^g*^^" ^till smaller ones, which are earned away by the cyelosis of the cndoplasm, and other masses of smaU size contmuc to be formed at the base of the tentacle. Round each of Jhcso lor the most part dissolved, bccommg reduced to a few granulations The c^nZt'ecu"f'^''' off fluid and diminishes in size, and the' contenJs^re eon formec L .nH " 'l'""' '^" ^^'^i^' JK'''^^'^' ^"^^^ '"^'^'^^^^ bodies are nnd nl' r '- Y] CO ourless bodies " which stain feebly mth nuclear staim, doirlT'^ ^'T P^°toPl^««^ of the prey ; " tinctin-bodies," staining deeply, and originatmg as described by Martin, from the chromatin of the prey ; and others, found in some acinetans, derived from the chlorophyll of green ciliates and algal spores devoured by the animal. If a Tocophrya bo starved the refrmgent bodies are slowly absorbed, and the protoplasm becomes quite clear (Pihpjev). Hence the refringent bodies tliat arise from the diges- tive vacuoles represent reserve-material ; there appears to be no defecation of indigestible residues. The nature and origin of the tentacles of acinetans have been much dis- cussed, and some authors have sought to derive them from cirri or cilia bchuberg (44) points out, however, that the structure of the tentacles is quite tJae opposite of that of the cilia ; in a cilium the axial portion is of firm con- sistence, the superficial layer is fluid, while in a tentacle the axis is fluid and the superficial sheath is of firm texture. Collin (877) considers that the pre- hensile tentacles are modifications or adaptations of a pseudopodium-fike process ; on the other hand, he regards the suctorial tentacles as organs of quite a different kind, more like the cytostomo of a eiliate than anything else ; they may be considered each as a cytostome which has gro\\Ti out from the body on a slender process or stalk (compare also Hickson, 826). In correlation with their sedentary habits, the organization of the Acinetaria is greatly simphfied as compared with the Cihata, and the remarkable structural and functional differentiation of the ectoplasm seen in the Ciliata is wanting altogether in Acinetaria, in which the ectoplasm is relatively a feebly-developed layer. Con- tractile vacuoles are usually present, one or more in number. As in Ciliata, the macronucleus exhibits a great variety of forms. One of the most remarkable is seen in the colonial form Dendrosoma, where the macronucleus is branched to the same degree as the colony, throughout which it extends continuously. The methods of reproduction are more varied, and exhibit a greater speciahzation, in the Acinetaria than in the Ciliata. Simple binary fission in the adult condition is rare in acinetans. Collin (881), hoAvever, has observed division into two or four within a cyst in Podophrya fixa. The fission usually takes the form of bud-forma- tion. The buds may be formed either on the exterior of the body or. in the interior in special brood-cavities, and they may be pro- duced in either case singly and successively or in batclies or relays of several at a time. The bud is usually a simple outgrowth of the cytoplasm containing a prolongation budded off from the macro- nucleus, and one of tlie daughter-nuclei derived from a division of the micronucleus. At first a simple cell without structural differ- THE INEUSORIA 459 entiation, the bud is set free with a complete or partial coat of cilia as a free-swimming "embryo," "larva," "swarm-spore" (Schwiirmer), or " gemmula." The larva often becomes parasitic within the body of another Infusorian (Fig. 192), multiplying there by binary fission. Finally it becomes free again, swims away, attaches itself in a suitable locality, and develops into the adult form. The cihated larvae of acinetans exhibit various types of ciliation, commonly classed as peritrichous (Fig. 193, A, B), holotrichous (Fig. 191, E), and hypotrichous. Collin (882) has studied recently the morphology of the different types of larvae. A B Fio. 192. — A and B, StylonycJiia mytUus infested by parasitic Acinetans. N, N , Macronuclei of the Stylonycliia ; P, parasitic Acinetan embryo from wiiich arise small ciliated larvaj (c.e) which swim off and develop into the adult free-living Acinetan. After Stein. The most primitive and commonest larval type of larva amongst the Acinetaria is very similar to a free-swimming stage of a Vorticellid. It has a principal axis round which the body is radially symmetrical, with an upper pole (posterior in movement) bearing a rudimentary adoral zone, and a lower (anterior) pole bearing a mass of secretion or a sucker, indicating the future point of fixation and representing the scopula of the Vorticellid ; the body is surrounded by several rings of cilia forming a zone more or less equatorial in position. Such a form, while retaining its radiate symmetry, may become cither lengthened or shortened to a remarkable degree' in the direction of the principal axis ; in the elongated forms the rings of cilia may increase in number until they cover the whole body, thus producing the holotrichous type. On the other hand, the body may become elongated in the morpho- logically transverse plane, and acquire a bilateral symmetry, with a dorsal surface representing the primitive upper pole and bearing the rudimentary adoral zone, and a ventral surface, with the sucker in the middle of it, repre- senting the primitive lower pole ; secondary anterior and posterior extremities are now distinguishable in relation to progression, but representing opposite 460 THE PROTOZOA The conjugation of the Acinetaria conforms in general to the type of the process seen in CiHata, as regards cytological details. Conjugation may take place between two individuals fixed near Fig. 193.— Free-swimming larva of Dendrosoma radians. A, Side veiw ; B, viewed from above ; 0, older larva with the first rudiments of the tentacles beginning to appear. After Hickson and Wadsworth (886). together (Fig. 190, C) ; then a lobe or outgrowth may be formed from one individual, which meets a similar outgrowth from the other, thus establishing contact. On the other hand, as in Peri- tricha, conjugation may take place between a fixed, ordinary indi- vidual and a free-swimming bud or larva liberated from another adult individual (Martin ; Collin, 879). In Dendrocomeies the macronuclei come into contact during conjugation, but separate again (Hickson and Wadsworth). Classification. — The Acinetaria are divisible into eight families (c/. Doflein, 7). 1. Hypocomidce, for the single, somewhat aberrant genus Hypocoina, which is free-swimming, ciliated on one surface, and wnth a single suctorial tentacle — possibly a persistent larval form (see Collin, 877). 2. Urnulidai. — With or without a house, with, one or few tentacles.' Rhyn- cheta, Vrnula (Fig. 191). 3. Metacinetidai. — With a stalked house opening at the upper end for the exit of the tentacles. Metacineta. B C THE INFUSORIA 461 4 Podophrvid(B.-Sta\ked or sessile, with no house and with numerous tentacles SvlKBrophrya, Podophrya (Fig. 190), Ephelota. 5 ^c'neiS -Stalked or sessile, with a house of simple form and wide aperture S witli numerous tentaeles, all knobbed. Tocophrya (Fig. 190), "^t'DeZl^omldce (TrichophryidcB).-Sess\lo, without a house; tentacles knobbed Arranged in tufts or branches. TricJwphrya, Dendrosoma, Lernco- Sr^Perez), Bhabdophrya (Cliatton and Collin), Astrophrya (Awermzew). 7 Dendrocometidce.-m^t forms mth numerous branched arms on the ends of which the suckers occur. Dendrocometes. . . . .„„i„„ 8 OvhryodendridcB.-MixTmo stalked forms with numerous short tentacles concentrated on proboscis-like processes. Vermiform individuals also occur (p. 455). Ophryodendron. . ^ Affinities of the Acinetaria.— The presence of cilia in the young stages, the possession of distmct vegetative macronuclei and generative mieronuclei, and the process of conjugation, similar in all essential details to that of tJie binata, can leave no doubt as to the position of the Acinetaria in the class Infusoria, and their affinities with the Cihata. Collin, in a series of interesting studies, has drawn attention to many points indicating a close relationship between Acinetaria and Vorticellids, more especially the structural homologies between the larv£e of the one and the free-swimming stages of the other group ; for example, the peritrichous arrangement of the cilia, the rudimentary adoral zone at the posterior pole, and the fixation by means of a scopula like organ at the anterior pole, points especially well seen in the larva of Tocophyra cyclopum. Bibliography. — ^For references see p. 502. CHAPTER XVIII AFFINITIES AND CLASSIFICATION OF THE MAIN SUBDIVISIONS— DOUBTFUL GROUPS In the foregoing chapters the Protozoa have been dealt with systematically, grouped in a somewhat conservative manner under the four old-estabhshed and generally-recognized classes. At the same time it has been pointed out that one class at least-namely the exclusively-parasitic Sporozoa— comprises two subclasses which are quite distinct from one another, and are descended, in all probability, from ancestors diflfermg greatly in characters and aflanities. And in the case of the three remainmg classes, con- sisting mainly of free-living, non-parasitic forms, two which exhibit more primitive characters— namely, the Sarcodina and Mastigophora —are connected with one another by transitional forms which render the distinction between them very arbitrary (p. 213) ; while the third, the highly-speciahzed Infusoria, are linked closely by structural characters and by transitional forms to the Mastigophora. Many authorities on the Protozoa have put forward schemes of classification which are intended to express the affinities and inter- relationships of the chief groups in a clearer and more satisfactory manner than the fourfold classification generally recognized. The systems proposed have taken the form either of subdividing the Protozoa into more than four classes or of uniting the recognized subdivisions into a smaller number of categories. Eolleston and Jackson (15) divide the Protozoa as a whole into three groups: (1) the Rhizopoda (= Sarcodina) ; (2) the Endoparasita (=Sporozoa); and (3) the Plegepoda, " referring to their mode of progression by means of a rapidly - repeated stroke {irXr^yrj) of vibratile processes," to comprise the Mastigophora and Infusoria. Dofiein (891) recognizes two principal stems in the Protozoan phylum: (1) the Plasmodroma, to include the Sarcodina, Mastigophora, and Sporozoa, organisms that make use of locomotor organs which represent true pseudo- podia, or their derivatives or modifications ; and (2) the Ciliophora, comprising the Ciliata and Suctoria, in which the locomotor organellas are cilia. The obvious criticism of this scheme is that, whatever opiiiion may be held as to the desirability of drawing a line between the Infusoria, so highly specialized in many respects, and other Protozoa, the distinctive character chosen is not a happy one, since whatever may be predicated of flageUa as derivatives of pseu- dopodia applies, apparently, with equal force to cilia. 462 CLASSIFICATION OF THE MAIN SUBDIVISIONS 463 Hartmann (892) recognizes six classes of the Protozoa : Class I. the Sarco- dim including four subclasses-namely, Rlnzopoda, Hehozoa, Radiolaria, wl Mvcetozoit Class II., the Cnidosporidia, including M.crosporidia, Sarco ;ToridrM^o;poridia, ^nd Actinomyxidia ; Class III the Mastigophora. ffiudin- the Rhizomastigina, Protomonadina, Binucleata, Chromomonadina Eu^lenofdca, and Phytomonadina, the order Binucleata including the Haemo- flacn^Ilatcs and tho Hc-emosporidia with the exception of the hjemogregarmes Class IV., the Telosporidia, including the gregarines, coccidia, and hsemo- gregarincs; Class V., the Trichonymphida; Class VI., the Infusoria. With regard to this classification, the order Binucleata has been dealt with at length above ; it only remains to say that the isolated position given to the Trichonympliida appears to express the defective state of laiowledge con- cerning the affinities of these peculiar parasites, rather than their true taxo- nomic importance. . ^ , , -n. , A number of radical changes in the classification of the Protozoa are pro- posed by Awerinzew (890). With Hartmann he unites the Hsemoflagellates and Hfemosporidia in an order Binucleata to be placed in the Flagellata. The class Sporozoa is to be entirely abolished. The order Amcebma (Amcebfea) is removed by him entirely from the Sarcodina, which will then comprise only the Foraminifera and some Heliozoa. The Amoebina are to be put with the Flagellata as the Amoeboflagellata, a group from which all other Protozoa are supposed to have arisen, and from which the Amoebina branch off in one direction, tho Flagellata and Dinoflagehata in another. The gre- garmes are believed by Awerinzew to be connected on the one side with tho Amoebina, on the other with the Coccidia. In the Neosporidia, the Sarco- sporidia are regarded as allied to Flagellata ; the Myxosporidia, Mierosporidia, and Actinomyxidia, are considered not to be Protozoa at all ; the Haplo- sporidia are to be placed provisionally as an independent group takmg origm from Amoebma. For the Infusoria, it is suggested that they take origin from amoeboflageUate ancestors rather than from true Flagellata. The object of what is termed a natural as opposed to an artificial system of classification is to endeavour to express by the arrange- ment of the groups the affinities of the living organisms concerned, and more especially the genetic relationships of one to another on the theory of evolution — that is to say, on the assumption or belief that forms now existing are descended from older ancestral forms, and that any two existing forms are descended from a common ancestral form more or less remote, according as the two existing forms in question have diverged more or less widely from one another. The foundation of a natural classification is therefore the 'phylogeny of the groups dealt with — that is to say, their pedigrees and lines of descent, so far as they can be traced. Phylogeny must, however, always be a matter of speculation, and to a large extent of personal opinion, rather than of direct observation. It is only possible to infer from the study of existing species what the ancestral forms may have been like, since it is unnecessary to point out that no form can be the ancestor of another species existing at the same time. The most that can be said of two co-existing species is that one of them may be beheved to have diverged much less in its characters from the common ancestral form than the other. When, therefore, a given form is said to have an amoebic or a cer- comonad ancestry, it is not intended to imply by that statement 4B4 THE PROTOZOA that the ancestor was Amoeba proteus or Cercomonas crassicauda but only that it was a form such that, if it existed at the present day, It would be referred by its characters to the genus Amoeba or Cercomonas, as the case miglit be. The data for drawing phylogenetic conclusions in Protozoa con- sist entirely of comparisons between the structure and life-history of the various existing forms. Paleontology gives no assistance since only skeletons are preserved as fossils. All that can be learned from the geological record is that the differentiation of the main groups must have taken place at an immeasurably remote period of the earth's history, since skeletons of Foraminifera and Radiolaria— groups of which the structure and life-history indicate a long pedigree— are found in the earliest fossiliferous strata. It is little wonder, therefore, that the phylogeny of the Protozoa is a subject on which the most opposite opinions are held, as is apparent from the classificatory systems cited above. There can be no finality in a phylogenetic theory, nor, consequently, in any scheme of classification put forward. Both the one and the other express merely the state of current knowledge, and may be expected to undergo modification as knowledge advances. It is impossible to discuss here at length the phylogeny and classification of the Protozoa, and only a few guiding prmciples can be put forward. From a general survey of the phylum, it may be claimed fixst of all that the Protozoa constitute a compact group with definite characters, not a mere receptacle into which can be put anything and everything of microscopic dimensions which is not a bacterium, a fungus, or a parasitic worm, as some writers seem to think. Common to all Protozoa in at least the principal stages of the life-cycle is the differentiation of the body into distinct nucleus and cytoplasm — that is to say, the possession of that type of organization to which I have proposed to restrict the appUcation of the term cell. Doubtless there are, or have been, transitions from this type to the simpler grade of organization characteristic of the bacteria and allied organisms, but such transitions must be sought for outside the phylum Protozoa. The essential unity and homogeneity underlying the innumerable differentiations of form and structure in the Protozoa may be taken to mean that the phylum as a whole is descended from a common ancestral form, and the first problem is, then, to attempt to form some notion of what the ancestor was like. In dealing with the more specialized forms, such as those constituting the Infusoria or the two principal subdivisions of the Sporozoa, it has been pointed out that each group appears to be derived either from flagellate or sarcodine ancestors. In reviewing the Mastigophora and Sarcodina, it was further pointed out that, greatly as the typical representa- CLASSIFICATION OF THE MAIN SUBDIVISIONS 465 tives of the two classes may differ, there are forms of which the systematic position is quite arbitrary. In such a form as Pseudo- spora, it becomes almost purely a matter of opinion or taste which phase of the life-cycle is to be regarded as the " adult " form determining the class in which the genus is to be placed. Thus, all paths of evolution in Protozoa ajjpear to lead back- wards to one or the other of the two forms that occur so frequently in the actual development as the earliest phases — the amoebula and the flagellula. Most of those who have speculated on the phylogeny of the Protozoa have, consequently, regarded the an- cestral form of the phylum as one combining amoeboid and flagellate characters. Biitschli (2) considered that the Rhizomastigina re- present more nearly than any other existuig group the primitive type of Protozoon. Since then, however, the life-cycle of the mastigamoebae has been studied, and it is seen that the adult amoeboid form is preceded in development by a simpler monad form (p. 266, Fig. 112), which makes it very doubtful if the niastig- amoeba itself can be taken as a primitive type. Awerinzew (890) also regards an " amoeboflagellate " type as the primitive stock of Protozoa, which gave rise to all existing groups, and became differentiated into the Amoebina on the one hand, the Flagellata on the other. If an organism possesses two kinds of locomotor organs — pseudo- podia and flageUa — it is reasonable to suppose that a still more primitive and ancestral form would have possessed only one of these two kinds of organs. It has been seen that there is a gradual transition from pseudopodia to flagella, the intermediate type of organ being a pseudopodium (axopodium) with a firm, rigid, or elastic secreted axis. The question then arises, Which end of the series is to be put first, the flagellum or the pseudopodium ? Inas- much as flagella are found commonly in bacteria, it might be argued that they represent the most prunitive type of locomotor organella, and that a simple flagellate monad would represent most nearly the ancestral type of organization in Protozoa. Then it must be sup- posed that the formation of pseudopodia is a secondary character, acquired by the ancestral form, and the pseudopodia themselves would represent either simple outgrowths of the naked body (lobo- podia) or modifications of flagella (axopodia). Havmg regard, however, to the manner in which flagella them- selves arise— as simple outgrowths from the body— and to the fact that their structure and mode of action are apparently of a much more specialized type than those of pseudopodia, the conclusion seems irresistible that pseudopodia preceded flagella in evolution. We may, then, regard as the most ancestral type in the Protozoa a mmute amoebula-form, in structure a true cell, with nucleus and 30 466 THE PROTOZOA cytoplasm distinct, which moved by means of pseudopodia • but It must be supposed that some of the, pseudopodia very soon under- went modifications which resulted in the acquisition of true flagella and thus arose at a very early stage of evolution the flagellula or monad-form. In all probabihty these earliest monads were forms with an amoeboid body, most nearly represented at the present day by such forms as Cercomonas (Fig. 114) or the flagellulae of Mycetozoa (Fig. 98). From such forms arose the Sarcodina and then: derivatives (Neosporidia) by loss of flagella and speciahzation of the amoeboid form in the adult, and the Mastigophora and their derivatives (Telosporidia, Infusoria) by speciahzation of the flagellar apparatus combined with the acquisition of a cortex and loss of amoeboid movement. If the foregoing phylogenetic speculations be accepted, it is clear that in a natural classification of the Protozoa the Sporozoa must be abohshed as a class, and the two groups comprised in them must either be raised to the rank of independent classes or dis- tributed amongst the others— the Telosporidia placed near the Mastigophora, the Neosporidia near the Sarcodma. The primary subdivision of the Protozoa, if it is to represent the first branching of the ancestral stem, should be one which places on one side the Mastigophora, Telosporidia (better RhabdogeniEe), and Infusoria, on the other the Sarcodma and Neosporidia (better Amoebogenise). Beyond this point it is scarcely profitable at the present time to push phylogenetic speculations farther. In conclusion, two groups of organisms require brief mention — the Spirochaetes and the Chlamydozoa — suice by many authorities they have been referred to a position in or near the Protozoa. The Spirochjetes. , Under the name " spirochaetes " are grouped a number of or- ganisms, free-living or parasitic, with flexible bodies of slender, thread-like form, concerning the nature and systematic position of which a great deal of confusion has existed of recent years, due chiefly to conflicting statements with regard to the facts of their structure and methods of reproduction. The group comprises five principal types, regarded each as of generic rank : 1. Spirochceta sens, strict., a name given by Ehrenberg in 1833 to a relatively large, free-living form, S. 'plicaiilis. Other species of the genus have been described. For a full account, see Zuelzer (904). 2. Cristispira, a name proposed by Gross (897) for a number of species parasitic in the digestive tract or crystalline style of LameUi- branch molluscs, and characterized by the possession of a crest or CLASSIFICATION OF THE MAIN SUBDIVISIONS 467 ridge, commonly but wrongly termed an " undulating membrane," running the length of the body. The type of the genus is C. bal- hianii, originally named by Certes Trypanosoma halbianii, from the crystalline style of the oyster. 3. Saprospira, Gross (898), for free-living, saprophytic forms similar in structure to Grisiispira, but without the crest. 4. Spiroschaudinnia, the name proposed by Sambon for the many species of minute spirochaetes parasitic in the blood of verte- brates and in various invertebrates. Such are ;S^. recurrentis { = obermeieri), parasite of human relapsing fever; iS. dutloni, parasite of African relapsing fever ; 8. gallinarum of fowls ; 8. 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 Grisiispira, 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. 8. duttoni, for example, is transmitted by a tick-- Ornithodoros moM&ato— which lives in the mud-floors of huts or in the soil in spots where caravans camp habitually. The spirocha^tes 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. pallidum, the spkocha3te of syphihs 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 DobeU (895)-consider that W/^^f '''' vahd reason for drawing a generic distinction between Trevonema Tvpet 4 and r^ndT^"' ^^''^'l^^ ' recurrentis, etc." Gross combines lypes 4 and 5 mider the name Spironema proposed by Vuillemin • but gTnus T;:;r:r ^^^^^^^ tUto,etdrltz:d^^i The forms parasitic in the blood of human beings and other vertebrates seSTJ-/^ '""^^'^l^,^' S^^teria of the genus s/iriUum, or at iTast o the iTrl^rfr T"' „1^i^« ^'^cent years, and the diseases caused by them Mere spoken of as spmlloses. Tlio chief points of difference between tS theeggLhrt^fs%'e^::ltT " ^"l^P--^-*^-* - to say. passed through in 468 THE PROTOZOA apparatus with kmetonucleus and trophonucleus, and a locomotor apparatus witli fiapllum and undulating membrane., Schaudiim further constructed a hypothetical form of " Urhcemoflagellat " connecting the spirocha;te 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 f omid realized in various groups of Protozoa as a transitory developmental condition (comparable somewhat to the gastrula- condition in the Metazoa), so also the spirochajte 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 monts wth regard to the structure of spirochajtes, and acknowledged that the trypanosome-type of structure was not to be made out in the minute parasitic spirochetes. Nevertheless, since his time the investigators of these organisms have been divided into two camps— those who hold fast to Schaudinn's theory of the spirochsetes as Protozoa, and those who class them with Bacteria, respectively ; it being generally assumed, for some unknown reason, that it they are not Protozoa they must be Bacteria, or vice versa. A third set of authorities compromise by placmg the spirochsetes in an intermediate position between the two groups. In considering the question of the affinities of the spirochetes, 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 itseK, continuing to do so mitil 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, nothmg 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 lirzysztalowicz 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 Leisliman (902) with regard to the development of S. duttoni in the tick. The spirochtete 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 spirocheetes again. The observations of Leishman have recently been fully confirmed by the in- vestigations on the development oi 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 Gristispira anodontm 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 aU cases, tlirough- 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. r „ , n Recently the structure of spirochtetes has been studied carefully by (.^ross (897, 898), Zuelzer (904), and Dobell (895), by means of proper cylological methods of technique. The results show a complete difference in every CLASSIFICATION OF THE MAIN SUBDIVISIONS 469 respect between spirochaetos and trypanosomes and other flagellates. In the M'ords of Dobell, " the nuclear and cytoplasmic structures are wholly different ; a trypanosomc has a flagollum, a spiroclitcte has none ; the crista is not an undulating membrane ; the cell-membranes arc not similar ; and, moreover, the method of division is quite different in the two organisms." Doflein (7) places the spirochsetes 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 Spirochceia Fig. 194. — Stages in the division of Cristispira peclinis. A, D, Two successive stages of the incurvation ; C, incurvation complete ; D, division of the body at the point where it is bent back ; E, F, separation of the two daughter- spirochastes. After Gross (897). {sic)," from which it would appear that he regards the spirochsetes as the last product of the hne of evolution that produced the trypanosonies and allied forms. For the various reasons that have been set forth above, it appears impossible to include the spirochsetes 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— ^■.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 Cyanophycese, 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 Avhat diseases are to be referred to Chlamydozoa. According to Hart- mann (909), undoubted chlamydozoal 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 ceU-inclusions in the infected cell. In order to maderstand 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 ceU-inclusions in the infected ceUs 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 ceU-inclusions were identified as the actual parasitic organisms causing the disease. They received zoological names, were referred to a defijiite 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 Cytoryctes ; of hydrophobia, Neuroryctes ; 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 J'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 otlier 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 vaccme-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, Avhich, 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 Gytoryctes 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 Jiydrophohi(B, which he regards as a true Protozoon, and which Calkins refers also to the Rhizopoda. Siegel (914) describes under the name Gytorhyctes organisms of a type perfectly different from those described by Calkins. He distinguishes four species— C?/to- rJiyctes vaccinice of vaccine and smallpox, G. luis of syphilis, G. scarla- tinoe of scarlet fever, and G. apMMrum 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 mauitained 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 (Gytoryctes) 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 behef that the true parasitic organism in these diseases consists of certain minute bodies— the Clilamydozoa or strongyloplasms * The chief cliaracteristics of the Chlamydozoa, according to ProAvazek and Lipscliiitz (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 the division of a centriole. Tavo dots are seen con- nected by a fine line like a centrodesmose, Avhich becomes draAvn 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 containmg the minute bodies themselves. 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^°-l^^'>> ^ mantle), thus producing in the case of vaccine the characteristic Guarnieri's bodies, in which the parasites 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 class 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 vaccme-lyniph (vaccination) produces a transitory local disturbance which confers partial immunity against infection with variola. It does not seem to be quite clear whether the organisms 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 tropica, of Oriental Sore, and L. donovani, of Kala-azar. _No evidence has been brought forward as yet, however, to show that an infection with Oriental Sore confers any immunity against Kala-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 Amceba 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 Garyoryctes. No Chlamydozoa are known, however, to occur as free-living, non- parasitic organisms, but this circumstance may be due to their extreme minuteness ; the species known 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 THE PROTOZOA tint Vel^^^^^^^ " " ^^'^ --^fl-d nature, and when 1 'r'"^ ^ membrane; consequently, wnen the two halves travel apart in tlie process of division the substance of the body is draM.^ out into a conncctingZ- Id until I S surface tension overcomes its cohesion. On the otTer hand whfch ■ ^^^^^^^^ cell St re or of any other characteri tt which mdicate any affinity to the Protozoa. Their type of organiza- tion seems to be the simplest possible in a livii/Ldy-f n ere gram o chromatm without cytoplasm, and without a membrane of a simpler type of organization than any bacterium, and perhaps represent more nearly than any other kno.vn organism the sLples possible form of hvmg being. Bibliography. ~FoT references see p. 504 Ite domum, saturce, venii Hesperus, ite capdlce. BIBLIOGRAPHY The references to literature are numbered consecutively, but are grouped according to the chapters. An asterisk (*) attached to a reference indicates that the ivork in question contains fidl references to the previous literature of the subject. Memoirs in lohich 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 Record,''' published annually by the Zoological Society of London ; the last volume published up to date is that for 1910 / the volume for 1911 will 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. Archivos do Institute Bacteriologico Camara Pestana (Lisbon). A.I.P. Annales de I'lnstitut Pasteur (Paris). A.K.G.A. Arbeiten aus dem kaiserlichen Gesundheitsamte (Berlin). A.P.EL Archiv fiir Protistenkunde (Jena). A.S.T.H. Archiv fiir Schiffs- und Tropenhygiene (Leipzig). A. T.M.P. Annals of Tropical Medicine and Parasitology (Liverpool). A. Z.E. Archives de Zoologie experimentale et generale (Paris). B. A.S.C. Bulletin Internationale de I'Academie des Sciences a Cracovie. B.B. Biological Bulletin (Woods HoU, Mass.). B.C. Biologisches Centralblatt (Leipzig). B. I.P. Bulletin de I'lnstitut Pastern- (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 fiir Bakteriologie, Parasitenkunde und Infections- krankheiten (Jena). C. R.A.S. Comptes-rendus hebdomadaires des Seances de I'Academie des Sciences (Paris). C.R.S.B. des Seances et Memoires de la Societe 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 Instituto 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. Sitzungsberichto der koniglich-preussischen Akademie der Wissen- schaften zu Berlin. S.B.G.B. — der Gesellschaft naturforschender Freunde zu Berlin. Q^;?' T Gesellschaft fiir Morphologic und Physiologic in Miinchen. b.M.I. Scientific Memoirs by Officers of the Medical and Sanitary Depart- mcnts of the Government of India (Calcutta). ^ A ■ y^i'Jiandlungen der deutschen zoologischen Gesellschaft (Leipzig). zl.A. Zoologischer Anzeiger (Leipzig). Zja.P. Zeitschrift fiir allgemoine Physiologio (Jena). Z.H. — fiir Hygiene und Infectionskrankheiten (Leipzig). /.w.Z. — fur wissenschaftliche Zoologie (Leipzig). 475 476 THE PROTOZOA CHAPTER I General Works on Protozoa. *(2! iS^Sr''-J'?i8^2 iSof ' P P-asitologie. Paris : Masson et Cie. w!* E^elmamr''^''' ^'^^gl^>«h''"dc Anatomic, 1. Leipzig: *!S ^^nqOQS^- P • S'^^'J- The Protozoa. New York : Macmillan and Co. * r 77 Protozoology. New York and Philadelphia : Lea and Fiebigcr n^s^IekSefE^er' ^-^^^^ I *(7) DoFLEiN, r\ (1911). Lehrbuch der Protozoenkunde. Third edition. Jena- Gustav Fischer. (8) Haetog, 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 Jsogue. *(10) Lang, A. (1901). Lehrbuch der vergleichenden Anatomic der wirbellosen Ihiere, 2te Auflage. Jena : Gustav Fischer. (11) Lankester, E. R. (1891). Protozoa. Encydopcedia Britannica, 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. Earth. 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 : (IG) GooDEY, T. (1911). A Contribution to our ICnowledge of the Protozoa of the Soil. P.R.S. (B.), Ixxxiv., p. 165. (17) Lattteeboen, R. (1901). Die " sapropelische " Lebewelt. 2.^., xxiv., p. 50. (18) Laveran, a., and Mesnil, F. (1899). De la Sarcocystinc, toxine des Sarco- sporidies. C.R.S.B., li., p. 311. (19) — and Pettit, A. (1911). Les trypanotoxines. B.8.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) RouDSKY, D. (1910). Le Trypanosoma lewisi Kent renforce. C.R.S.B., Ixix., p. 384. (23) — (1911). La possibilite de rendre le Trypanosoma lewisi virulent pour d'autres rongeurs que le rat. G.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 Hutchinson, 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 Sarcosporidicn. A.P.K., xx., p. 97. (26) — and Braun, H. (1911). Ein Protozocntoxin (SarcosiJoridiotoxin). A.P.K., xxii., p. 351. (27) Wendelstadt and Fellmer, T. (1910). Einwirkung von Kaltbliiterpas- sagen auf Nagana- und Lewisi-Trypanosomcn. Zeilschr. f. Immunitdts- forschung, v., p. 337. (28) Winter, F'. W. (1907). Untersuchung iiber Peneroplia pertusus (Forskal). A.P.K.. X., p. 1. BIBLIOGRAPHY 477 CHAPTER III In addition to the general works cited under Chapter I., see especially : (29) Heeon-Allen, E.. and Eaeland, A. (1909). A New Species of Technitella. Journ. Quekett Microsc. Club (2), x. p. 403. n^w. in 7pllpn (30) KoLTZOFF N. K. (1903). Formbostimmende clastische Gebilde in Zeilen. E.G.. xkiii., p. 680. 1 •■ OCA (31) — (1906). Die Gestalt der Zelle. Arch, mtlcr. Jwai. Ixvii., p. ^64. (32) Pkowazek, S. v. (1908). Biologic der Zellen, I. ii.C. xxvm., p. 782. (33) (1909). Theorie der Cytomorphe. Z.A., xxxiv., p. liZ. (34) Rhumbler, L. (1898). Physikalische Analyse von Lebenserschemungen der Zelle, I. ' Arch. EntwicMungsmech., vii., p. 103. (35) _ (1902). Die Doppelschalen von OrbitoUtes. A.P.K., i., p. 193. (36) Vebwobn, M. (1888). Biologische Protisten-Studien. Z.w.Z., xlvi., p. 45o. CHAPTER IV In addition to Nos. 34 and 35, see : *(37) BuTSCHiJ, 0. (1894). Microscopic Foams and Protoplasm. (Translation by E. A. Minchin.) London : A. and C. Black. (38) Faure'-Feemibt, E. (1908). La Structure des Matieres Vivantes. B.S.Z.F., xxxiii., p. 104. *(38'5) — (1910). Les Mitochondries des Protozoaires et des CeUules sexuelles. Arch. d'Anat. Microsc, xi., p. 457. *(39) Fischer, A. (1899). Fixirung, Farbung und Bau des Protoplasmas. Jena : Gustav Fischer. (40) Rhumblee, L. (1902). Der Aggregatzustand und die pliysikalischen Beson- derheiten des lebenden Zellinhalts. Z.a.P., ii., ^d. 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) ScHTTBERG, 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) Araoao, H. de B. (1910). Ueber Polytomella agilis. M.I.O.G., ii., p. 42. (46) Awebinzew, 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 Gerebratul\t,s 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 Ai-ten parasitischer Ciliaten. M.I.O.G., iii., p. 130. *(49) Chatton, E. (1910). La structure du Noyau et la Mitose chez les Amcebiens A.Z.E. (5), v., p. 267. (50) Collin, B. (1909). La Conjugaison d' Anoplophnja branchiarum (Stein) {A. circulans, Balbiani). A.Z.E. (5), i., p. 345. *(51) DoBELL, C. C. (1909). Chromidia and the Bmuclearity Hypothesis. THE PROTOZOA (60) ^Fb™, £ (1810,. ■a,p„,„ ch,„„,die,, MitoohondHcs. (66) FEANyA, C, and Athias, M (1907) r^orr , a ,50, Gr=rrri?rsi^^^^ tiiie. A.P K xix p metachromatiques ou Grains de Volu- (6?) ^Tltm' (i^ODi: Poly-energide Kerne. B.C., xxix., pp. 481 and 491 62 _ ind CWs C°?f9\o %f ' P^f-t-¥me. Jena : G?.^stav FrscSer ifK-v, Ta ^- Flagellatenstudien. M.I.O.C ii r, 64 "Tp.l! x!7366 ^i-l^-'^oplast, Caryosom und^'cfe'ntrosom. (64) H™g/r.'(1898).' Kemtheilung, Richtungskorperbildung und Befruch- ^''^ ~ 'i^Xs^Stv^^i,:^^ Kemvermehrung bei Arcella ^aris. Ku,- ifivl ~ !!oo?!' ''J'^ Zelltheorie. A.P.K.. i., p. 1. ~ ivm.! p. 7^^' Wechselverhaltnis von Kern und Protoplasma. S.B.G.M.P., ~ iLv^^ixii?! p^^?9°°'''^'''^''^'P^'^* """"^ ^^"^ Dualismus der Kernsubstanzen. (69) Jahn E (1904). Kernteilung und Geisselbildung bei den Schwarmern von /'7n^ T DcMtec/i. £oi. Ges., xxii. p 84 (70) Janicki C. (1910) Parasitische Flagellaten, I. ' Lophomonas Ualtarum, Jj. striata. Z.w.Z., xcv., p. 243. (71) — (1911). Der Parabasalapparat bei parasitisohen FlageUaten. B.C.. XXXI. I p. i5^J.* ' (71-5) — (1912) Parasitische Arten der Gattung Paramceba. Verh. Naiur- jorsch. Ges. Basel, xxin. (72) Legeb, L., and Dtjboscq, 0. (1911). Deux Gregarines des Crustaces. A.Z.E. (5), VI., Notes et Revue," p. lix. (73) Maier H. N. (1903). Der feinere Bau der Wimperapparate der Infusorien. A.P.K., 11., p. 73. (74) Mesnil, F. (1905). Chromidies ct Questions connexes. B.I.P., iii., p. 313. (75) MiNCHiN, E. A. (1911). Some Problems of Evolution in the Simplest Forms oi Liie. Journ. Quekett Microsc. Club (2), xi., p. 165. (76) Naglee, K. (1911 ). Protozoen aus einem Almtumpel, I. Amaba liartmanni, n. sp. Anhang : Zur Centriolfrage. A.P.K., xxii, p. 56. 77) POPOFE M. (1909). Die Zellgrosse, ihi-e Fixierung und Versrbung. Arch. Zdljorschung , iii., p. 124. (78) Reichenow, E. (1910). Hcemogregarina stepanowi. Die Entwicklungsge- schichte einer Hamogregarine. A.P.K. xx. p 251 (79) Robeetson, M. (1911). The Division of the Collar-CeUs of the Calcarca Heteroccda. Q.J. M.S., Ivii., p. 129. (80) — and Minchin, E. A. (1910). The Division of the Collar-Cells of Clalhrina coriacea. Q.J. M.S., Iv., p. 611. (81) ScHAXJDiNN, F. (1896). Der Zeugungskreis von Parawia26« ejttard?. S.B.A.B., p. 31. Reprinted, Schaudinn's Arbeiten, 1911, p. 115. (82) — (1896). Das Centralkorn der Heliozoen. V.D.Z.G., vi., p. 113. (With discussion by Lauterborn and Biitsclili.) (83) Siedlecki, M. (1905). Die Bedeutung des Karyosoms. B.A.S.C., p. 559. (84) Wenyon, C. M. (1911). Oriental Sore in Baghdad, together with Observa- tions on a Gregarine in Stegomyia fasciata, the Hasmogrcgarines of Dogs, and the Flagellates of House Flies. Py., iv., p. 273. (85) ZuELZEE, M. (1904). Difflugia urceolata. A.P.K. , iv., p. 240. ■ (86) — (1909). Wagrierella borealis. A.P.K., xvii., p. 135. BIBLIOGRAPHY 479 CHAPTER VII In addition to the works cited horc, see also Nos. 45, 48, 49, 50, 56, 68, 60, 62, 64, 66, 69, 70, 71, 71-5, 78, 79, 80, 81, 82, and 86 above. (87) AraCtAO, H. de B. (1904). Amosba diplomitotica. M.I.O.G., i., p. 33. (88) AwEHiNZEW, S. (1904). Teilung von Amoeba proteus. Z.A., xxvii., p. 399. (89) Haetmann, M., and Chagas, C. (1910). Schlangenhamogregarinen. A.P.K., XX., p. 351. (90) — — (1910). Die Kernteilung von Amosba hyalina. M.I.O.C., ii., p. 159. (91) Heetwio, R. (1903). Korrelation von Zell- und Kerngrosse. B.C., xxiii., pp. 49 and 108. (92) — (1908). None Probleme der Zellenlehre. Arch. f. Zellforschung, i., p. 1. (93) Lebedbw, W. (1908). Trachelocerca phcenicoptej-us. A.P.K., xiii., p. 70. (94) MoROFF, T. (1908). Die bei den Cephalopoden vorkommenden Aggrecjata- Arten. A.P.K., xi., p. 1. (95) Nagler, K. (1909). Entwicklungsgeschichtliche Studion iiber Amoben. A.P.K., XV., p. 1. (96) — (1911). Caryosom nnd Centriol beim Teilungsvorgang von Chilodon uncinatus. A.P.K., xxiv., p. 142. (97) Prowazek, S. v. (1903). Die Kernteilung des Entosiphon. A.P.K., ii., p. 325. (97'5) Reichenovv, E. (1909). Hcematococcus pluvialis. A.K.G.A., xxxiii., p. 1. (98) ScHAtJDiNN, F. (1894). Kerntheilung mit nachfolgender Korpertheilung bei Ammba crystalUgera. S.B.A.B., 1894, p. 1029. Reprinted, Schaudinn's Arbeiten, 1911, p. 95. (99) — (1900). Der Generationswechsel bei Coccidien. Zool. Jahrbucher {Abih. f. AnaL), 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) SwARCZEWSKY, B. (1908). Die Fortpflanzungserscheinungen 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 fiS 74, 75, 81, 85, 92, 93, 99, and 101. ' ' (102) Baitsell, G. a. (1911). Conjugation of Closely Related Individuals of btylonychia. Proc. Soc. Exper. Biol. Med., viii. p 122 (103) BoTT, M. (1907). Fortpflanzung von Pdomyxa. A.P.K. 'viii v 120 (104) Calkins, G. N. (1904). Studies on the Life-History of Protozoa, IV. J ,M/,Zi., i., p. 423. * (105) — (1906). The Protozoan Life-Cycle. B.B. xi p 229 ^'''^-(Tiss 'a:^:k^i^' - (107) ^^J^.^^^^^^P^^- (1911)- La Conjugaison des Infusoires cilies. G.R.A.8.. linn! (^911). La Fecondation des Infusou-es cilies. G.R.A.8. clii r, 1703 (109) Dehorne, a (1911)^ Permutation nucleaix-e dans la Conjuga^soJ'de pidium colpoda. G.R.A.S., clii., p. 1354 (110) DOBELL^ C. C. (1911). The Principles of Protistology. A.P.K.. xxiii.. (111) DoFLEiN, F. (1907). Die Konjugation der Infusorien. 8.B.G.M.P.. xxiii.. ^'''^ ^IZS^-m: sessuaie negli (113) - aOO^. Die^Con^igation und sexuelle Differenzierung der Infusorien. ^'''^ ^\Sn^-' Sdon ^-^'^"^ «f Sex. Revised (115) Hamburger, C. (1908). Die Conjugation von Ster^tor ca^ruUus. Z.w.Z.. (116) Haetmann, k. (1909). Autogamio bei Protisten. A.P.K.. xiv., p. 264. ' THE PROTOZOA (117) M.^(1910).^ A^^^^^^^^^ Br. Hart.ann's " Autoga.io bei Proto- ''''' ^™'P^iS;.e^S^!i:^^^ '^-^^r. . (119) — a905). Das Problem der sexuellcu Diilerenzierung. V.D.Z.G., 1905 ''"Sc'i!S\, ^-Z-'- (122) MA^-|«^ K^(^1889).^LeEajeuni8sem^^ ehe. les Cili6s. (123) MuLsmv^k^l9n).^^Fo;tpflanzungserscheinungen bei MonocysUs roslrata. ^'''^ "^""S^^rf^ A Biometrieal Study of Conjugatiou in Paramecium. (125) PopoFF. M. (1908). Die GametenbUdung und die Conjugation von Car- chesium folyfinum. Z.w.Z., Ixxxix p 478 (126) P^^NDTL, H. (1906). Die Konjugation von Didinium nasutum. A.P.K., (127) PeowIzek, S. V (1905). Der Erreger der Kohlliernie, Plasmodiophora brassicm. A.K.G.A., xxii., p. 396. 77 (1907). Die Sexualitat bei den Protisten. A.P.K. ix v 2'> S ^nono^'"' I- Copulation von ^dmo^z-Z/^. S-iJ^J .B., p. 83. (IM) — (1902 . lirankheitserregende Protozoen, II. Plasmodium vivax. A. K.G.A., XIX., p. 109. nilol ~ !Jnn!l' ^^o^tpflanzung einiger Rliizopoden. J./f.G. J., xix., p. 547. (1^2) — (1904). Generations- und Wirtswechsel bei Tnjpanosoma und Spiro- chmte. A.K.G.A., xx., p. 387. Reprinted, with " Nachtrag," in Pritz bchaudinn s /l?-6ei• (366) Chatton, E. (1906). Les Blastodinides. O.R.A.S., cxliii. p 981 (367) — (1907). Nouvel Aper^u sur les Blastodinides [Apodinium mycetoides) O.R.A.S., cxliv., p. 282. (368) — (1910). Sur I'Existence de Dinoflagelles parasites coelomiques. Les Syndmium chcz les Copepodes pclagiques. O.R.A.S., cli. p. 654 (369) — (1910). Paradinium poucheti, Flagelle parasite' A'Acartia ' clausi. L.E.S.B., Ixix., p. 341. (.370) DoGiEL, V. (1906). Die Poridinion. Mitth. Zool. Stat. Neapel, xviii.. p. 1 (371) Dtjboscq, 0., and Collin, B. (1910). La Reproduction selaido d'uA Pro- tisto parasite dos Tintinnidos. O.R.A.S., cli. p 340 (372) JoLLOS, V (1910). Dinoflagellatonstudien. a'.P.K., xix., p. 178 ''''' ""^MlLtlSd, xlirfm • ' ""^^ ^^""^ theVtoflagellata. THE PROTOZOA ^''"^ ""'S; S.^'p/LT^- ^'y'^^'^'^y TriposoUma. Univ. CcUifornia Pm. \lm ~ !!on?!" k*''^^*"'"^ «f fo"2/a«^«^ f^r|:^^(19il). .Arzneifeste Mikroorganismen. I. T. lewisi. ,AAc^ TT ^•^■^■P-K- (I Abth. Ong.), Ixi., p. 102. (446) Hambtogee. C (1911). Einige parasitische Flagellaten. Vcrh. Heidelherq. JSaturhist.-Med. Ver. (n. F.) xi p 211 (447) Hartmann M. (1910). Eine w'eitere Art der Schizogonie bei ScMzotrypanum cruzi. A.P.K., XX., p. 361. ^ (448) HiNDLE, E. (1909). Life-History of T. dimorphon. Univ. California Pull. Zdool., VI., p. 127. ~ (1910). Degeneration Phenomena of T. gambiense. Py., iii., p. 423 (450 — (1910). A Biometric Study of y. firawiftiewse. /6icZ., p. 455. (4ol) — (1911). The Passage of T. gambiense through Mucous Membranes and Skm. Ibtd., iv., p. 25. (452) JoLLOS, V. (1910). Bau und Vormehrung von Trypanoplasma helicis. A.P.K., xxi., p. 103. (453) Keysselitz, G. (1904). Trypanophii grobbeni. A.P.K., iii., p. 367. (454) — (1906). Generations- und Wirtswechsel von Trypanoplasma horrdi. A.P.K., vii., p. 1. (455) — (1907). Die undulierende Membran bei Trypanosomen und Spirochiiten A.P.K., X., p. 127. (456) — and Mayer, M. (1908). Die Entwicklung von T. brucei in Olossina fusca. A.S.T.H., xii., p. 532. (457) Kleine, F. (1909). Positive Infectionsversuche mit T. brucei durch Olossina palpalis. Deutsch. Med. Wochenschr., xxxv., p. 469. Die Entwicklung von Trypanosomen in Glossinen. Ibid., p. 924. Die Aetiologie der Schlafkrankheit. Ibid., p. 1257. Tsetsefliegen und Try- panosomen. Ibid., p. 1956. (458) — (1910). Trypanosomenbefunde am Tanganyika. Ibid., xxxvi., p. 1400. (459) — and Taute, M. (1911). Ergiinzungen zu imseren Trypanosomenstudien. A.K.G.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 naoh Ostafrika entsandten Kommission. A. E.G. A., xxxi., p. 1. (461) Laveran, a. (1911). Identification et Qassification des Trypanosomcs des Mammiferes. A. I. P., xxv., p. 497. (462) — (1911). Les Trypanosomcs, ont-ils des Formes latentes chez leursHotes vertebres ? O.R.A.S., cliii., p. 649. (464) — and Mesnil, P. (1902). Des Trypanosomcs des Poissons. A.P.K., i. p. 475. (465) — and Pettit, A. (1910). Des Trypanosomcs du Mulot et du Campagnol (T. grosi et T. microti). G.R.S.B., Ixviii., p. 571. (466) (1910). Le Trypanosome du Lerot {Myoxus nilda) et la Puce qui parait le propager [T. blanchardi). Ibid., p. 950. (467) Lebailly, C. (1906). Les Hdmatozoaircs parasites des T616ostecns matins. Arch. Parasitol., x., p. 348. (468) Lebedefp, W. (1910). T. rotatorium, Gruby. Ilerlwig's Festschrift, i., p. 397. (469) Machado, a. (1911). Zytologische Untersuchungcn iibcr T. rotatorium, Gruby. M.I.O.C., iii., p. 108. (470) Manteufel (1909). Studien iiber die Trypanosomiasis der Ratten. A.K.O.A., xxxiii., p. 46. BIBLIOGRAPHY 491 (471) Martin, C. H. (1910). Trijpanoplasma congeri — I. The Division of the Active Form. Q.J. M.S., Iv., p. 485. (472) Maktin, G., Lebcetjf, A., and Roubatjd, E. (1908). Transmission du " Nagana " par les Stomoxcs ct les Moustiques. B.S.P.E., \., p. .35.5. (473) Mathis, C, and Legeb, M. (1911). Parasitologic ct Pathologie humaines et animales au Tonliin. 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 Guyana. G.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., lii., 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 (Ceratophyllus fasciatus). P.R.S. (B.), Ixxxii., p. 273. (481) (1911). Transmission of T. ^ewm by the Rat-Flea. Brit. Med. 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.S., Iv., p. 113. (484) Moore, 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 Hindle, E. (1908). Life-History of T. lewisi. A.T.M.P., ii., p. 197. *(487) Neresheimee, E. (1911). Die Gattung Trypanoplasma. Vide Prowazek (14), p. 101. (488) Neumann, R. 0. (1909). Protozoische Parasiten im Blut von Meeresfischen. Z.H., Ixiv., p. 1. (489) NovY, F. G., and McNeal, W. J. (1905). Trypanosomes of Birds. Journ. Infect. Diseases, ii., p. 256. (490) and Torrby, H. N. (1907). Trypanosomes of Mosquitoes and Other Insects. Ibid., iv., p. 223. (491) Ottolenghi, D. (1908). T. IruceiundT. equinum. G.B.B.P.K. (I. Abth. Orig.), xlvii., p. 473. (492) — (1909). Die Entwicklung einiger pathogener 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 Trypanosomes. J.H., v., p. 191. (495) — and Avari, C. R. (1909). On the Seasonal Prevalence of T. lewisi in Mus rattus and in Mus decumanus. Pi/., ii., p. 305. (496) PoLicAED, A. (1910). Sur la Coloration vitale des Trypanosomes. G.R S B Ixviii., p. 505. (497) Prowazek, S. v. (1905). Studien fiber Siiugetiertrypanosomen. A.K.Q A- xxii., p. 351. (498) (1909). Kritische Bemerlmngcn zum Trypanosomenproblem. AST H xni., p. 301. • • •. (499) Robertson, M. (1906). Certain Blood-inhabiting Protozoa. Proc. R Phys. Soc. Edinburgh., xvi., p. 232. (500) — (1907). A Trypanosorao found in the Alimentary Canal of Pontobdella muncata. Ibid., xvii., p. 83. irnJ! ~ i!onnl- ^ifo-Cyclc of T. viltatm. Q.J.M.S., liii., p. 665. ~Sl/(rl!S^^^^ the Alimentary Tract of PontobdeUa (503) - 09U). ^jTransmission of Plagellatos living in the Blood of Fishes. Phil. (504) ^°°^^™J^E•\'w 'T.lewisimllcematopinusspinulosus. G.B.B.P.K. THE PROTOZOA f?nS g^f^^^^sc^, F. (1909). Trypanosomcn-studien. A.P.K. xv p 263 (o0(,) RouBATJD E (1909). Lcs Trypanosomes pathogcncs ct la oSsinapatdis (o08) H. (19^01). ^ La Fonction ot Eolation d« p;tit Noyau des Trypano- (500) Stephens, J. W. W., and Fantham, H. B. (1911). Peculiar Morphology of ?B )'"^SSif™° 2'8°^ ^ °^ Sleeping Sickness {T. rhodesiense). P.ks. (510) Stockman, s""(1910): A Trypanosome of British Cattle. Journ. Comp. wempeit^., xxiii., p. 189. (all) Strickland, C. (1911). Mechanism of Transmission of T. lewisi by the Rat- ■ -^eo'- ^0"»->i-. 1911, p. 1049. (512) - and Swellengrebel, N. H. (1910). On T. lewisi and its Relation to /ciov c ^®^tam Artiiropoda. Py.. iii., p. 436. (513) Stuhlmann, F. (1907). Die Tsetsefliegen (Olossinafusca und Gl. tacUnoides). A.K.tf.A., XXVI., p. 301. (514) Swellengrebel, N. H. (1909). Ban und ZeUteilung von T. gambiense e?Mi«W9?i. Tijdschr. Ned. Dierk. Ver. (2), xi., p. 80. inirl ~ /in ^i^ation and Staining of T. lewisi. Pij., iii., p. 226. YK^nl ~ * 1 J- Noi'^nal 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 1 . 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) — (1911). Transmission of T. leivisi by Rat-Fleas. Three New Hcrpeto- monads. Journ. Inject. Diseases, viii., p. 125. (521) Tatjte, M. (1911). Die Beziehungen dcr Glossina morsitans zur Schlaf- krankhoit. Z.H., Ixix., p. 553. (522) Thirotjx, 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. (5:6) Werbitzki, F. W. (1910). Blepharoplastlose Trypanosomen. G.B.B.P.K. (L 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 ccxlebs) and the Redpoll (Linota rufescens). Q.J. M.S., Iv., p. 641. (528) Yakimoef, W. L., Kohl-Yaki.\ioef, N., and Korssak, D. W. (1910). T. korssaki of Mus agrarius, Piroplasmoses of Mus agrnrius, Reindeer, Yak, and Bears. G.B.B.P.K. (I Abth. Orig.), Iv., p. 370. (529) Zttpitza, M. (1909). Die Vogel- und Fischtrypanosomen Kameruns. A.8.T.H., xiii., Beiheft 3, p. 101.. (c) Crithidia, Leptomonas, Herpetomonas, etc. See also No. 84. (530) BoDET, G., and Rotjbaud, E. (1911). La Presence au Dahomey et Trans- mission du Leptomonas davidi. G.R.8.B., Ixx., p. 55. (531) Chatton, E. (1909). Un Trypanosomide nouveau d'une Nycteribie, et les Relations des Formes Trypanosoma, Herpetomonas, Leptomonas et Grithidia. G.R.S.B., Ixvii., p. 42. (532) — and Alilaire, E. (1908). Coexistence d'un Leptornonas et d'un Trypano- soma chez un Muscide non vulnerant, Drosophila confusa. G.R.S.B., Ixiv., p. 1004. (533) — and Leger, A. (1911). Eutrypanosomes, Leptomonas et Leptotrypano- somes chez Drosophila confusa (Muscide). G.R.S.B., Ixx., p. 34. (534) (1911). Quelques Leptomonas de Muscides et leurs Leptotrypano- somes. Ibid., p. 120. (535) Ditnkerly, J. S. (1911). Life-IListoTy ot Lept. musccB-domesticce. Q.J. M.S., Ivi., p. 045. BIBLIOGRAPHY 493 (536) Flu, P. C. (1911). Die im Darm der Stubenfliege vorkommendcn proto- zoiiren Gebildo. G.B.H.P.K. (I Abtli. Orig.), Ivii., p. 522. (537) Fkanca, C. (1911). L'Existence en Portugal de Lept. davidi dans le Latex de JEuphorbia peplus et E. segetalis. B.S.P.E., iv., p. 532. (538) — (1911). Notes sur Lept. davidi. Ibid., p. 669. (539) Geoegbwitch, J. (1909). Le dcvcloppemcnt de (?r« ^mene, ii., p. 113. ^ cSx^a/r*^- TyP^r^r^^ ^^""^ SPI^^'^ I'^^*"^^^^ P^tes of (677^ nqo?f ^^r^'^A^,''^ Kala-Azar. Q.J.M.S.. xlviii., p. 367. ^0/7) - (1907). The Miiroy Lectures on Kala-Azar. Brit. Med. Journ.. irn&\ T> ^^^^^^y 23, March 2 and 9. flow, R- (1909). Development of the Parasite of Oriental Sore in Cultures. y^.J.M.b., Im., p. 747. (579) THmoux, A., and Teppaz, L (1909). La Lymphangite epizootique des /^Q.^^ t7 ^^^^^^""^ ^1 Senegal. ^./.P., xxiii., p. 420. ' ^ ^ ^ (080) ViSENTiNi A. (1910) La Morfologia ed il Qclo di Sviluppo deUa Leishmania. ,-o■,^ d. Chn. Med. d. R. Univ. Roma. (081) 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. O.R.ii.B., Ixx., p. 130. (582-5) DuNKERLY, J.S. ( 1912). Thelohania and Prowazekia in Anthomyid Flies. G.B.B.P.K. (I Abth. Orig.), Ixii., p. 136. (583) Habtmann, M. (1911). Die FlageUatenordnung Binucleata und die Gattung Prowazekia. A.P.K.. xxii., p. 141. (584) Martini, E. (1910). Pr. cruzi und ihro Beziehungen zur Atiologie von ansteckenden Darmkrankheiten zu Tsingtau. Z.H. Ixvii. p 275 (585) Naegler, K. (1910). Pr. jiarva. A.P.K., xxi., p. 111. (586) Walker, E. L. (1910). Trypanoplasma ranoe. Journ. Med. Research, xxiii. (n.s. XVIII.), p. 391. (587) Whitmore, E. R. (1911). Pr. asiatica. A.P.K.. xxii., p. 370. CHAPTER XIV SPOROZOA— TELOSPORIDIA (a) General Works. *(588) Hagenmulleb (1899). Bibliotheca Sporozoologica. Ann. Mus. Nat. Hist. Marseille (2), i. *(589) Minchin, E. A. (1903). The Sporozoa. A Treatise on Zoology (Laukester) (London, A. and C. Black), p. 150. (590) Woodcock, H. M. (1910). Sporozoa. Encydcp. Brit., eleventh edition, XXV., p. 734. Coccidia. Ibid., vi., p. 615. Gregarines. Ibid., xii., p. 555. Hsemosporidia. Ibid., xii., p. 806. Endospora. Ibid., ix. p. 383. (b) Gregarines. See also Nos. 72, 84, and 123. (591) AwERiNZEW, S. (1909). Die Vorgiinge der Schizogonie bci Grcgarmen aus dem Darm von Aniphiporus sp. A.P.K., xvi., p. 71. (592) BEAtrcnAMr, P. de (1910). Uue Gregarine nouvclle du Geiu-e Porospora. G.R.A.S., cli., p. 997. (593) Berndt, a. (1902). Die im Darme der Larve von Tenebrio ?no^«^ ^ ^^^^^"^^ J/^ff^hr. f. Infektionskrankheiten der Hatistiere, yiii. p 39 (753) Yakimoee, W. L., Stolnikoff, W. J., and Kohl-YakiW N (igil) h. Achromaticus vesperuginus. A.P.K. , xxiv., p. 60. ' (g) Incertse Sedls. (754) NicoLLE, C., J^^Manceaux, L. (1909). Un Protozoaire nouveau du Gondi. (755) Patella, V.' (1910)."' Corps de Kurloff-Demel dans quelques Mononucleaires du Sang des Cobayes. La Genese Endotheliale des LeucoZTMono nKa^ q ''''^'^''^^'i^ ^ang (Siena, Imprimerie St. Bernardin) p 211 (756) Seidelin, H (1911). Protozoon-like Bodies in Yellow-Fever Patients Journ. Pathol. Bacterial., xv., p. 282 fatients. ~p?229; B^letin, i., CHAPTER XVI SPOROZOA— NEOSPORIDIA A. Cnidospoeidia (a) General Works. (758) AXTERBAOK, M. (1910). Die Cnidosporidien. Leipzig : Werner Klin khardt. . (b) Myxosporidia. (759) Awer™w^_ S. (^1909). Die Sporenbildung bei Ceratomy.a drepanopsett.. ^'''^ ~ %''pT^S S'''"" ^^"^•^'^^-^ Oottus (?62! So™' F W\ ' ""^"^-^ P-rado.a. O.B.A.S., elii., p. 631. ^ ^ £%.'), xi.,^r28i. ^ool. JahrbUcher (Ibtk. f. Anat. u. 500 THE PROTOZOA (763) Gp(19J8)- Die Entwicklung von Myxobolus pfeifferi. I. and IL (764) ^^^J;^ il^^^^^^^^ (1906). La Paroi sporale dcs Myxosporidies. (765) (1907). Coccomyxa morovi. C.R.A.S. cxlv p 85 (7C5-5) Meecier, L (1910). La sexualite chez les Myxo;poridies et chez les Micro sporidies. Acad. Boy. Belgique. Mim. 8,o. (2), ii., No. 6 (766) Plehn, M. (1904). Die Drehkrankheit der Salmoniden (Lentospora cere- Oralis). A.P.K., v., p. 145. (767) ScHRODEK, 0. (1907). Entwicklungsgeschichte der Myxosporidien. bphasromyxa labrazesi (sabrazesi). A.P.K., ix., p. 359. (768) — (1910). Die Anlage der Sporooyste (Pansporoblasten) bei Sphceromyxa sabrazesi. A.P.K., xix., p. 1. > f j (c) Actinomyxidia. (769) Catillery, M., and Mesnil, F. (1905). Sphceractinomyxon stolci. A.P K vi., p. 272. (d) Microsporidia. (770) AwEEiNZEw, S., and Feemor, K. (1911). Die Sporenbildung bei Glugea anomala. A.P.K., xxiii., p. 1. (771) Chatton, E., and Kkemef, A. (1911). Les Protistes du genre Octosporea. B. S.Z.F., xxxvi., p. 172. ■ (772) Hesse, E. (1904). Le Developpement de Thelohania legeri. O.R.S.B. Ivii., p. 571. (773) — (1905). Myxocystis mrazeki. O.R.S.B., Iviii., p. 12. (774) LiSGER, L., and Duboscq, 0. (1909). Perezia lankesterim. A.Z.E. (5), i., Notes et Revue, !>. Ixxix. (775) — and Hesse, E. (1910). Cnidosporidies des Larvos d'Ephemerds. C. R.A.S., cl., p. 411. (776) Meecier, L. (1908). Neoplasie du Tissu Adipeux chez les Blattes Parasitees par une Microsporidie. A.P.K., xi., p. 372. (777) Mrazek, a. (1910). AufPassung der Myxocystiden. A.P.K., xviii., p. 245. (778) Perez, C. (1904). Une Microsporidie parasite du Garcinus mwnas. C.R.S.B., Ivii., p. 214. (779) — (1905). Microsporidies Parasites des Crabes d'Arcachon. Bull. Stat. Biol. Arcachon, viii. (780) — (1908). Duboscqia legeri. C.R.S.B., Ixv., p. 631. (781) Schroder, 0. (1909). Thelohania chcetogastris. 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) Stempell, W. (1904). Nosema anomalum. A.P.K., iv., p. 1. (785) — (1909). Nosema bombycis. A.P.K., xvi., p. 281. (786) — (1910). Morphologie der Microsporidien. Z.A., xxxv., p. 801. (787) Weissenberg, R. (1911). Einige Mikrosporidien aus Pischen (Nosema lophii, Olugea anomala, 01. Hertwigii). S.B.O.R., p. 344. (787-5) Woodcock, H.M. (1904). On Myxosporidia in Flatfish. Trans. Liverpool Biol. Soc, xviii., p. 126. (e) Sarcosporidia. See also Nos. 18, 25, and 26. (788) Betegh, L. v. (1909). Entwicklungsgange der Satcosporidien. C.B.B.P.K. (lAbth. Orig.), lii., p. 566. (788-5) Crawley, H. (1911). Sarcocyslis rileyi. Proc. Acad. Philadelphia, 1911, p. 457. (789) Darling, S. T. (1910). Experimental Sarcosporidiosis in the Guinea-Pig. J.E.M., xii., p. 19. , ^ , . „ , (790) Erdmann, R. (1910). Kern und metachromatische Korper bei Sarko- sporidien. A.P.K., xx., p. 239. (791) (1910). (Sarcocysiis wiMm in der Muskulatur. S.B.G.B., -p. 317. (792) FiEBiGER J. (1910). Sarkosporidien. Verh. Zool.-Bot. Oes. Wien. Ix., p. (73). BIBLIOGRAPHY 501 (793) Laveran, a., and Mesnil, F. (1899). La Morphologic des Sarcosporidics. G.R.S.B., li., p. 245. (794) Neore, L. (1910). Le Stadc intestinal do la Sarcosporidia do 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., Ihid., Iv., p. 373. (797) Teichmann, E. (1911). Die Teilungen der Keime in der Cyste von Sarco- cystis tenella. A.P.K., xxii., p. 239. (798) VtriLLEMiN, P. (1902). Sarcocystis tenella. G.R.A.S., cxxxiv., p. 1152. (799) Watson, E. A. (1909). Sarcosporidiosis : Its Association with Loco- Disease, etc. Journ. Comp. Pathol. TherapeuL, xxii., p. 1. B. Haplosporidia. (800) Beattie, J. M. (1906). Rhinosporidium kinecdyi. Journ. Pathol. Bacteriol., xi., p. 270. (801) Cattllery, M., and Chappellier, A. (1906). Anurosporidium pdseneeri. O.R.S.B., Ix., p. 325. (802) — and Mesnil, F. (1905). Les HaiDlosporidies. A.Z.E. (4), iv., p. 101. (803) Chatton, E. (1907). Caullerya meanili. O.R.S.B., Ixii., p. 529. (804) — (1908). Blastulidium pcedophthorum. O.R.S.B., Ixiv., p. 34. (805) Crawley, H. (1905). Ccelosporidium hlatellce. Proc. Acad. Philadelvhia Ivii., p. 158. (806) King, H. D. (1907). Eertramia hujonis. Ihid., lix., p. 273. (807) Laveran, A., and Pettit, A. (1910). Une Epizootie des Truites. G.R.A.S. cli., p. 421. (808) MiNCHiN, E. A., and Pantham, H. B. (1905). Rhinosporidium kinealvi. Q.J.M.S., xlix., p. 521. ^ < f B (809) RiDEwooD, W. G., and Pantham, H. B. (1907). N eurosporidium cevhalo- disci. Q.J. M.S., li., p. 81. (810) Robertson, M. (1908). A Haplosporidian of the Genus Ichthyosporidium. Proc. R. Phys. Soc. Edinburgh, xvii., p. 175. (811) — (1909). - An Ichthyosporidian causing Disease in Sea-Trout. P.Z S 1909, p. 399. ' (812) Steimpell, W. (1903). Die Gattung Polycaryum. A.P.K., n., p. 349. ^A^^^N, E. (1906). Bertramia kirkmanni. Ami. Natal. Govt.' Mus. i. p 7 (814) Wright, J. (1907). Rhinosporidium kinealyi. New York Med. ' Journ ' December 21. C. iNCERTiE SeDIS. iaifil Ly^^Vhocystis johnstonei. A.P.K., xiv., p. 33.'^. (Sib; — (lyu). Die Entwicklungsgeschichte von Lymphocystis johnstonei. .ij. * -L * j\. . J XXII., p, ivy. (817) Chatton, E (1906). La Biologie, etc., Aqs Amcebidium. A.Z.E. (4) v Notes et Revue, p. xvii. ^ ' ~ ^"'^^Vordla perplexa. G.R.S.B., Ixii., p. 42. oon 7^ (1910). Gastrocystis gUruthi. A.Z.E. (5), v., Notes et Revue p cxiv (820) GiLRUTH,J. A.(1910). Gastrocystis gilruthi! Proc. Roy. Soc VictoriaTn.s.) xxiii., p, ly. ^ lloll ^- (1908). GapUlus intestinalis. Biologica, il, p. 1. (822) Kras^silstschik, J. M. (1909). Neue Sporozoen bei Infekten. A.P.K.. ^'''^ "^''S^s^tSvt'^p'L"'' Cl^ytridiopsis. A.Z.E. (5), i. ^'''^ ^""ZZo^, Siii"? p.^;f3'^- Trans. Biol. Soc. 502 THE PROTOZOA CHAPTER XVII INPUSORIA (a) General Works. (825) H™, M. (1910). I„ta„™. «„:,d„p. Brit., .leventh edition, sv. (b) Ciliata. 115'l2t°122°m'!vl^','i!; fj?',!^"'' 'i!'- »»• 104, 106.109, 111.113. o;2^^^™^^\f I^- (1911)- IcMhyopUhiriua multifiliis. A.P K xxi v 61 (829) Ca^lleey. m and Mesnil, F. (1§03). La Structure ntLt; d^n^lnfui ^S'?n^ m o^^^T > *^ Actimes (Fcettingeria actiniarum). C.R.S.B., Iv., p. 806 — ^^'^;;^^^^^^^^PP^^Yms'''^ {Rhizocaryum concavum). /Qoilf C- ^l^^^)- Infusoires astomes. (5), iii. p. 341 (831-5) Chatton E. (1911). Perikaryon czslicola and Conchovhrys davidom. Not'^s et Revue, p. viii. (832) Collin, B (1909). Deux Formes nouvelles d'Infusoires Discotriches. A. A. Ml. (5), 11. JNotes et Revue, p. xxi. (833) Dobell C. C. (1909). Infusoria parasitic in Cephalopoda. Q.J. M.S.. liii., (834) Fatxre-Feemiet, E (1905). L'Appareil 6xateur chez les Vorticdlidce. A.P.K., VI., p. 207. (835) — (1907). Mitochondries et Sph6roplastes chez les Infusoires cilies. U.R.S.B., Ixii., p. 523. (836) — (1908). Tintinnidium inquilinum. A.P.K., xi., p. 225. (837) — (1908). L'Ancijstropodiutn maupasi. A.P.K., xtii., p. 121. (838) — (1909). Le Maeronucleus des Infusoires cilies. B.S.Z.F.. xxxiv., p. 55. (839) — (1910). Le Mycterothrix tuamotuensis. A.P.K., xx., p. 223. (840) GoNDEE, R. (1905). Kemverhaltnisse bei den in Cephalopoden schmarot- zenden Infusorien. A.P.K., v., p. 240. (841) Hamburgee, C. (1903). Trachdius omim. A.P.K., ii., p. 445. (842) — (1904). Die Konjugation von ParamcBcmm ftMrsarm. ^.P.Z., iv., p.l99. (843) — and Buddenbeock, v. (1911). Nordische Ciliata mit Ausschluss der Tintinnoidea. Brandt and Apstein, Nordisches Plankton. (844) Joseph (1907). Kemverhaltnisse von Loxodes rostrum. A.P.K.. viii.. p. 344. (845) Kasanzeff, W. (1910). Loxodes rostrum. A.P.K., xx., p. 79. (846) KiEENiK, E. (1909). Chilodon hexastichus. B.A.S.G., p. 75. (847) KoFOiD, C. A. (1903). Protophrya ovicola. Mark Anniversary Volume, p. 111. (848) Legee, L., and Duboscq, 0. (1904). Les Astomata representent-ils un Groupe nature! ? A.Z.E. (4), ii.. Notes et Revue, p. xcviii. (849) (1904). Les Infusoires endoparasites. A.Z.E. (4), ii., p. 337. (850) Martini (1910). Uber einen bei amobenruhrahnlichen Dysentericn vor- kommenden Ciliaten. Z.H., Ixvii., p. 387. (851) Mast, S. 0. (1909). The Reactions of Didinium nasutum. B.B., xvi., p. 91. (851-5) Maupas, E. (1888). La Multiplication des Infusoires cilies. A.Z.E. (2), vi., p. 165. (852) Metoalf, M. M. (1907). Excretory Organs of O?)oitma. ^.P.Z., x., pp. 183, 365. (853) — (1909). Opalina : Its Anatomy, etc. A.P.K., xiii., p. 195. (854) Meuniee, a. (1910). Microplankton des Mors de Barents ct de Kara. Duo d'Orleans, Gampagne Arctique de 1907. Brussels. BIBLIOGRAPHY 50^ (855) MiTBorHANOW, P. (1905). La Structure, etc., dos Trichocystes des Para- mecies. A.P.K., v., p. 78. (856) Neresheimee, E. R. (1903). Die Hohc histologischer DiEferenzierung bei heterotrichen Ciliaten. A.P.K., ii., p. 305. (857) Nebesheimer, E. (1907). Die Fortpflanzung der Opalinen. A.P.K., Suppl. i., p. 1. (858) — (1908). Fortpflanzung eines parasitischen Infusors (Ichthyophthirius), S.B.Q.M.P., xxiii. (859) Prowazek, S. v. (1904). Der Encystierungvorgang bei Dileptus. A.P.K., iii., p. 64. (860) — (1909). Conjugation von Lionotus. Z.A., xxxiv., p. 626. (861) — (1909). Formdimorphismus bei Ciliaten Infusorien. ilf./.O.C, i., p. 105. (862) Roux, J. (1899). Quelques Infusoires cilies des Environs de Geneve. Rev. Suisse ZooL, vi., p. 557. (863) ScHEWiAKOFF, W. (1893). Die geographische Verbreitung der Susswasser- Protozoen. Mem. Acad. Imp. St.-Petersbourg (vii.), xli. (864) Schroder, 0. (1906). Campanella umhdlaria. A.P.K., vii., p. 75. (865) — (1906). Epistylis plicatilis. Ibid., p. 173. (866) — (1906). Vorticella monilata. Ibid., p. 395. (867) — (1906). Stentor ccervZeus und St. rceselii. A.P.K., viii., p. 1. (868) ScHUBOTZ, H. (1908). Pycnothrix monocystoides. Denkschr. Ges. Jena, ^ui., p. 1. (869) ScHWBYBE, A. (1909). Tintinnodeenweichkorper, etc. A.P.K., xviii., p. 134. (870) SiEDLECKi, M. (1902). VHerpetophrya 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 cydopum. A.Z.E. (4), viii., Notes et Revue, p. xxxiii. (879) — (1909). La Conjugaiaon gemmiforme chez les Acinetiens. O.R.A.S., cxiviii., p. 1416. (880) — (1909). Les Formes hypertrophiques et la Croissance degenerative chez quelques Acinetiens. G.R.A.S., cxlix., p. 742. (881) — (1909). Sur deux Acinetiens. Ibid., p. 1407. (882) — (1909). La Symetrie, etc., des Embryons d'Acmetiens. A.Z.E. (5), ii., Notes et Revue, p. xxxiv. (883) FiLiPjEv, J. (1910). Tocophrya quadripartita. A.P.K., xxi. p. 117 (884) Hartog, M. (1902). Notes on Suctoria. A.P.K., i., p. 372. (885) HicKSON, S. J., and Wadswobth, J. T. (1902). Dendrocometes paradoxus. Q.J. M.S., xlv., p. 325. (886) (1909). Dendrosoma radians. Q.J. M.S., liv., p. 141. (887) ISHiKAWA, C. (1897). Fine in Misaki vorkommende Art von Ephelota. Journ. Coll. Sci. Tokyo, x., p. 119. (888) Martin, C. H. (1909). On Acinetaria. Parts I. and II. Q.J MS liii p. 351. Part III. Ibid., p. 629. ' " (889) Perez, C. (1903). Lernoeophrya capitata. C.R.S.B., Iv., p. 98. THE PROTOZOA CHAPTER XVIII . (a) Classification. (890) A..^.^^,s. (19^10)^^ Die Stellung i. Systo. und die Klassifizierung der p.4i r akd No 675 ^^^^^"^ ^^r Protozoen. F», E), 447 (Fig. 187) Paramoeba, 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 coll, 403 Parthenogenesis, 137 Parthenogonidia, 267 Partial karyogamy, 126, 153, 453 Pathogenic, 19 — amoebte, 226 Paulinella, 214 Pearl-stage, 334 P^brine, 24, 411 Pectinelte, 442 Peduncle, 31 Pellicle, 32, 45, 435 Pelomyxa, 78, 144, 150, 205, 214, 227 — palustris, 227 (Fig. 91) Peltomyces, 243 Peneroplis, 15, 235 Peranema, 274 — trichophorum, 273 Peranemidai, 274 Peresia, 418 Perforate, 231 Peridiniales, 276 Peridinidw, 278 Peridinium, 278 — divergens, 278 (Fig. 122) Perldium, 241 Perikaryon, 439 Periplast, 45, 259 Peripylaria, 251, 255 Peristome, 433, 442 Peritricha, 433, 438, 440, 441, 442, 448 Peritrichous larvee, 459 Peritromidce, 440 Pcritromus, 440 Pernicious malaria, 358 Peroral membrane, 445 Phacus, 274 — triqueter, 274 (Fig. 118). PhEEnocystea, 412 Phteodaria, 256 Phteodium, 252 Phosphorescence, 201, 278 Phototaxis, 202, 205 Phylogeny, 463 Physarum didermoides, 242 Physodes, 244 Phytoflagollata, 274 Phytomonadina, 274 Phyiomyxince, 243 Piroplasma, 24, 357, 379, 393, 394 384, 385 (Fig. 162) — caballi, 379 Piroplasma canis, 382, 383 (Fig. 161), 384, 385 (Fig. 162), 387 — donovani, 393 — hominis, 379 Piroplasmoses, 378 Piroplasms, 378, 390 Plagiotomidm, 439 Planont, 398, 408, 413, 423 Planorbulina, 232 (Fig. 93, 9) Plasmodiophora, 243 — brassicw, 149 (Fig. 76), 243 Plasmodium, 100, 128, 240, 398, 423 Plasmodium, 357 — brasilianum, 364 — cynomolgi, 364 — diploglossi, 358 — falciparum, 358, 359, 360 (Fjg. 156) — inui, 364 — kochi, 364 — malarioB, 358, 359 — pithed, 359, 364 — prcBCor, 358 — relictum, 358 — vivax, 137 (Fig. 72), 358, 359, 360 (Fig. 156) — vassali, 364 Plasmodioma, 462 Plasmogamy, 128 Plasmotomy, 100 Plastin, 73, 103 Plastinoid gramiles, 41, 195, 340 Plastogamj, 128, 209 Plogepoda, 462 Pleistophora, 418 — longifilis, 413 (Fig. 171), 415, 416 — periplanetoe, 416 — species, 413 Pleodorina californica, 267 Pleuronema, 55, 439, 442 — chrysalis, 56 (Fig. 27) Pleuronemidm, 439 Podophrya, 461 — fixa, 456 (Fig. 190, C), 458 — gemmipara, 108 (Fig. 55) — mollis, 456 (Fig. 190, A) Podophryidw, 461 Polar bodies, 143 — capsule, 399 (Fig. 163) — cones, 117 — filament, 399 — masses, 110 — plates, 117 Polycaryum, 424 Polychromophilus, 364 Polycystid, 326 Polycyttaria, 256 Polycnergid nuclei, 121, 151. 255 Polykaryon, 121, 255 Polymastigidai, 272 Polymastigina, 271 Polymastigoto, 259 Polymastix, 272 Polymoiphism, 162, 163, 297. 311 Polyspora, 339 Polysporea. 409, 418 Polysporulea, 424 PolystomeUa, 210 — crispa, 139, 234 (Fig. 95), 235. 236 (Fig. 96) Polythalamous, 36, 232 Polytomella agilis, 86 (Fie. 43) Polytrema, 231 33 514 THE PROTOZOA Polytricha, 439 Polyzoic, 349 Poneraviceha, 224 Pontobddla muricala, 291, 303 Ponloviyxa flam, 218 Porospora, 337, 340 — gioantca.H (.Fig. 35), 336, 339 (Fig. 150) — legeri, 336 Porosporidm, 341 Pouchetia, 62 — cormita, 61 (Fig. 31) Prehensile tentacle, 457 Proboscidiform individuals, 455 Prohoscidium, 442 Prococcidia, 352 Proflagollata, 469 Promitosis, 109 Pronucleus, 127 Propagative eel), 405 — phase, 21, 166 Propulsive pseudopodium, 401 Prorocentracoee, 276 Prorocentrum, 278 Prorodon, 439 — teres, 32 (Fig. 14), 444 (Fig. 186, B, C), 446 Proteomyxa, 217, 268 Proteosoma, 358, 364, 365, 3.93 Protista, 4, 5 Protoblast, 426 Protococcacoije, 15 Protoentospora ptychoderce, 229 Protokaryon, 75, 87, 108 Protomorito, 327 Protomonadina, 270 Prolophrya, 452 — ovicola, 452 Protopliyta, 8 Protoplasm, 29, 40 Protozoa, 2, 10, 464 Prowazek's bodies, 470 Prowazekia, 260, 271, 281, 319, 321, 322 — asiatica, 319 — cruzi, 319 — parva, 319, 320 (Fig. 141) — weinbergi, 319, 320 (Fig. 141) Pseudochlamys-Btago, 170, 177 Pseudoplasmodida, 243 Pseudoplasmodium, 242 Peeudopodiosporo, 169 Pseudopodium, 30, 46, 90, 199, 214, 400, 465 Pseudospora, 213, 218, 249 Psorosperm, 165, 323 Pterocephalus, 173, 327, 329, 330, 339 — gracilis, 174 (Fig. 79), 332 (Fig. 145), 334 (Fig. 147) — nobilis, 339 Pulsellum, 52, 259 Pusulc, 277 Pycnothrix, 452 — monocystoides, 443, 446, 447, 452 Pyramimonas, 275 Pyronoid, 63,188, 261 Pyrodinium, 201, 278 Pyrosoma, 379 Pyxinia, 329, 330 Quartan malaria, 358, 359 Radiolaria, 218, 249 Radium-rays, oHects of, 205 Rainey's corpusclos, 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 Reservoir-vacuole, 262 Respiration, 195 Reticulosa, 217, 218 Reticuloso, 48 Reticuliun (nuclear), 75, 103 — (protoplasmic), 41 Rhabdogeniee, 325, 406 Phabdophrya, 461 — trimorpha, 455 Rhaphidiophrys, 245 Rhootaxis, 207 Rhinosporidium, 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 Rontgen-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. 170). — rileyi, 420 — tenella, 419, 420, 421 (Fig. 175) Sarcocyte, 327 Sarcode, 40 Sarcodina, 11, 213 Sarcosporidia, 20, 419 Scaiotricha, 440 Scarlet fever, 470 Schaudinnella, 355 Schewiakovella schmeili, 425 Schizocystidm, 339, 341 Schizocystis, 339 — gregarmoides, 336, 338 (Fig. 149) Schizogenea, 418 Schizogony, 166, 324, 392 SchizogrogarintE, 328, 339 Schizokinete, 373 Schizont, 166, 181, 324 Schlzontocyte, 344 Schizotrypanuvi, 285, 307, 392 — cruzi, 28, 295 (Fig. 128), 290, 30!i. (Fig. 132), 307 Schizozoito, 344, 428 Sclerotium, 166, 240 Scopula, 441, 456, 459 Scyphidia, 440, 441 Secondary nuclei, 66 INDEX Socrotion, 197, 19S Selenidiidco, 341 Selenidium, 339 — caullcryi, 336, 337 (Fig. 148) Selenococcidium, 352, 354 — intermedium, 344, 350 (Fig. 155), 351 Senility, 131, 135, 155 Sensory organs, 201, 446 Soparation-spindle, 104 Septate, 326 Serumsporidia, 425 Sox, 154 Sexual differentiation, 160, 170, 176 — phases of trypanosomes, 305 Shell, 33, 45, 232 (Fig. 93) SiedlecJcia, 339, 352 Silicoflagellata, 274 Sleeping sickness, 26 Smithia, 380 — microti, 380 Soma, 130 Somatic number, 143 Sorophora, 243 Sorosphmra, 243 Sorus, 242 Souma, 304 Spasmonome, 448 Species, 141, 162 Spermatocyte, 143 Spermatozoon, 125 Sphcoractinomyxon, 409 — stolci, 409, 410 (Fig. 170) Sphferollaria, 255 Sphcoromyxa sabrazesi, 404 (Fig. 167), 405 SphcorojjJirya, 461 Sphserozoa, 256 Sphere, 95 Spheroplast, 41, 448 Spicule, 36 Spindio (nuclear), 103 Spirigera, 442 Spirillacea, 467 Spirillar forms, 319 Spirillum, 467 Spirochceta, 466 ■ — plicatilis, 466 — ziemanni, 371, 468 Spirochsetes, 466 Spirochona, 440 Spirochonidce, 440 Spiroloculina, 232 (Fig. 93, 5) Spironema, 467, 469 Spironemacea, 469 Spiroschaudinnia, 467 — anserina, 467 — duttoni, 467, 468 — gallinarum, 467 — obermeieri, 467 — recurrentis, 467 Spirostomum, 196, 197, 208, 438, 439, 445 — amUguum, 431 (Fig. 180) Spongomonas, 270 — splendida, 84 (Fig. 41) — uvella, 85 (Fig. 42) Sporal residuum, 349 Sporangium, 240, 241 Spec, 165, 166, 323 Sporo-formation, 166 Sporetia, 150 Sporoblast, 325 Sporocyst, 165 Sporocyst-mothor-cell, 403 Sporoduot, 335 Sporogony, 181, 325 Sporomyxa, 243 Sporont, 166, 181, 325, 325 Sporoplioro, 242 Sporoplasm, 405 Sporozoa, 12, 323, 462, 466 Sporozoito, 169, 324 Sporulation, 122, 165, 166 Spumollaria, 255 StannomidcB, 238 Starvation, 195, 210 Stemonitis fiaccida, 82 — fusca, 240 (Fig. 98) Stemm-pseudopodium, 401 Stempellia, 418 — mufabilis, 418 Stenophora, 329 Stentor, 61, 202, 211, 437, 438, 439, 441, 445, 446 — cceruleus, 444 (Fig. 186, A, I) — niger 444 (Fig. 186, G) — roeselii, 10 (Fig. 8) StentoridcB, 439 Stephanosphwra, 267, 276 Stercomarium, 238 Stercome, 194, 233 Stigma, 61, 205, 262 Stomatophora coronata, 328 Streaming movements, 199 Strongyloplasmata, 470 Stylonychia, 438, 440 — histrio, 444 (Fig. 186, H) — mytilus, 211, 459 (Fig. 192) Stylorhynchus, 173, 329, 330, 339 — longicollis, 174 (Fig. 79), 327 (Fig. 142), 339 Suctoria, 455 Suctorial tentacle, 190, 456 Sulcus, 276 Surface-tension, 200 Surra, 26 Swarm-spore, 169, 396 Symbiosis, 15 Symbiotic algee, 197 Synactinomyxon, 409 Syngamy, 126, 438 Synkaryon, 127 Syphilis, 467 Syzygy, 330 Tachyblaston, 460 Tactic, 202 Tactile bristles, 443 — organs, 201 Tconiocystis, 327, 339 Taxis, 202 Technitella thompsoni, 34 Tcloblast, 426 Telomyxa, 418 — glugeiform.is, 418 Telosporidia, 325, 395, 466 Temperatm-e, effects of, 206 Tentaculif era, 455 Tertian malaria, 358, 359 Tost, 33 Testacoa, 217, 219 Teiramyxa, 243 Tctratrichomonas, 272 Totrazoio, 349 Thalamophora, 219 516 THE PROTOZOA TItalassicolla, 255 — pclaoica, 30 (Fig. 13) Thalassophysa, 255 Thccamoebre, 219, 229 2'fieileria, 379 — parva, 380, 382, 386 Thelohania, 418 — chcBtogasiris, 416 (Fig. 173) — conlefeani, 412 — mcenadis, 416 Thelyplasni, 129 Thormotaxis, 202, 203 Thigmotaxis, 207 Thyroid extract, effects of, 204 Tinctin-body, 458 Tintinnidm, 439, 441, 443, 447 Tocophrya, 461 — cyclopum, 461 — limhata, 460 — quadripartita, 210, 456 (Fig. 1£0 B) 460 Toddia, 387 — bufonis, 387 Tolerant, 21 Tonicity, effects of, 207 Total liaryogamy, 126, 453 Toxocystis homan, 426 Toxoplasma, 319, 387 — canis, 387 — cuniculi, 387 — gondii , 387 — talpcB, 387 Trachelidm, 439 Trachelius, 439 — ovum, 441, 448 Trachelocerca, 439, 448, 453 — phcenicopteriis, 120 (Fig. 66), 449, 450 (Fig. 188) Traclioma, 470 Tractellum, 52, 259 Trailing flageilum, 53, 260 Transmission of trypanosomes, 289 Transmutation of energy, 199 Treponema, 467 — pallidum, 467, 468 — pertenue, 467 Triad inomyxon, 409 Trichia varia, 241 (Fig. 101) Triohitcs, 442 Trichocyst, 46, 435, 447 (Fig. 187) Trichodina, 440, 441 Trichomaslix, 260, 271 Trichomonas, 17, 36, 56, 258, 260, 271 — eberlhi, 8 (Fig. 5), 36 — hominis, 272 — vaginalis, 272 Trichonympha, 276 — hertwigi, 276 Trichonymphida, 463 Trichonymphidw, 89, 276, 454 Trichophrya, 461 Trichophryidat, 461 Trichorhynchus, 446 Trichosphmrium, 51, 216, 229 — sieboldi, 73 (Fig. 34), 182 (Fig. 81) Trimastigamceba, 268 Tripylaria, 251, 256 Tritoblast, 426 Trizoic, 349 Trophic phase, 324 Trophochromatin, 71 Trophochromidia, 150 Trophonucleus, 78, 85, 280, 288 Trophozoite, 324 Tropical malaria, 358 Trypanomouad, 282, 298, 299 Trypanomorpha, 308 TrypanopMs grobbeni, 309 Trypanoplasma, 56, 78, 87, 260, 271 281 287, 308,309, 321,322 ' * — abramidis, 310 (Fig. 134) — borreli, 312 — congeri, 312 — dendrocceli, 309 — gryllotalpai, 309, 310 — gurneyorum, 78 (Fig. 36) — helicis, 309, 311, 312 — intestinalis, 312 — keysseliisi, 310 (Fig. 134) — rano), 319 — vaginalis, 309 Trypanosoma, 270, 280, 283, 308 320 321 ' , > — balbianii, 467 — blanchardi, 25 — bnicii, 19, 25, 2G, 27 (Fig. 12), 285, 291, 296, 305, 306, 308 — cazalboui, 304 — cruzi, 285, 295, 296 — cuniculi, 25, 26 (Fig. 11) — dimorphon, 304 — drosojihilm, 315 (Fig. 137) — duttoni, 25, 26 (Fig. 11) — elyomis, 25, 26 (Fig. 11) — equinum, 285, 305 — equiperdum., 22, 26, 285 — evansi, 26, 27 (Fig. 12), 285 — gambiense, 19, 26, 27 (Fig. 12), 285, 291, 296, 297, 303, 304, 305, 300 — gramdosuvi, 288, 297, 298 (Fig. 129) — grayi, 304 — hippicum, 285 — inopinatum, 290 — lewisi, 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) — noctuw, 59 (Fig. 30), 137, 144, 158. 283, 297, 305, 306, 308, 391 — pecaudi, 304 — percm (myonemoe), 58 (Fig. 28) — rabinowitschi, 25 — raicB, 291, 303 — remaki, 9 (Fig. 6) — rhodesiense, 26, 286 — rotatorium, 59 (Fig. 30), 283, 297, 307 — sanguinis, 283 — vittato}, 303 — vivax, 27 (Fig. 12), 287, 291, 304 Tryranosomes, syngamy, 136 TrypanosomidoB, 268, 270 Trypanotoxin, 20 Trypanozoon, 308 Ultramicrosoopic stages, 306 Unciform individuals, 455 Undulating membrane, 55, 2C0, 286, 287, 443 Undulina ranarum, 283 Unicellular, 1, 3 INDEX 517 Urceolarinco, 440 Urhasmoflagollat, 468 Vrnula, 457, 460 — epislylidis, 457 (Fig. 191), 460 UmuUdco, 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 individtials, 455 Vestibule, 433 Volutin, 68, 195, 289 Volvocidce, 267, 275 Volvox, 3, 131, 206, 267, 276 Vorlicella, 440, 441, 445, 446 V or licella microstoma, 172 (Fig. 78) — monilata, 446 VorticeliidcB, 440 Vorticellinco, 440 Waanerella, 48, 51, 92, 120, 245, 246, 248 — borealis, 93 (Fig. 48). 247 (Fig. 102) Wrong hosts, 291 Xonophya, 34, 238 Xenophyophora, 218, 237 Yaws, 467 Yellow cells, 252 Zoochlorellse, 15, 252 Zoomyxa, 243 Zoospore, 169, 262 Zoospm-idoB, 218 Zoothamnium, 440 Zooxanthellse, 15, 252 Zygote, 125 THE END BII-LINQ AND SONS, LTD , PKINTBR3, GUILDFORD MEMORANDA MEMORANDA 519 MEMORANDA Mr. Edward Arnold's List of Tecbnical & Scientific Publications Extract from the LIVERPOOL DAILY POST "During recent years Mr. Edward Arnold has placed in the hands of engineers and others interested in applied science a large number of volumes which, independently altogether of their intrinsic merits as scientific works are very fine examples of the printers' and engravers' art, and from their appearance alone would be an ornament to any scientific student's library Fortunately for the purchaser, the publisher has shown a wise discrimination m the technical books he has added to his list, with the result that the contents of the volumes are almost without exception as worthy of perusal and study as their appearance is attractive." The Dynamical Theory of Sound. By Horace Lamb, D.Sc LL.D., F.R.S., Professor of Mathematics in the Victoria University of Manchester, viii + 304 pages, 86 Illustrations. Demy 8vo I2S. 6d. net (inland postage 5d.). An Introduction to the Theory of Optics Bv ^t''?"r?T^''"'''!^''"/.^.^-V^'^-°-' ^•^•S- Honorary Professor of Physics at the University of Manchester. Second Edition (Revised) xvii^^2 pages. Demy 8vo., 15s. net (inland postage 5d.). The Becquerel Rays and the Properties of T^'^nf Tl' r^iT ?c"-- J- ^-^-S- Professor of Physics at the Imperial Col lege of Science and Technology. Second Edition (Revised and Enlarged), vi + 215 pages. Demy 8vo., 8s. 6d. net (inland postage 5?) Physical Determinations. Laboratory Instruc- tions for the Determination of Physical Quantities. By W. R. Kelsey B.Sc. Second Edition, xii + 329 pages. Crown 8vo., 4s. 6d. Advanced Examples in Physics. Bv A O Allen M.A., B.Sc. Assistant Lecturer in Physics at Leeds Univershv With Answers. Crown 8vo., 2s. (inland postage 5d ) university. Notes on Practical Physics. By A. H. Fison ^lioi^lTnl^J'^Sr^^^^ '^'-^'^ °^ ^^y^ Hospital An Introduction to Practical Physics • for Colleges and Schools. By E. H. Barton D Sp fr q p- / 55 ^?gure^:'c;own?va''y6r °' """"^"^^^ Nottingham. ^'l^fJ^'^^^J^^^^^ Of Mathematical Functions SnJdo^n;^rmy\^o;,t^rLr^^^ King. Co^l ege.* LONDON: EDWARD i^R^^^^TsT.^ MADDOX STREET. W. 2 Mr. Edward Arnold's List of Logarithmic and Trigonometric Tables (To Five Places of Decimals). By J. B. Dale, M.A. 2s. net. Mathematical Drawing. Including the Graphic Solution of Equations. By G. M. Minchin, M.A., F.R.S., and J. B. Dale, M.A. ys. 6d. net (inland postage 4d.). Graphs and Imaginaries By J. G. Hamilton, B.A., and F. Kettle, B.A. Crown 8vo., is. 6d. Homogeneous Co-ordinates. By W. P. Milne, M.A., D.Sc, Mathematical Master, Clifton College. Crown 8vo.,5s. net. An Introduction to Projective Geometry. By L. N. G. FiLON, M.A.., F.R.S., Assistant Professor of Mathematics, University College, London. Crown 8vo., ys. 6d. Vectors and Rotors (with Applications). By O. Henrici, Ph.D., F.R.S., LL.D., and G. C. Turner, B.Sc. 4s. Gd. The Strength and Elasticity of Structural Members. By R. J. Woods, M.E., M.Inst.C.E. Second Edition. xii+ 310 pages. Demy Svo., los. 6d. net (inland postage 4d.). BY THE SAME AUTHOR. The Theory of Structures. xii + 276 pages. Demy Svo. , los. 6d. net (inland postage 4d.). Reinforced Concrete Design. By O. Faber, B.Sc, A.M.I.C.E., etc.. Chief Engineer to Messrs. Trollope and Colls, Ltd. ; and P. G. Bowie, A.C.G.I., Assistant Engineer to Messrs. Trollope and Colls, Ltd. xx + 332 pages. With 158 Illustrations. 12s. 6d. net (inland postage sd.). The Calculus for Engineers. By John Perry, M.E., D.Sc, F.R.S., Professor of Mechanics and Mathematics in the Royal College of Science. Tenth Impression. Crown 8vo., 7s. 6d. The Balancing of Engines. By W. E. Dalby, M.A., B.Sc, M.Inst.C.E., M.I.M.E., Professor of Engineering, City and Guilds (Engineering) College. Second Edition. xii+ 283 pages. Demy Svo., los. 6d. net (inland postage 4d.). Valves and Valve Gear Mechanisms. By W. E. Dalby, M.A., B.Sc, M.Inst.C.E., M.LM.E. xviii + 366 pages. Royal Svo., 21s. net (inland postage sd.). Machine Sketches and Designs for Engineering students. By A. Cruickshank, A.M.I.Mech.E., and R. F. McKay, M.Sc. Demy 4to., is. 6d. Petrol Engine Construction and Drawing. Bv W. E. DoMMETT, Wh. Ex., A. M.I. A. E., Admiralty Prizeman. ^ [/n the Press. Technical and Scientific Publications Steam Turbine Design. With especial reference to the Reaction type, and including chapters on Condensers and Propeller Design. By John Morrow, M.Sc, D.Eng. (Armstrong Col- lege, Newcastle-on-Tyne). viii -i- 472 pages. Demy 8vo. i6s.net. Steam Boilers and Boiler Accessories. By W. Inchley, B.Sc. , Lecturer in Electrical and Mechanical Engineering, University College, Nottingham. Fully illustrated. Crown 8vo., cloth. Heat Engines. By H. A. Garratt, Assoc. M, Inst. C.E., M.I.N.A., Principal of the L.C.C. School of Engineering and Navigation, xii + 332 pages, 173 Figures, and a Chart of Properties of Steam. Crown 8vo., 6s. (inland postage 4d.). Heat and Steam. Notes and Kxaniples on Steam Engines and Turbines for Engineers and Engineering Students. By Eng.-jLieut. S. G. Wheeler, R.N. viii-t-224 pages, 85 Figures. Crown 8vo., 4s. 6d. net (inland postage 4d.). Hydraulics. For Engineers and Engineering students. By F. C. Lea, M.Sc, A.M.Inst.C.E., Lecturer in Applied Mechanics and Engineering Design, City and Guilds (Engineering) College, London. Second Edition. 15s. net (inland postage 5d.). Hydraulics. By R. Busquet, Professeur a I'Ecole Industrielle de Lyon. Translated by A. H. Peake. 7s. 6d. net. The Practical Design of Motor-Cars. By James GcNN. viii + 256 pages. Demy 8vo. ios.6d.net. Power Gas Producers : their Design and Application. By Philip W. Robson, sometime Vice-Principal of the ■ Municipal School of Technology, Manchester. iv-|- 247 pages. Demv 8vo., los. 6d. net (inland postage 4d.). The Foundations of Alternate Current Theory ^y^-^-^^^^^^^-^, D.Sc. (Lond.), M.LE.E. xii+ 300 pages. Demy 8vo., 8s. 6d. net (inland postage 4d.). r o y Electrical Traction. By Ernest Wilson, Whit °^ Electrical Engineering in the Siemens Laboratory, King s College, London; and Francis Lydall, B.A. B Sc Two volumes, sold separately. Vol. L, Direct Current ; Vol II 'Alter- nating Current. 15s. net each (inland postage sd. each). A Text-Book of Electrical Engineering;. Bv Dr. A. Thomalen. Translated by G. W. O. Howe, M.Sc Second Edition, viii + 464 pages. Royal 8vo. , 15s. net (inland postage 6d. ) Alternating Currents. A Text-Book for students of Engineering:. By C. G. Lamb. M.A., B.Sc, A.M I E E Second Edition. 333 pages. ' los. 5d. "nel 4 Mr. Edward Arnold's List of Electric and Magnetic Circuits. By Ellis H Crapper, M.LE.E Head of the Electrical Engineering Department in the University College. Sheffield, viii + 380 pages. los 6d. net. Applied Electricity. A Text-Book of Electrical Engineenng for "Second Year" Students. By J. Paley Yorke. becond Edition, xii + 420 pages. 7s. 6d. (inland postage 4d.). Exercises in Electrical Engineering. By T. Mather, F.R.S.. M.LE.E., and G. W. O. Howe, M Sc M I E E viii+ 72 pages, is.6d.net. Physical Chemistry: its Bearing on Biology and Medicine. By J. C. Philip, M. A., Ph.D., B.Sc, Assistant Professor of Chemistry in the Imperial College of Science and Technology. Illus- trated. 7s. 6d. net. Lectures on Theoretical and Physical Chemis- try. By Dr. J. H. Van 'T Hoff, Professor of Chemistry at the University of Berlin. Translated by R.^ A. Lehfeldt, D.Sc. Part I. Chemical Dynamics. 12s. net. Part II. Chemical Statics. 8s. 6d. net. Part III. Relations Between Properties and Composition. 7s. gd. net. A Text-Book of Physical Chemistry. By R. A. Lehfeldt, D.Sc, Professor of Physicsat the Transvaal University College, Johannesburg, xii + 308 pages. Crown 8vo., 7s. 6d. (inland postage 4d. ). Organic Chemistry for Advanced Students. By Julius B. Cohen, Ph.D., B.Sc, Professor of Organic Chemistry in the University of Leeds, and Associate of Owens College, Manchester. viii4-632 pages. Demy 8vo., 21s. net (inland postage 6d.). Organic Chemistry for Advanced Students. Part II. By the same Author. [/» Preparation. The Chemistry of the Diazo-Compounds. By John Cannell Cain, D.Sc, Editor of the Publications of the Chemical Society. Demy 8vo. , los. 6d. net (inland postage 4d. ). The Chemical Synthesis of Vital Products and the Inter-relations between Organic Compounds. By Raphael Mel- dola, F.R.S. , V.P.C.S., F.I.C, etc. ; Professor of Chemistry in the City and Guilds of London Technical College, Finsbury. Vol. I., .\vi + 338 pages. Super royal 8vo., 21s. net (inland postage 5d.). Organic Analysis : Qualitative and Quantita- tive. By H. T. Clarke, B.Sc, A.I.C., Lecturer in Stereo-Chemistry in University College, London. With Introduction by Professor J. Norman Collie, Ph.D., LL.D., F.R.S. viii + 264 pages. Crown 8vo., 5s. net. Elements of Inorganic Chemistry. By the late W. A. Shenstone, F.R.S. New Edition, revised and partly rewritten by R. G. DuRRANT, M.A., Assistant Master, Marlborough College, cloth, 5s. (inland postage 4d.). Technical and Scientific Publications 5 A Course of Practical Chemistry. Being a Revised Edition of "A Laboratory Companion for Use with Shenstone's 'Inorganic Chemistry.'" By the late W. A. Shen- STONE, F.R.S. xii + 136 pages. Crown 8vo., cloth, is. 6d. Outlines of Inorganic Chemistry. With special reference to its Historical Development. By E. B. Ludlam, D.Sc, Head of Chemical Department, Clifton College. With Introductory Note by Professor Sir W. Ramsay, K.C.B., F.R.S. Crown 8vo., 4s. 6d. Outlines of Experimental Chemistry. By E. B. Ludlam, D.Sc, and H. Preston. Demy 8vo., 2s. Exercises in Chemical Calculation. By H. F. Coward, D Sc., Chief Lecturer in Chemistry, Municipal School of Technology, Manchester ; and W. H. Perkins, M.Sc, Assistant Lecturer in Chemistry, University of Leeds, viii + 152 pages. 2s. 6d. net. A History of Chemistry. By Dr. Hugo Bauer, Royal Technical Institute, Stuttgart. Translated by R. V. Stanford, B.Sc. (Lond.). Crown 8vo., 3s. 6d. net (inland postage 4d.). Physical Chemistry for Beginners. By Dr. Ch. M. Van Deventer. With a Preface by Dr. Van 'T Hoff. Translated by R. A. Lehfeldt, D.Sc. Crown 8vo., cloth, 2s. 6d. The Principles of Applied Electrochemistry. By A. J. Allmand, D.Sc. 136 Figures. Demy 8vo., cloth. Experimental Researches with the Electric Furnace. By Henri Moissan. Translated by A. T. de Mouilpied, M.Sc, Ph.D. xii + 307pages. Demy8vo., los. 6d. net (inland postage 4d.)! Electrolytic Preparations. Exercises for use in the Laboratory by Chemists and Electro-Chemists. By Dr Karl Elbs, Professor of Organic and Physical Chemistry at the University of Giessen. Translated by R. S. Hdtton, M.Sc. 4s. 6d. net. Introduction to Metallurgical Chemistry for Technical Students. By J. H. Stansbie, B.Sc. (Lond.), F.I C, Lecturer m the Birmingham University Technical School. Second Edition xii + 252 pages. Crown 8vo., 4s. 6d. (inland postage 4d.). On the Calculation of Thermo-Chemical Con- stants. By H. Stanley Redgrove. B.Sc. (Lond.), F.C.S. iv+ 102 pages. Demy 8vo., 6s. net (inland postage 4d.). ^ ^ Electroplating. By W. R. Barclay, A.M.I. E.E Silver Medallist, City and Guilds of London; Lecturer on Electro- A mTfp .University of Sheffield; and C. H. Hainsworth, ver'^tfof^sLeffieT"' '^"''^"'^ Engineering in the^Uni- Manual of Alcoholic Fermentation and ' the Allied Industries. By Charles G. Matthews, F I C F C S ptr- xvi + 295 pages. Crown8vo..7s. 6d.net (inland postage 4d.).' " ' 6 Mr. Edward Arnold's List of The Chemistry of Breadmaking. By James Grant. M.Sc.Tech., F.I.C., F.C.S., Head of the Fermentation Industries Department in the Municipal School of Technology, Manchester ; Ex- aminer in Chemical Technology in the Victoria University, Manchester. viii+ 224 pages. Illustrated. 5s.net. An Introduction to Bacteriological and En- zyrne Chemistry. By Gilbert J. Fowler, D.Sc, Lecturer in Bacterio- logical Chemistry in the Victoria University of Manchester. Illustrated. Crown 8vo., 7s. 6d. net. Modern Methods of Water Purification. By John Don, A.M.Inst. Mech.E., and John Chisholm, A.M. Inst. Mech.E. xvi + 368 pages. 96 Illustrations. Demy 8vo. , 15s. net. Smoke : A Study of Town Air. By J. B. Cohen, F.R.S., Professor of Organic Chemistry in the University of Leeds; and A. G. Roston, B.A., B.Sc, Science Tutor in the Depart- ment of Agriculture, University of Leeds. 5s. net (inland postage 4d.). Practical Photo-micrography. By J. Edwin Barnard, F.R.M.S., Lecturer in Microscopy, King's College, London. Illustrated. Demy 8vo. , 15s. net. The Chemistry and Testing of Cement. By C. H. Desch, D.Sc, Ph.D., Lecturer in Metallurgical Chemistry in the University of Glasgow. Illustrated. los. 6d. net (inland postage 4d.). Wood. A Manual of the Natural History and Industrial Applications of the Timbers of Commerce. By G. S. Boulger, F.G.S., A.S.I., Professor of Botany and Lecturer on Forestry in the City of London College. Second Edition, xi + 348 pages, with 48 Plates and other Illustrations. Demy 8vo., 12s. 6d. net (inland postage 5d.). A Class Book of Botany. By G. P. Mudge, A.R.C.Sc, and A. J. Maslen, F.L.S. With over 2co Illustrations. Crown 8vo., 7s. 6d. Elementary Botany. By E. Drabble, D.Sc, Lecturer on Botany at the Northern Polytechnic Institute. 234 pages, with 76 Illustrations. Crown 8vo., cloth, 2s. 6d. An Experimental Course of Chemistry for Agricultural Students. By T. S. Dymond, F.I.C. 2s. 6d. The Development of British Forestry. By A. C. Forbes, F.H.A.S., Chief Forestry Inspector to the Department of Agriculture for Ireland. Author of "English Estate Forestry," etc. Illustrated. Demy 8vo., cloth, los. 6d. net. English Estate Forestry. By A. C. Forbes, F.H.A.S. x + 332 pages, Illustrated. Demy 8vo., 12s. 6d. net (inland postage 5d.). Technical and Scientific Publications 7 Astronomical Discovery. By Herbert Hall Turner, D.Sc, F.R.S., Savilian Professor of Astronomy in the University of Oxford, xii + 225 pages, with 15 Plates. Demy 8vo., cloth, los. 6d. net (inland postage 5d.). An Introduction to the Study of the Protozoa. With Special Reference to the Parasitic Forms. By E. A. Minchin, F.R.S., Professor of Protozoology in the University of London. With 194 Figures and Bibliography. Demy 8vo., cloth. [/» the Press. The Evolution Theory. By Dr. August Weis- MANN, Professor of Zoology in the University of Freiburg in Breisgau. Translated, with the Author's co-operation, by Professor J. Arthur Thomson, and Margaret Thomson. Two vols., xvi + 416 and viii + 396 pages, with more than 130 Illustrations. Royal 8vo., cloth, 32s. net. The Chances of Death and Other Studies in Evolution. By Professor Karl Pearson, M.A., F.R.S. 2 vols. With Illustrations. Demy Svo., 25s. net (inland postage 6d.). Hereditary Characters. By Charles Walker, M.Sc. M.R.C.S., Director of Research in the Glasgow Cancer Hospital. Demy Svo., 8s. 6d. net. The Life of the Salmon. With reference more especially to the Fish in Scotland. By W. L. Calderwood, F.R.S.E., Inspector of Salmon Fisheries for Scotland. Illustrated. 7s. 6d, net. A Text-Book of Zoology. By G. P. Mudge, A.R.C.Sc. (Lond.), Lecturer on Botany and Zoology at the London School of Medicine for Women, and Demonstrator on Biology at the London Hospital Medical College. Illustrated. Crown 8vo., 7s. 6d. House, Garden, and Field. A Collection of Short Nature Studies. By L. C, Miall, F.R.S. viii + 316 pages. Crown 8vo., 6s, (inland postage 4d.). Animal Behaviour. By C. Lloyd Morgan, LL.D., F.R.S., Professor of Psychology in the University of Bristol, viii + 344 pages. Second Edition. 7s. 6d. net (inland postage 5d.). BY THE SAME AUTHOR. Psychology for Teachers. New Edition, entirely rewritten. xu + 308 pages. Crown 8vo., cloth, 4s. 6d. An Introduction to Child-Study. By W B Drummond, M B. C.M., F.R.C.P.E., Medical Officer ind Lecturer on Hygiene to the Edinburgh Provincial Committee for the Training of Teachers. 348 pages. Crown 8vo., 6s. net (inland postage 4d.). The Child's Mind: its Growth and Training By W. E. Urwick, M.A. Crown 8vo., cloth, 4s. 6d. net. 8 Mr. Edward Arnold's Technical & Scientific Book, ARNOLD'S GEOLOGICAL SERIES^ The Geology of Coal and Coal-Mining. By The Geology of Ore Deposits. By H. H Thomas and D. A MacAlister, of the Geological Survey of Greai Britai^n. Illustrated, ys. 6d. net (inland postage 4d ) ^ The Geology of Building Stones. By T. Allen Howe, B Sc. Curator of the Museum of Practical Geology. Illustrated 7s. 6d. net (inland postage 4d.). ^ ^uuiira:ea. The Geology of Water Supply. By H. B. Wood- ward. F.R.S. Illustrated. Crown 8vo., 7s. 6d. net (inland postage 4d ) Geology of the Soil and Substrata. By H B Woodward, F.R.S. Crown 8vo. [Inthe Press A Text-Book of Geology. By P. Lake, M.A Royal Geographical Society Lecturer in Regional and Physical Geograohv at the University of Cambridge; and R. H. Rastall, MA FGS Demonstrator in Geology in the University of Cambridge. Illustrated" Demy 8vo., i6s. net. The Dressing of Minerals. By Henry Louis, M.A., Professor of Mining and Lecturer on Surveying, Armstrong College' Newcastle-on-Tyne. x + 544 pages. With 416 Illustrations. 30s.net. ' Traverse Tables. With an Introductory Chapter on Co-ordinate Surveying. By Henry Lodis, M.A and G. W. Caunt, M.A. Flexible cloth, 4s. 6d. net (inland postage 3d.). Oil-Finding: An Introduction to the Geological Study of Petroleum. By E. H. Cunningham Craig, B.A., F.G.S., late of H.M. Geological Survey. With an Introduction by Sir Boverton Redwood, Bart. 13 Plates, and 18 Illustrations. 8s. 6d. net. Winding Engines and Winding Appliances : Their Design and Economical Working. By G. McCulloch, A.M.I.M.E., Inspector of Machinery in the Department of Mines, West Australia; and T. Campbell Fdters, M.I.M.E. Fully Illustrated. Demy 8vo. [In the Press. Mines and Minerals of the British Empire. By Ralph S. G. Stokes, xx + 403 pages, 70 Illustrations. Demy Svo., 15s. net (inland postage 5d.). Geological and Topographical Maps : their Interpretation and Use. By A. R. Dwerryhouse, D.Sc, F.G.S., Lecturer in Geology in the Queen's University of Belfast, viii + 133 pages, with 90 Figures. 4s. 6d. net. LONDON : EDWARD ARNOLD, 41 & 43 MADDOX STREET, W.