MARINE BIOLOGICAL LABORATORY. Received Sept .....18, 19 37 Accession No. 47 317 Given by....Lea....&..J , e.bi.ger . . Place, P)aila.de.lp.h.i.a.., Pa....... *^* flo book op pamphlet is to be removed from the Lab- oratory tuithout the permission of the Trustees. RARYU £--#"'»' /-^ THE BIOLOGY OF THE PROTOZOA BY GARY N. CALKINS, Ph.D., Sc.D. PROFESSOR OF PROTOZOOLOGY, COLUMBIA UNIVERSITY SECOND EDITION, THOROVOHLY REVISED ILLUSTRATED WITH 223 ENGRAVINGS AND 2 COLORED PLATES LEA & FEBIGER PHILADELPHIA 19 3 3 Copyright LEA & FEBIGER 1933 PRINTED IN U. S. A. TO MY WIFE WHOSE UNSELFISH DEVOTION HAS MADE THIS BOOK POSSIBLE ( L ! B f? A R % PREFACE TO SECOND EDITION. In writing' this volume the author has made no effort to give a complete account of the Protozoa. As indicated by the title, it is rather a study in biology illustrated by the unicellular animals. The concept of a changing organization brought about by continued metabolism was developed in the first edition. This conception has been amplified in some respects, strengthened and condensed in others, and furnishes the basis for an interpretation not only of life histories but of the significant biological phenomena of cell division, maturity, sex differentiation, fertilization and senescence as well. To strengthen this conception a considerable change in the order of presentation has been introduced. After the first intro- ductory chapter we plunge at once in Chapter II into the sub- stances and structures of the fundamental organization. This is followed in Chapters III and IV by the development of these sub- stances and structures into cytological derivatives (Chapter III) and taxonomic structures (Chapter IV) of the derived organization. In Chapter Y the general physiological activities are considered in anticipation of Chapter VI on reproduction. The problem of gen- eral vitality and its significance in fertilization and the accompany- ing phenomena of sex differentiation, maturation, reorganization, adaptation and variations are treated in Chapters VII, VIII and IX. The special chapters on taxonomy, together with more elab- orate keys to genera, are transferred from the middle of the book to the end in Chapters XI, XII, XIII and XIV. Parasitism and disease should be considered in any work on general biology. These topics were omitted in the first edition but are introduced here in Chapter X. Another innovation is the elimination of all references to chlorophyll-forming flagellates, the protozoan flagellates being limited to the Zoomastigophora. Reorganization or de-differentiation of the derived taxonomic structures at periods of division, endomixis and fertilization wherebv vi PREFACE the protoplasm is restored to the condition of the fundamental organization with a renewed potential of vitality, is treated as a special attribute of Protozoa and as an important distinction between Protozoa and Metazoa. Through such reorganizations either by division alone as in the Zoomastigophora and in occa- sional forms here and there throughout the Protozoa, or by the more drastic means of endomixis and fertilization, the protoplasm is able to continue at an optimum of vitality. With this conclusion and with the recognition of an internal self-regulating mechanism for reorganization, resulting in the continuation of vitality, we are in accord with the essence of Weismann's conclusion that protoplasm of Protozoa is potentially immortal. On the other hand, we can- not agree with Weismann in his further conclusions that natural death is unknown in Protozoa, and that every individual is a potential germ cell. G. X. C. New York City. CONTENTS. CHAPTER I. Introduction Size, Form and Appearance of Protozoa . ...'... 26 A. Form-relations of Protozoa 30 B. Protoplasmic Structure . 39 CHAPTEE II. The Fundamental Organization. I. Nuclear Substances and Structures of the Fundamental Organization 49 1. Chromatin . . 54 2. Other Substances of the Nucleus 57 Intranuclear Kinetic Elements 60 (a) Endobasal Bodies 60 1. Large Homogeneous Endobasal Bodies til 2. Endobasal Bodies With Centrioles 63 3. Nuclei With Pole Plates and Without Endo- basal Bodies 65 II. Cytoplasmic Elements of the Fundamental Organization ... lis 1 . Chromidia 69 2. Volutin Grains 72 3. Mitochondria 73 1. ( iolgi Apparatus 77 5. Silver Line System so CHAPTER III. Derived Organization. I. Cytological 83 A. Derived Nuclei and Derived Nuclear Structures .... 84 1. The Formation of a Nucleus 84 2. Multiple and Dimorphic Nuclei 84 3. Nuclear Derivatives During Division 88 (a) Origin of Chromosomes and of Intranuclear Spindles at Division ... 88 lb Origin of Fertilization (Meiotic) Chromo- somes 100 B. Derived Organization; Cytoplasmic Changes . . 104 1. Cytoplasmic Chromatin . ... 104 2. Cytoplasmic Kinetic Elements 104 .'••/."■ 7 CONTENTS CHAPTER IV. Derived Organization. Taxonomic Structures I. Derived Structures of the Endoplasm. II. Differentiations of the Cortex Metaplastic (a) Cortical Differentiations for Support and Protection (b) Motile Organoids 1. Flagella .... 2. Pseudopodia ... Rhizopodia Filopodia 3. Cilia 4. Composite Motile Organs Membranulae Membranelles Undulating Membrane; Cirri . . in ( )ther Organoids Adapted for Food-getting (d) Oral and Anal Cortical Modifications (e) Contractile Vacuoles 133 135 136 139 141 115 US 150 152 155 155 155 157 157 162 1(14 170 CHAPTER V. General Physiology. A. Respiration B. Excretion of Metabolic Waste .... ('. Irritability I). Nutrition 1. Food-getting Secretions and Digestive Fluids Digestion of Carbohydrates and Fats Saprozoic Nutrition 2. Products of Assimilation 174 17(i 179 183 is:; 193 198 199 203 CHAPTER VI. Reproduction. I. Equal Division and Evidence of Reorganization A. Division in Mastigophora B. Division in the Sarcodina C. Division in Infusoria in) Evidence of Nuclear Reorganization . (Id Evidence of Cytoplasmic Reorganization II. Unequal Division (Budding or Gemmation) A. Exogenous Budding B. Endogenous Budding III. Multiple Division (Spore-formation i IV. Development 209 210 213 215 217 218 225 226 228 233 241 CHAPTER VII. Vitality. I. Isolation Cultures ....." II. Organization and Differentiation 1. I nter-di visional Differentiations 2. Cyclical Differentiations (a) Cyclical Differentiations Peculiar to Youth (b) Cyclical Differentiations Peculiar to Old Age (c) Cyclical Differentiations Peculiar to Maturity 2 1s 260 260 266 266 269 271 CONTENTS IX CHAPTER VIII. Phenomena Accompanying Fertilization. I. The Environmental Conditions of Fertilization (a) Ancestry (b) Environment II. Internal Conditions at the Period of Fertilization . III. The Process of Fertilization A. Meiotic Phenomena {-class I. Heliozoa Haeckel Si ili-class II. Radiolaria Haeckel . Class II. Rhizopoda von Siebold Sub-class I. Proteomyxa Lankester Sub-class II. Mycetozoa de Bary Order I. Acrasida van Tieghem Order II. Phytomyxida Schroter Order III. Euplasmodida Lister Sub-class III. Foraminifera d'Orbigny Sub-class IV. Amoebaea .... Order 1. Amoebida (Gymnamoebida) Ehrenberg Order 2. Testacea Key to Actinopoda Sub-class 1. Helizoa Haeckel . Order I. Aphrothoraca Hertwig . Order II. Clamydophora . Order III. Chalarothoraca Order IV. Desmothoraca Sub-class 2. Radiolaria Joh. Midler . Class II. Rhizopoda von Sieb Sub-class I. Proteomyxa .... Sub-class II. Mycetozoa de Bary Order I. Acrasida van Tieghem . Order II. Phytomyxida Order III. Euplasmodida Lister . Sub-order 1. Exosporea Rostaf Sub-order 2. Myxogastres Fries Sub-class^III. Foraminifera D'Orb. . Sub-class IV. Amoebaea Btitschli Order I. Amoebida Aut. . Order II. Testacea M. Schultze 436 437 438 442 443 445 447 449 449 450 453 455 456 459 460 460 460 460 461 461 461 461 462 462 462 463 463 463 466 466 466 467 CHAPTER XIII. Special Morphology and Taxonomy of the Infusoria. Classification of the Infusoria Infusoria Class I. Ciliata Perty 1852; Btitschli 1889 186 488 CONTEXTS xi Infusoria — Class I. — Continual. Sub-class I. Holotricha Stein 1850 188 Order 1. Astomida IS'i Order 2. Gymnostomida -490 Sub-order 1. Prostomina (Prostomata Schewiakoff) 490 Sub-order 2. Pleurostomina Schew. 1886; Em. Kahl 491 Sub-order 3. Hypostomina (Hypostomata Schewiakoff) 491 Key to Genera 491 Order 2. Gymnostomida 491 Sub-order 1. Prostomina 491 Sub-order 2. Pleurostomina (Tribe Pleurostomata Schewia- koff; Kahl) 497 Sub-order 3. Hypostomina Schewiakoff 1896; Em. Kahl 498 Order 3. Trichostomida Butschli 1889 499 Order 4. Hymenostomida 503 Sub-class II. Spirotricha Butschli 1889; Em. Kahl 1931 ... 508 Order 1. Heterotrichida Stein 508 Order 2. Oligotrichida Butschli 1889 512 Order 3. Ctenostomida (Lauterborn) Kahl 1931 . . 516 Order 4. Hypotrichida Stein s. str 516 Sub-class III. Peritricha Stein 521 Sub-class IV. Chonotricha Wallengren 522 Class II. Suctoria Butschli 523 CHAPTER XIV. Special Morphology and Taxonomy of the Sporozoa. Class I. Telosporidia Schaudinn 533 Sub-class I. Gregarinina . . 534 Order 1. Eugregarinida Doflein Emend 540 Order 2. Schizogregarinida Leger (1892) 541 Sub-class II. Coccidiomorpha Doflein 541 Order 1. Coccidiida Leuckart, Em 541 Sub-order 1. Eimeriina 541 Sub-order 2. Hemosporidia Danilewsky, em. Doflein 542 Sub-order 3. Babesiina 543 Order 2. Adeleida 544 Class II. Cnidosporidia Doflein 515 Order 1. Myxosporidia Butschli 548 Order 2. Actinomyxida Stole 551 Order 3. Microsporidia Balbiani 552 Class III. Acnidosporidia Cepede 555 Key to Subdivisions and Genera of Sporozoa 558 Bibliography ... 571 \ ft 5 BIOLOGY OF THE PROTOZOA. CHAPTER I. INTRODUCTION. A protozoon is a minute animal organism, usually consisting of a single cell, which reproduces its like by division, by budding, or by spore formation and whose protoplasm has passed, or will pass, through various phases of vitality collectively known as the life cycle. The maze of microscopic life to which the scientific world was first introduced by Anton von Leeuwenhoek in 1075 included a heterogeneous collection of animals and plants. Crustacea, rotifers, minute worms, diatoms and desmids, as well as the more minute Protozoa, were all grouped together during the eighteenth and nine- teenth centuries, first under the nondescript term animalcula and later under the more ecological term Infusionsthiere of Ledenmiiller (1763). The correct zoological position of the higher types was recognized before the middle of the nineteenth century and the group of strictly unicellular forms was first definitely outlined by von Siebold in 1848 under the name Protozoa, a term substituted by Goldfuss (1820) for Oken's suggestive Urthiere (1805), while the old name Infusoria has been retained for one of the subdivisions of the group. The haziness in classification of the older zoologists has not entirely disappeared in the light of modern knowledge and we are confronted today by the difficulties of distinguishing between Bacteria, unicellular Algae and unicellular animals or Protozoa. It is no reflection on modern science that we are unable clearly to differentiate between these three groups. To accept the problem as insoluble at the present time is merely to admit and apply our conviction that evolution is now, and has been in the past, the pri- mary biological principle underlying the diversities of forms and functions of living things. Few biologists today will refuse to accept the view that higher types of animals — Metazoa— have been derived from forms in the past which were more or less similar to present-day Protozoa; or the view that higher plants have been evolved from unicellular plants. The variations and adaptations 18 BIOLOGY OF THE PROTOZOA which have been the stepping stones in this evolution have been and are still in progress among all types of unicellular things, so that no artificial definition of Bacteria, of Protozoa, or of Algae will distinguish with strict accuracy either of these groups from the others. Haeckel (1866) undertook to avoid the difficulty by com- bining all unicellular forms under the common name Protista, but this is, obviously, only another name for the aggregate and an artifice for concealing the real difficulties which we should like to overcome. Minchin (1912), on the ground of structural characters, would distinguish Protozoa from Bacteria by the assumption that the latter are not of " cellular grade" because of the absence in many Bacteria of a typical cell nucleus. Here again, however, the old difficulty shows its head, for in this sense, many well-recognized Protozoa are not, while many Bacteria are, of cellular grade (see Dobell, 1911). The problem after all has mainly an academic inter- est, and the chief practical value to be gained by its solution would be to set the limits of a text-book or monograph. We may reason- ably expect to find therefore, in treatises on Protozoa, some types which with equal right should be included in works on lower plants and on Bacteria. In this connection the greatest difficulty lies in the separation of one group of the flagellated Protozoa from the unicellular algae. We are still tied firmly to the old tradition that animals move and plants are quiescent, and a chlorophyll-bearing organism, if actively moving, is ipse facto an animal. Were I to advocate this as the main distinction between animals and plants, there would be, undoubtedly, a storm of protests from all biologists. And yet, what other characteristics do chlorophyll-forming organ- isms have to justify us in claiming them as animals? At the present time there is a double taxonomic system, one botanical, the other zoological for these questionable forms, and these systems are widely different. We can avoid the resulting confusion by adopting one or the other system of classification. My own conviction is that zoologists should follow the historical precedent furnished in the last century by the elimination from Protozoa of filamentous algae, desmids and diatoms, and now transfer to the botanists the entire aggregate of so-called Protozoa in which the ability to form chlorophyll is a characteristic. (See also p. 412.) It is less difficult to distinguish between Metazoa and Protozoa; the occurrence of a gastrula stage in the development of a question- able form is sufficient to place it unmistakably with the higher animals. Protozoa, indeed, are often associated in cell aggregates called colonies, the individual cells being held in place by proto- plasmic connections, by stalk attachments, or by fixation in a com- mon gelatinous matrix. In some questionable cases, e. g., Mago- sphaera, these colonial aggregates resemble tissues of Metazoa in their structural appearance, but tissue cells are dependent upon INTRODUCTION 19 other parts of the animal for fulfilment of their vital activities while every cell of a colonial protozoon may be self-sufficient and inde- pendent, and differentiation among them is limited, at most, to reproductive and somatic cells (e. g., Epistylis, Zoothamnium and other vorticellids) . While the single protozoon is to be compared structurally with a single isolated unit tissue cell of a metazoon as a bit of protoplasm differentiated into cell body, or cytoplasm, and nucleus, it is a very different unit physiologically. In its vital activities it should be compared, not with the unit tissue cell, but with the entire organism of which the tissue cell is a part. All animal organisms perform the same fundamental vital activities of nutrition, excretion, irri- tability with movement and reproduction, which are fundamental attributes of living animal protoplasm. In the higher types of Metazoa these primary activities are performed by complex organ systems, nutrition for example, involving not only the digestive system but the muscular, nervous, circulatory and respiratory systems as well. Each organ has its particular part to play in the economy of the whole and each cell is differentiated for the purpose of its specialized function. Tissue cells, therefore, are physiologic- ally unbalanced cells since they are preeminently specialized for secretion, or contraction, or irritability, etc. Division of labor in a physiological sense here reaches its highest expression. In the lower Metazoa the organ systems are less highly special- ized; fewer organs are present to perform the same fundamental vital activities and the tissue cells have relatively more kinds of work to do for the organism as a whole. Thus the supporting and covering cells of a coelenterate combine the functions of respiration, irritability, muscular contraction, excretion and circulation with the primary functions of an epithelium. Each of them is more nearly balanced physiologically than a single cell of the higher types, but it still needs the activities of other cells, and the organism is again the sum-total of all its cellular parts. In the protozoon, finally, we find a cell which is physiologically balanced ; it is still a cell and at the same time a complete organism performing all of the fundamental vital activities within the con- fines of that single cell. Whitman, in his essay on "The Inadequacy of the Cell Theory" (1893), clearly expressed the inconsistencies in the common use of the designation "cell" for this variety of struc- tures, and later writers, notably Gurwitsch (1905) and Dobell (1911), have followed in a similar vein. As organisms the Protozoa are more significant than as cells. In the same way that organisms of the metazoan grade are more and more highly specialized as we ascend the scale of animal forms, so in the Protozoa we find intracellular specializations which lead to structural complexities difficult to harmonize with the ordinary 20 BIOLOGY OF THE PROTOZOA conceptions of a cell. In perhaps the majority of the Protozoa the fundamental vital activities are performed, as in the simpler Ameba or simple flagellates, by the protoplasm as a whole and without other visible specializations than nucleus and cell body. In other forms, Mac. Mic.-- C.V.- Fig. 2. — Diplodinium ecaudatum, a parasitic ciliate in cattle. A, anal canal and defecatory vacuole; C. V., one of the two contractile vacuoles; M, motorium with fiber to circumpharyngeal ring; Mac., macro nucleus ; Mic, micronucleus ; S, skeletal layer. (After Sharp.) however, intracellular differentiations lead to intracellular division of labor which in some types becomes as complicated as are many of the organisms belonging to the Metazoa. Thus Diplodinium ecaudatum, one of the Infusoria, according to Sharp (1914), has intracellular differentiations of extraordinary complexity (Fig. 2). INTRODUCTION 21 Bars of denser ehitinous substance form an internal skeleton; special retractile fibers draw in a protrusible proboscis; similar fibers closing a dorsal and a ventral operculum; other fibrils, func- tioning as do nerves of Metazoa, form a complicated coordinating system; cell mouth, cell anus and a fixed contractile vesicle or excreting organ are also present. All of these are differentiated parts of one cell for the performance of specific functions, and all perform their functions for the good of the one-celled organism which measures less than -jto mcn m length. Analogous, if not so com- plete intracellular differentiations are present in the majority of Infusoria, while many of the flagellates, notably the Hypermastigida, have an almost equally elaborate make-up. In all such cases the single cell is a complicated mechanism and the cooperating parts have the same relation to the organism as a whole as do the organs of a metazoon. Compared with an Amoeba proteus or other simple rhizopod such complex organisms are highly specialized and show the extent to which intracellular differentiation may be carried. As Gurwitsch, Hartmann, Dobell and others have pointed out, the application of the term cell which designates a structural unit with specific physiological activity in Metazoa seems to be inappropriate, and, as Whitman argued, inadequate. A significant difference between Protozoa and Metazoa lies in the phenomenon of reversibility. Differentiations in the protozoan organism are reversible and the derived organization is restored to the fundamental organization (see p. 83) at periods of division, parthenogenesis and fertilization. This does not occur in Metazoa where differentiated cells derived from the fundamental organiza- tion of the egg are irreversible and the "somatic" individual dies. Cell aggregates or colonies are likewise highly variable in their functional specialization. AYhile many of them consist of fortuitous groups of cells with dimensions varying with the number of indi- viduals joined together {e.g., Ophrydium versatile, Poteriodendron petiolatum, etc.), others are definite in form, number of cells and in arrangement. Here the colony as such has a distinct individ- uality and in some cases (e. g., Zoothamnium alternans) under- goes a definite developmental cycle. Again some colonies com- posed of otherwise independent cells do not react as separate individuals but the colony reacts as a coordinated whole. Thus Zoothamnium arbuscula, composed of many hundreds of individual cells in a colony which may attain a diameter of 1 inch, reacts as a unit organism if any one of the component cells is irritated. The entire aggregate contracts into a small ball, so minute that it is scarcely visible. The concerted action is due to the con- traction of stalk myonemes which are continuous throughout the entire aggregate, like the coenosarc of some hydroid colonies. 22 BIOLOGY OF THE PROTOZOA For such colonies of protozoa, as for analogous colonies of hydroids, the expression "individual of a second order" has been applied. Between the limits of the simplest and the most complex of uni- cellular organisms are the great majority of the (estimated) 15,000 or more known Protozoa. In each of the main subdivisions sim- plicity as well as extreme complexity of organization is represented, each subdivision including a series of representative forms ranging from one extreme to the other. Differentiation in the different subdivisions do not follow the same lines of development, however, so that we are able to classify Protozoa according to a fairly natural system. These diverse lines of development make it difficult to treat this branch of the animal kingdom in any general way; the wide range in habitat from the purest waters of lake or sea to the Fig. 3. — Types of Protozoa. A, Amoeba proteus, a rhizopod; B, Peranema tricho- phora, a flagellate; C, Stylonychia mytilis, a ciliate; D, a polycystic! gregarine; E, Tokophrya quadripartita, a suctorian. (A, after Calkins, B, C, E, after Butsehli; D, after Wasielewsky.) foulest ditch, and adaptations to environments varying in charac- ter from a mountain stream to the semifluid substance of an epithe- lial, nerve or muscle cell, has brought about manifold varieties of structure. To describe all of these modifications under a few headings, or to attempt to formulate general laws from the different and often highly complicated life histories, is out of the question. The general trends of differentiation, however, permit of grouping the different kinds of Protozoa in four types which were first out- lined by the French microscopist Felix Dujardin in 1841. Three of these types— Sarcodina, Mastigophora and Infusoria— are based upon the nature of the locomotor organs— pseudopodia, flagella and cilia respectively— while a fourth type— Sporozoa— includes organ- isms which are invariably parasitic in mode of life and are essentially without motile organs (Fig. 3). INTRODUCTION 23 DISTRIBUTION OF PROTOZOA. Protoplasm is an aggregate of fluid colloidal substances in which water plays a conspicuous part; exposed to the air it dries and desic- cation is fatal to the majority of Protozoa, although it is possible that some forms, like certain rotifers, may reabsorb moisture and again become active. If after losing its water the protoplasm is surrounded by impervious membranes, further evaporation is pre- vented and within such capsules the protoplasm remains alive. This is the condition of encystment and many kinds of Protozoa, protected by their cyst membranes, may live for long periods in a dried state (Fig. 4). Because of their light weight these cysts may be carried in the air and blown by the winds with dust, until surrounded 1 2 3 Fig. 4.— Types of cysts. Eughjpha alveolata, testate rhizopod; Podophrya fixa, suctorian; and Chilomastix mesnili, a parasitic flagellate, urn, undulating mem- brane. (First and second, original; third, after Kofoid and Swezy, University of California Publications in Zoology, 1920.) again by water the organisms emerge from their cysts and are active once more for a few hours. Such encysted forms account in part for the surprising protozoan fauna in uncovered sterilized water in which food substances come from similarly protected germs of Bacteria and minute plant forms. Similar encysted forms may be present on the blades of dried grass, leaves and other vegeta- tion. In the infusions formed by soaking such dried vegetation in water various species of monads (Monas, Oicomonas, Bodo) and of ciliates (Colpoda, Oxytricha, Stylonychia, Urostyla, Gastrostyla and Vorticella) and the rhizopod Ameba make their appearance in the order given (Woodruff, 1912). Puschkarew (1913) concluded that air-borne cysts play only a minor role, however, in the spread of Protozoa. It was found that, on the average, there are only 2| protozoon cysts per cubic millimeter of air and that these are limited 24 BIOLOGY OF THE PROTOZOA to 13 species and represent the same types for the most part as those listed by Woodruff. Protozoa are very apt to stick to solid sub- stances when they encyst and are carried, in the dried state, with such substances, which accounts in part for the appearance of Protozoa in all kinds of infusions. Similar adhering cysts may be carried from place to place by birds and other flying creatures or by land animals, thus helping to maintain a common type of proto- zoan fauna in pools and casual waters. The commonest species of Paramecium, viz., P. aurelia and P. caudatum, are widely distrib- uted over the earth and are almost universally used in general laboratory work as examples of ciliated Protozoa. Their mode of distribution, however, has been a continued puzzle for their sup- posed inability to form cysts has been generally recognized. Re- cently, however, Cleveland (1927), upon injecting unknown species of Paramecium in the rectum of a frog, found that a definite cyst membrane is formed bv manv of the Paramecia. After a few Fig. 5. — Paramecium caudatum, stages in encystment. The final product may be easily mistaken for a sand grain. (After Michelson, Arch. f. Protistenkunde, courtesy of G. Fischer.) . days division within the cyst and ex-cystation were observed. Michelson (1928), furthermore, has described encystment of Para- mecium caudatum under conditions of slow desiccation entailing loss of peristome, vacuoles and cilia. When fully dried the crumpled cyst wall resembles a small sand grain and as such may be over- looked (Fig. 5). Some forms to which Lauterborn (1901) has applied the term "sapropelic fauna" appear to be able to live without free oxygen. Thus Frontonia leucas, Prorodon ovum, Spirostomum ambiguum, Pehmyxa palustris, P. binucleata, etc., which usually live in rela- tively clear waters, may also live in the sulphurous medium of putrefying vegetable and animal matter, while certain species of ciliates of fantastic form seem to require this peculiar habitat for their vital activities (Dactylochlamys pisciformis, Lauterb., Saprodi- nium dentatum, Lauterb., Discomorpha pectinata, Levand., Pelodt- nium reniforme, Lauterb.). Doflein, following the suggestion made earlier by Bunge, believed that the anaerobic parasitic forms of the INTRODUCTION 25 digestive tract may have had their initial start toward parasitism when living as such sapropelic forms. 1 Protozoa are distributed over the entire world. Wherever there is moisture, there will these unicellular animals be found unless conditions of heat or of chemical composition are inimical to life. Oceans and their tributaries, lakes, ponds, pools and ditches, mountain streams and wells contain them, their numerical abund- ance depending on the available food. They are present, not only in permanent waters, but also in casual puddles of field and road, in droplets caught in the axils of leaves or in hollows of rocks, in rain w T ater of roof or pail and in damp moss. In many cases they are active for only an hour or more until their world dries up, when they may be saved again by encystment, but some forms retain their activity in ordinary garden earth where they are supposed to play an important part in connection with Bacteria of the soil (Cutler and Crump, 1920; Goodey, 1916). The majority of such soil-dwelling forms belong to the Sarcodina and Mastigophora, Gruber's Amoeba ierrieola being a typical case, while other genera and species are discovered from time to time (Bodo, Prowazelcia, Spironema, Oicomonas, Cercomonas, D hn a stig amoeba punctata and many others (see Soil-dwelling Protozoa, Chapter X, p. 353). While excessive heat kills them, excessive cold does little harm beyond retarding vital activities and the melted ice of glaciers may teem with them. They may live, not only in the exposed waters of the earth's surface, but also as parasites in the fluids of other living protoplasm or its products. They may be found in the warm blood of birds and mammals, or in the cold blood of fishes, amphibia and reptiles; in the digestive tract of every type of animal; in the saliva and urine of different types and in the living protoplasm itself of plants, other Protozoa and of tissue cells. No type of animal life is free from the possibility of association with Protozoa either as commensals, or svmbionts or parasites (see Chapter X, p. 358). The common Protozoa of our own ponds and pools are exactly the same in genera and species as those found in similar places in Europe, Asia, Siberia, Africa, South America and Australia; they are cosmopolitan, and the temptation to describe new species because they happen to have been found in some hitherto unexplored local- itv has no justification from the facts of geographical distribution. This is particularly applicable to the fresh water forms but does 1 The suggestive experiments and conclusions of Avery and Morgan (1924) give reason for the belief that the inability of some organisms to live in free-oxygen hold- ing media is due to the absence in such forms of a peroxidase capable of breaking down hydrogen peroxide. The latter accumulates under ordinary aerobic conditions and is detrimental to forms which are unable to provide the peroxidase. The limi- tation of free oxygen may be the explanation of successful artificial cultivation of forms — for example Spirostomum ambiguum — which grow best under partly anaero- bic conditions (see Bishop, 1923). 26 BIOLOGY OF THE PROTOZOA not apply equally to the deep sea types. The littoral fauna of salt water, like the fresh water forms, appears to have a cosmopolitan distribution according to the observations of Gourret and Roesser (1886), of Levander and of Hamburger and Buddenbrock in Europe, while in North America the brackish waters are particularly rich in number and variety of Protozoa. The pelagic and deep sea forms appear to be unequally distributed; some types are apparently limited to the Indian Ocean; others to the Atlantic, while many tropical genera and species, especially of Radiolaria and Foramini- fera, are not found in the polar seas and vice versa. Some strictly pelagic forms, on the other hand, notably Tintinnidae, are found on or near the surface of sea water in all parts of the world. Observations are sufficiently numerous to show that not only is there a certain climatic distribution of salt water forms, but a ver- tical distribution as well. Certain genera and species of Radiolaria and Foraminifera are present in the surface waters but are rarely found at the depth of from 600 to 3000 feet, while some families, notably the Challengeridae and Tuscaroidae, are present only in the extreme depths of the sea. Many species are sufficiently adaptable to live either in fresh, brackish or salt water; indeed most of the common forms of rhizo- pods, flagellates and ciliates seem to be equally at home in either. Many types, however, sometimes entire groups of Protozoa, are not so ubiquitous; the sub-class Radiolaria for example, comprising more species than any other entire class of Protozoa, is exclusively marine, while another large sub-class of the Sarcodina, the Fora- minifera, comprises only a few fresh water representative species. Many more types of Choanoflagellates are present in salt than in fresh water. Ciliates are poorly represented in the deep sea, although one family— Tintinnidae— is wonderfully rich in salt water forms while fresh water forms are uncommon. Heliozoa, another sub-class of the Sarcodina, on the other hand, are typically fresh water forms with relatively few salt water representatives. The distribution of parasitic forms belonging to all groups of the Protozoa obviously follows the distribution of their hosts, and we know too little on this subject to generalize; where animals are segregated the opportunities for parasitism are enhanced while some climatic conditions are more advantageous than others for the spreading of germs. Thus the blood-dwelling parasites are more common in the tropics than elsewhere, the biological condi- tions favorable to the intermediate transmitting hosts being largely responsible for their numbers and variety. SIZE, FORM AND APPEARANCE OF PROTOZOA. Although Protozoa belong unquestionably to the microscopic world their sizes vary within wide limits. Some are large enough INTRODUCTION 27 to be picked up with forceps (Porospora gigantea, a gregarine, up to 16 mm.) and many of the larger ciliates are easily visible to the unaided eye (Bursaria truncatella, Spirostomum ambiguum) while many smaller types can be seen by the trained eye as mere white specks which, in some cases, may be identified by their characteris- tic movements (e. g., Paramecium, Frontonia, Dileptus, Amphileptus, Loxophyllum, etc.). At the other extreme in size are types which are barely visible even with the most powerful lenses of the micro- scope. From 8 to 16 such forms have ample room for existence in a red blood corpuscle (Babesia canis), or 200 to 300 may live simulta- neously in a single infected liver or spleen cell of man (Leishmania B k Fig. G. — Dileptus gigas, two sister cells. A, normal individual; B, individual starved for several days. (From Calkins.) donovani). Between these two extremes of size lie the majority of Protozoa. Their measurements are usually expressed in terms of " microns " or thousandth parts of a millimeter which are represented by the symbol n, each micron being 2t|-oo- °^ an ^ ncn - Thus Leish- mania donovani measures from 2 n to 4 /x, Paramecium caudatum upward of 200 //, Bursaria truncatella, 1500 /x, etc. The same species frequently shows remarkable variations in size due to environmental conditions or to different stages in the life history. Thus normal specimens of Paramecium caudatum may measure from 175 /j, to 250 /x when fully grown and similar variations are characteristic of all species. Environmental factors, especially 28 BIOLOGY OF THE PROTOZOA food conditions, are frequently responsible for changes in size and character of a species, often rendering them difficult to recognize and affording tempting opportunities for swelling the list of syn- onyms by new names for the abnormal forms. Thus Dileptvs gigas when starved has a very different size and character from the normal form (Fig. 6). Again, different normal stages in the life history of a given species are not infrequently mistaken for different species, largely because of difference in size. Thus Uroleptus metritis (see Fig. 1), in its adult vegetative condition, measures about 150 (J., but immediately after conjugation not only is it reduced Fig. 7. — Uroleptus mobilis Engelm. Old age specimens showing degeneration of macronneleus M and loss of micronuclei. See frontispiece. (After Calkins.) by one-third in size, but its internal structure is entirely different from that of the usual form, while during the period of old age it frequently measures less than 75 // (Fig. 7), and has a different appearance from the more youthful stages. Even more striking examples of normal dimorphism are shown by the rhizopod Dimastig amoeba and by the ciliate Glaucoma (Dalla- sia) frontata. Species of the former usually appear as small earth- dwelling ameboid rhizopods, but with the addition of water they develop flagella and become actively moving ellipsoidal flagellates. Glaucoma frontata in its usual vegetative state is a more or less quiescent tailed form (Fig. 8), but under certain environmental INTRODUCTION 29 conditions not yet fully understood it becomes an active tailless navicular organism which divides repeatedly, giving rise to minute M07 Fig. 8. — Glaucoma (Dallasia) frontata. Vegetative individual. A, anus; BC, buccal cavity; CV, contractile vacuole; LS, "ladder" system; LU, left undulating membrane; M, mouth of buccal cavity; MOT, region of motorium; RU, right undu- lating membrane; T, "tongue" in buccal cavity. (After Calkins and Bowling, Arch. f. Protistenkunde, courtesy of G. Fischer.) 30 BIOLOGY OF THE PROTOZOA individuals one-sixteenth the original size (Fig. 200, p. 485). To the uninitiated such variations in forms and habits offer great temp- tation to swell the list of synonyms. A. Form-relations of Protozoa. The forms of Protozoa are highly varied and depend to some extent upon the mode of life, to some extent upon the mode of reproduction and to some extent upon their lifeless skeleton elements, but in the last analysis they depend upon the physical consistency of the protoplasm. Fluid types, if not confined by resistant cell membranes, readily change in form according to environmental conditions, or by virtue of forces coming from metabolic activities within. Amoeba proteus and other species of Ameba are amorphous and are constantly changing in shape, a characteristic phenomenon to which the term ameboid movement is applied, and the same protoplasm may be spherical in form, or flattened on the substratum, or extended in various ways. Many forms, under certain pressure conditions in the surrounding medium due to evaporation or reduced volume of water, will suddenly burst and disappear leaving no trace whatsoever of their previous presence. This phenomenon has been repeatedly mentioned by earlier observ- ers in connection with types of Protozoa belonging to all classes, and the term diffluence was applied to it by Dujardin. In such cases the fluid protoplasm is usually confined by a resisting membrane or cortex which remains intact during the ordinary phases of activ- ity, but when the pressure from within becomes too great for the resistance of the membrane the latter collapses, the cell disappear- ing with all the characteristics of a miniature explosion. Another evidence of the difference in density between different species of Protozoa is the reaction after cutting with a scalpel. Some species, for example Paramecium cavdatum, are extremely difficult to cut successfully owing to the fluid character of the inner protoplasm which, as soon as the cortex is cut, flows out and disin- tegrates; in my experience not more than 20 per cent out of more than 1000 operations on Paramecium caudatum have been success- ful, but the percentage is greatly increased by preliminary treat- ment with neutral red. Other forms of ciliates on the other hand may be cut in any plane, Uronychia transfuga and Uroleptus mobilis for example, reacting to such operations with all the physical properties of a piece of cheese. The more fluid Protozoa, when the form is not maintained by resistant cortical differentiations, react to physical properties of the surrounding medium. When forces on all sides are equal, as in suspended water-dwelling types like Actinophrys sol, Actinosphae- rium, many Radiolaria, etc., the form is spherical, or spherical also in parasitic forms enclosed in the protoplasm of the host cell as is the case with the majority of Coccidia. In all types, under certain environmental conditions, or when continuously irritated, there is INTRODUCTION 31 a tendency to become globular and this is the form assumed by the great majority of Protozoa when they encyst. The spherical or homaxonic type, furthermore, is characteristic, not only of free floating forms, but also of the most generalized representatives of all classes of Protozoa. While density or consistency of the protoplasm is thus one of the factors determining form in Protozoa, its effect in the majority of types is offset by the presence of definite membranes, shells, tests and skeletons; by specialized protoplasmic differentiations; or by foreign bodies. Thus the density of the sluggish Pelomyxa palustris Fig. 9. — Euglypha alveolala (A), and Cochlio podium, sp. (B). (After Calkins.) is due to the enormous number of crystals of mud and sand, shells of diatoms and peculiar refractile bodies resembling glycogen in make up. Membranes of living substance, as in Cochlioyodium (Fig. 9) and the majority of flagellates and ciliates, of lifeless chitin as in Allogromia oviforme (Fig. 10) or the lifeless materials secreted by the cell and deposited on it are responsible for the forms assumed by many Protozoa. Even delicate types such as Clathrvlina elegans and the majority of Heliozoa retain their forms by virtue of the protecting shells of lifeless materials deposited on a chitinous mem- brane. The protoplasmic bodies of many of the fresh water shelled 32 BIOLOGY OF THE PROTOZOA rhizopods are relatively dense like that of the naked Amoeba verru- cosa and are more or less globular or pyriform in shape. On such a protoplasmic basis the shells of Dlfflugia species, Euglypha, Cyylw- : \ , Ik ' 1 i •; n D Fig. 10. — Allogromia oviforme, foraminiferon with chitinous monothalamous shell and reticulose pseudo podia. (£>) a recently captured diatom; OS) chitinous shell. (From Calkins after M. Schultze.) deria, Centropyxis, Arcella, etc., are deposited and these, once formed, are never changed (Fig. 11). Only rarely are these shelled rhizopods flattened or discoid as in Hyalodiscus (see Chapter XII). The typical form in many shell-bearing or skeleton-forming rhizo- INTRODUCTION 33 pods may be due in its last analysis to the finer structure of the pro- toplasmic body in which the skeleton or shell parts are deposited. Dreyer (1892) has given evidence to show that the form and size - . \ 4^1 &&*\ \ X Fig. 11. -Pseudodifflugia sp. circular mouth opening and mosaic shell (.4). B, division stage. (Original.) Fig. 12. — Schematic figure illustrating the modifications of skeletons according to mechanical principles of deposition. (After Dreyer.) of the elements making up the skeletal or shell parts depend upon the alveolar make up of the protoplasm, the interalveolar deposits of silica, etc., taking the form of spicules as in Heliozoa and many 3 34 BIOLOGY OF THE PROTOZOA Radiolaria, of bars, hexagons, rings, fenestrated capsules, etc. (Fig. 12). Freely moving types are usually monaxonic. The type form of a freely moving flagellate or holotrichous ciliate is ellipsoidal, the cell being drawn out with its main axis extending in the direction of movement. Attached forms are usually polyaxonic or radially sym- metrical, the variations in form depending upon the nature of the B Fig. 13. — Diphasic rhizopods. A, B, C, heliozoa-like and flagellated stages of Dimorpha mutans, (After Blochman.) D, E, F, Dimastigamoeba gruberi, ameboid and flagellated stages; E, origin of blepharoplast (bl) from endosome; r, rhizoplast. (After C. W. Wilson.) attaching portion. Some for example are attached by the proto- plasm of the posterior end of a cylindrical body (e. g., Cothurnia, Vaginicolla, etc.); others by the more or less stalk-like attenuated end of the body (e. g., Scyphidia, Podophrya, etc.); and others by chitinous stalks of variable length (Vorticella species) which may be more or less branched (Poteriodendron, Epistylis, Carchesium, Zooth- amnium, etc.). In the same individual the form may change with INTRODUCTION 35 change in mode of life, well illustrated by Bimorpha mutans (Fig. 13), by Bimastigamoeba gruberi or Trimastigamoeba. Fantastic types such as Biscomorpha pectinata or Tripalmaria dogieli (Fig. 14) are not uncommon and no evident connection between such bizarre forms and their mode of life is apparent. Methods of food-getting and the nature of the food are also potent factors in determining form. Many of the diatom- and desmid- eating eiliates, whose food lies on the bottom, are characteristically flattened forms with the mouth on the under, or physiological ventral, surface (holotrich- ous eiliates belonging to the genera Chilodon, Orthodon, Opisthodon, Chlamy- dodon, Loxophyllum, etc., and the major- ity of the hypotrichous eiliates) . Special food-getting, or current-directing, organs frequently modify the form as in the collared flagellates (Choanoflagellates) and in types like Folliculina ampulla (Fig. 94, p. 169), Bursaria truncatella (Fig. 94, p. 169), cephalont gregarines, Pleuronema (Fig. 199, p. 482), etc. Shift- ing of the position of the mouth in re- sponse to different food requirements, as Biitschli has shown, has undoubtedly been the cause of some form changes. Thus the proboscis-bearing species and the asymmetrical Chilodon types may owe their characteristic forms to such a shifting of the oral region (Fig. 15). The monaxonic types, while typically ellipsoidal in form, are frequently characterized by a spiral twisting of the cell body, espe- cially in the rapidly moving forms. In some cases, notably in the flagellates Streblomastix, Spiromonas, Holomastigotes, etc., and in the eiliates Aegyria, Paramecium, Metopus sigmoides, etc., the spiral twist is highly characteristic (Fig. 16). Bilateral symmetry is of rare occurrence among Protozoa; indeed there seem to be few significant cases, that of Giardia being the best known (Fig. 17). Here the two nuclei, the motor complex and the eight flagella are arranged in the neatest bilateral manner. One possible mode of origin of such bilaterally symmetrical types is indicated by Uroleptus mobilis (Fig. 18). Here two individuals, after conjugation, fused to form a single double and bilaterally symmetri- cal individual which persisted through 367 generations (see also Fig. 127, p. 245). Form may be dependent also upon the mode of reproduction. Fig. 14. — Tripalmaria" dogieli (minor). Gut parasite of the horse with three bundles of cilia and internal skeleton. X 520. (After Strelkow, Arch. f. Pro- tistenkunde, courtesy of G. Fischer.) Fig. 15. — Diagrams illustrating shifting of the mouth in ciliates from terminal to lateral or ventral surface (A, B, C, D). E, Prorodon griseus, corresponds with A; F, Am.phileptus claparedi, corresponds with B or C; and G, Nassula microstoma, corre- sponds with D. (E and F, after Butschli; G, after Calkins.) A Fig. 16.— Types of spirally wound Protozoa. A, Streblomastix strix. (After Kofoid and Swezy.) B, Lacrymaria sp. (Original) ; C, Heteronema sp. (Original.) (36) INTRODUCTION 37 In this connection we have to do only with the multinucleated and with the colonial forms of Protozoa, for in ordinary division the daughter cells separate completely and reproduction has no effect on the form assumed. Thus the foraminiferon Allogromia oviforme gives rise by what is termed budding division to a free daughter L-__/ M-— *i — m Fig. 17 Fig. 18 Fig. 17. — A bilaterally symmetrical flagellate, Giardia muris Grassi. AX, axostyle; B, blepharoplast; BB, basal body; C, centriole; E, endosome; N, nucleus; PL, parabasal body; RH, rhizoplast. (After Kofoid and Swezy.) Fig. 18. — A bilaterally symmetrical ciliate from Uroleptus mobilis. A double individual formed by fusion of two individuals after conjugating. With two mouths and adoral zones (a. z.); two sets of cirri (/); and two sets of macronuclei (M) and micronuclei (m). For structure of single individual see Frontispiece. (Original.) cell which builds an independent test for itself while the other cell remains in the old test. In other forms of Foraminifera, however, the bud of protoplasm does not become separated from the parent bulk of the cell but takes a position in relation to the other portion which possibly depends upon the physical conditions of the proto- 38 BIOLOGY OF THE PROTOZOA plasm. New shells are deposited about the buds and chambered individuals result (Fig. 19). Repetition of the process gives rise to distinct types of polythalamous or many-chambered Foraminifera, depending upon the position assumed by the bud (Nodosarine, Frondicularian, Rotaline types, etc.). Dogiel (1929) interprets the duplication (polymerization) of organelles such as contractile vacuoles, macro- and micronuclei, flagella groups, particularly of Polymastigida, somatella formation (see p. 233), multiple nuclei and kinetoplasts of Calonymphidae (see p. 115), etc., as evidence of gradations in cellular differentia- tions in Protozoa leading to a multicellular condition which is fully established in Metazoa. I D V Fig. 19. — Types of shells of Foraminifera. A, B, side and ventral aspects of Cornu- spira sp. ; C, and D, types of Nodosaria. (After Carpenter.) In colonial types the form of the aggregate is determined by the manner in which the individuals are held together after division. The different types are described as spheroid, catenoid, arboroid and gregaloid colonies. In the majority of spheroid colonies, the associated cells are held together by a gelatinous matrix secreted by the individual cells. The typical form of such colonies is spher- ical as in the genus Proterospongia, among the flagellates, or Ophryd- ium versatile among the ciliates. In catenoid colonies the individuals are attached end to end as in some species of ciliates (e. g., Hapto- phrya), or side by side as in the flagellate Rhipidodendron. In arboroid colonies the individuals are attached by longer or shorter stalks in a branching, often bush-like colony [Clathrulina elegans, Poteriodendron petiolatum (Fig. 139, p. 418) , Codosiga eymosa (Fig. 20), Epistylis umbellaria (Fig. 143, p. 280), Carchesium polypinum, Zooth- amnium arbusctda, etc.] In the majority of these arboroid colonies each individual is borne on its own stem which branches from a common stalk. In some cases, however, especially amongst the flagellates, each stalk bears a cluster of individuals as in Cladomonas INTRODUCTION 39 fruticulosa, Anthophysa vegetans (Fig. 21) or Phalansterium digi- iatum (Fig. 22). In Rhipidodendron splendidum the gelatinous branches, colored brown or red by oxide of iron, are arranged in parallel rows, spreading out fan-like as they increase with divi- sion of the cells, the aggregate forming an organ-pipe-like arboroid colony. Gregaloid colonies, finally, are fortuitous aggregates of previously independent individuals found mainly amongst the rhizo- pods and Heliozoa, or in parasitic flagellates under adverse envir- onmental conditions (Spirochetes, Try panosomes) . The origin of gregaloid colonies is not connected in any way with the manner of reproduction. Fig. 20. -Type of flagellate colony. Codosiga cymosa Kent, an arboroid colony of collared flagellates. The combination of all of the above factors effective throughout past ages has resulted in fixed, complex forms which, as in Metazoa, are today associated with the germinal make-up of the protoplasm or genotype, and are transmitted by inheritance. B. Protoplasmic Structure.— All protoplasm contains the same fundamental chemical elements — C, H, N, O and P— which are necessary for the performance of vital activities. With these are associated mineral elements of one kind or another— Na, K, Ca, Mg, Fe, S, etc., usually as salts of different kinds, and water. In its last analysis form depends upon the chemical and physical combinations of these elements which indicate specific protoplasmic 40 BIOLOGY OF THE PROTOZOA organizations and interactions of different protoplasmic substances and which form the physical basis of inheritance. A minute frag- ment of Uroleptus mobilis is difficult to distinguish from a similar fragment of Dileptus gigas, yet the former develops into a perfect Uroleptus, the latter into Dileptus. The encysted forms of many types are impossible to identify until the cysts are opened and vital processes begin again. These facts indicate that the finer or ulti- mate composition of protoplasm is different in different forms and 4 A . . . . 24 . 3 Phytostearin 3.2 Asparagin J Phosphatids 1.3 Nucleoproteid 32 . 3 Other organic stuffs . . . 3.5 Free nucleic acid . . . . 2.5 Mineral stuffs 3.4 INTRODUCTION 45 the Protozoa must be traced back to the chemical nature of the proteins and to their relations and interactions with other substances in protoplasm. Types which have a similar chemical and physical make up, with similar metaplastids and plastids, are practically identical in form and structure and we recognize them as distinct species. Variations in chemical composition, be they ever so little, must result in different chemical reactions and products, and in corresponding variations in form and structure of the organism, and these variations furnish the basis for classification. Under normal environmental conditions the reactions among the varied substances in protoplasm of the same species, with their products and arrangement of these products, are individual and invariable. Furthermore, the entire organism partakes of this indi- viduality. A fragment of Stentor obtained by cutting or by shaking cannot be distinguished from a similar fragment of Dileptus, yet the former regenerates into a perfect Stentor, the latter into a per- fect Dileptus. Or an encysted Uroleptus mobilis is morphologically identical with an encysted Didinium nasutum; both are apparently homogeneous balls of undifferentiated protoplasm; the one emerges from the cyst and develops with the characteristic differentiations of Uroleptus, the other of Didinium. In short, the homogeneous ball representing Uroleptus is as specific and different from the homogeneous ball representing Didinium, as the adult Uroleptus is different from the adult Didinium. We may speak of this undiffer- entiated chemical and physical make-up as the fundamental organ- ization of the species, in a sense similar to the architectonik of Driesch. The adult characteristics result from the interactions of the specific proteins, carbohydrates, salts, water, etc., among themselves and with the environment, and represent what we may call the derived organization. Organization in the above sense is not only specific but is con- tinuous from generation to generation, and has come down through the ages subject, however, to modifications and changes through interaction with the environment or through changes coming from within as in amphimixis. While organization is continuous the actions and reactions going on within it are discontinuous. More or less prolonged periods of rest are characteristic of all living things, best exemplified in the case of spores, eggs, encysted Protozoa and seeds. At such times the organization is static; the chemical substances making up the specific organization are present but quiescent, or at least, in the absence of water, relatively inactive. A striking illustration is afforded by the phenomenon of desiccation in some types of animals, e. g., rotifers, which has been known for decades. For some years I had on my shelf a bottle of minute amorphous granules which appeared like specks of dust under the microscope. After placing a 46 BIOLOGY OF THE PROTOZOA few of these granules in water each of them would become an active, living rotifer in an hour or so. Here organization was present but inactive, and activity began with the absorption of water and with oxidation. The rotifer in the active state is the same rotifer that it was in the dried condition, so far as organization is concerned, but it differs in that the organization is now in action. It is a difference of the same nature as that between an automobile stand- ing in the garage, and the same automobile travelling 30 miles an hour. The organization is in action in both moving rotifer and mov- ing automobile; is static in the dried rotifer and in the standing machine. The automobile simile, however, will not stand analysis. The parts of the machine are little changed by activity and the organ- ization remains the same throughout its period of usefulness. With a living thing, on the other hand, the chemical and physical make up changes with every activity and, as a result of such activities, the protoplasmic organization itself will change. An encysted Uroleptus is a motionless and apparently a homogeneous ball of protoplasm; an hour later it is an elongate, cigar-shaped organism with special- ized motile organs in the form of membranelles and cirri, and its contractile vacuole pulsates with rhythmical regularity as it moves actively about in the water. The organization has undergone a change in this brief period; the first indication is the swelling and enlargement of the cyst wall, evidently by the absorption of water; oxidation probably occurs and substances already present, or new substances formed as a result of this initial oxidation, are responsible for the newly-developed structures or derived organization not present before. Such structures, however, are the morphological expression of the adult organization and their formation corresponds to the development and differentiation of the metazoon egg. Continued activity involves other and still more subtle changes in organization; some of these are evident in individual life between division periods; others are evident only in a long series of individuals constituting a life cycle. These will be more fully treated in Chap- ters VII and VIII. Other changes in organization may be brought about by environ- mental conditions; or they may be brought about by changes in one or more of the substances constituting the protoplasm of the species, as when amphimixis introduces a new combination of chro- matin into the organization. These are undoubted factors in the phenomena of adaptation and probably play a part in the orig- ination of new species and types. Consideration of these and of similar activities in living proto- plasm lead to questions regarding the nature of life and the nature of vitality. Should we use the two terms life and vitality as syno- nyms? We are very apt to speak of life as activity, or to say that INTRODUCTION 47 life is a series of reactions, integrations and disintegrations. These may be manifestations of life but they are incomplete manifesta- tions and do not tell the whole story. An encysted protozoon, a spore, a seed, a resting egg, or a dried rotifer, shows no more evi- dence of activity than does a parked car, yet each has life and in a proper environment would manifest activity. An emulsion of oil, salts and water manifests activity strikingly similar to the move- ments of an Ameba, yet such an emulsion has no life. The encysted protozoon or the dried rotifer has protoplasmic organization which the oil emulsion has not, and with absorption of oxygen and water becomes animated. Life thus is incontestably bound up with organization of protoplasm and, for descriptive purposes at least, we find a distinct advantage in a clear discrimination between this concept and the concept vitality. Whatever name we give it, however, brings us no nearer to a conception of what life actually is, for it cannot be measured and endures until the organization is disintegrated. With vitality the case is different; here we have to do with protoplasm in motion and the activities can be measured from beginning to end of a life cycle. While organization has evi- dently been continuous from the first protoplasm, vitality has been intermittent or discontinuous. Organization may exist without vitality and has always the potential possibility of vitality, but vitality is impossible without organization. I would define vitality, therefore, as the sum total of actions, reactions and interactions between and amongst the substance* making up the organization of protoplasm and between these and the environment, while life may be defined as protoplasmic organization manifesting vitality or with a potential of vitality. CHAPTER II. THE FUNDAMENTAL ORGANIZATION. Weismann's conception of a metazoon as made up of germinal and somatic protoplasm is equally true of a protozoon. Here, however, the two are combined in the make-up of a single cell, and Weismann was not entirely right in considering all Protozoa as equivalent to the germinal protoplasm only of Metazoa. In gen- eral the derived organization of a protozoon is a combination of the fundamental organization which retains its fundamental germinal characteristics and the derivatives from it which characterize the adult or fully differentiated individual. Like the metazoan somatic plasm, these derivatives have a limited existence, and again like somatic plasm, new ones are formed from the germinal protoplasm with each successive act of reproduction. An essential difference between the somatic structures of Protozoa and those of Metazoa, is that such structures in Protozoa are reversible while in Metazoa they are irreversible. It is important to make the attempt at least to distinguish between the fundamental or germinal protoplasm and the structures which are derived from it. The latter, as in Metazoa, provide the structural features by which species are differentiated and classified. Although with our present knowledge it is impossible to analyze protoplasm and to discover the nature of the ultimate fundamental organization which involves the differences between species, it is possible by experiment and upon a morphological basis to determine what protoplasmic parts are necessary for perfect development. Thus, in the experiment with fragments of Stentor or Dileptus (see p. 45), we find that no development occurs if nuclei are not included in the fragments, and nuclei without cytoplasm are equally impotent. So, too, in all encysted Protozoa, we invariably find a combination of nuclei and cytoplasm. The legitimate inference is that both nucleus and cytoplasm are necessary for continued vitality and that interactions between these two primary components are necessary for the formation of the structures of the derived organization. This is such a fundamental biological truth that it seems hardly necessary to emphasize it here. It is difficult to distinguish upon a morphological basis between the visible differentiations of the fundamental organization and structures of the cell which should be included more properly in THE FUNDAMENTAL ORGANIZATION 49 the derived organization. Some substances are found in all Protozoa and these may be considered the raw materials from which the derived organization is manufactured. Although they are intimately related, it is convenient to describe the constituents of the nucleus and those of the cytoplasm under separate headings. I. NUCLEAR SUBSTANCES AND STRUCTURES OF THE FUNDAMENTAL ORGANIZATION. The term "nucleus" is ordinarily applied in a morphological rather than a physiological sense. If the activities of the component parts of the nucleus are absolutely necessary for the maintenance of life of the cell, then, in some cases such as Holosticha, Trachelocerca, or Vile phis, such activities must be performed by substances which appear to be identical with chromatin but which are distrib- uted throughout the cell. On the other hand, it is highly probable that some functions are possible by virtue of the physical prop- erties of a definite, but permeable, nuclear membrane, as in the tissue cells of Metazoa. It is this type of membrane-bound nucleus that we find in the vast majority of Protozoa. Certain constantly recurring substances are characteristic of protozoan as of metazoan nuclei, but some types of arrangement and combination of these substances are typical of Protozoa and are rarely found in Metazoa. The most universal of these nuclear constituents are (1) chromatin, which is sometimes called nuclein or identified as such; (2) nuclear sap or nuclear enchylema filling the spaces of the linin reticulum; (3) nuclear membrane which forms a permeable partition between cytoplasm and nucleoplasm; (4) plastin, often so called without being specifically identified as such; also termed paranuclein, or pyrenin. Plastin by itself forms true nucleoli which are comparatively rare in Protozoa. In addition to these, kinetic elements are characteristic of the majority of protozoan nuclei, and these in the present work will be called endobasal bodies. It must be frankly admitted that very little is known in regard to the chemical nature of these various constituents of the nuclei in Protozoa and much confusion exists in the literature owing to the promiscuous use of these terms in relation to structural elements of the nucleus without knowledge of the actual chemical make up. In their resting stages the nuclei of Protozoa present a bewildering variety of forms and structures, differing in this respect from the much less variable tissue nuclei of the Metazoa. Because of these manifold differences students of the Protozoa have experienced great difficulty in grouping nuclei for purposes of description. They agree, however, in recognizing two primary nuclear types, the 4 50 BIOLOGY OF THE PROTOZOA vesicular and the massive. Nuclei of the massive type more clearly resemble the nuclei of spermatozoa being filled with small chromatin granules, but they rarely present the homogeneous appearance of a spermatozoon nucleus, the individual granules, although closely packed, being recognizable (Fig. 23). In vesicular nuclei the chromatin granules may be distributed more or less evenly through- Fig. 23. — Types of vesicular and massive nuclei. A, vesicular type of Pelomyxa binucleata; B, of Polystomcllina crispa; both with multiple endosomes; C, nucleus of Actinosphacrium eichhornii with granular plastin (p); D, E, F, macro- and micro- nuclei of Paramecium caudatum, the latter in different stages of vegetative mitosis. (A, B, after Doflein; C, after Hertwig; D, E and F, original.) out the nucleus, or they may be segregated in "net-knots" or either alone or combined with other nuclear substances may be combined in one large central globular mass to which Minchin gives the name endosome as an equivalent for the term Binnenkdrper , or they may be aggregated in several such globular masses or multiple endo- somes distributed throughout the nucleus or plastered to the nuclear membrane. THE FUNDAMENTAL ORGANIZATION 51 Endosonies may consist entirely of chromatin as appears to be the case in nuclei of some Microsporidia (Glugea and Thclohania), or some flagellates (Prowazekia, Belar, 1920, etc.). Or they may be composed of chromatin and plastin in various combinations. Thus in Actinosphaerium eichhornii in some stages of nuclear activ- ity, the chromatin component is in the form of an incomplete ring which partially encloses the plastin portion (Fig. 23, C). In other cases the plastin is entirely surrounded by a cortex of chromatin which may be dense and compact as in the case of many types of rhizopods and Sporozoa or loosely aggregated as in nuclei of End- amoeba intestinalis (Fig. 24). The distributed granules of deeply staining material which represent the substitute for a nucleus in Dileptus gigas are similarly composed of a plastin core and a chromatin cortex, / X the former increasing enormously after / - '^. ; « ^ treatment of the animal with certain /• -'.'';;" I * ' 1 kinds of food such as beef broth. Here | « v ; , '■■ the term endosome is scarcely applicable " ^ ;-;| since the bodies in question are not in- {'% ',-.* \ ;j M " :, '■". ' side a nuclear membrane, but they appear ( ^Nfe • 'c ■*' to be morphologically equivalent to these intranuclear structures. After treatment with beef broth the body of Dileptus is enormously distended due to the swelling x t v %,'<& of these cytoendosomes (Fig. 25). \'% '*^-^^ The centrally placed intranuclear body ^4§ . is generally described under the name karyosome, a term which has been so r Jj G \f:~ E ' ldamoch ^ intes - . *. ' . , , „ ,, -r, hnahs; (e) endosome; (c) cor- widely used by students or the rrotozoa tex of chromatin. and for so many obviously different structures that it is practically synonymous with endosome or Binnenkorper. Thus Minchin describes it as a combination of chro- matin and plastin; Doflein defines a karyosome as a centrally placed, sharply outlined and constant constituent of the nucleus, which may contain no chromatin or may be a combination of other substances with chromatin and which divides during nuclear division, to form two corresponding daughter structures. Hartmann's (1911) defini- tion is more limited, a karyosome in his use of the term being an endosome (Binnenkorper) containing a centriole. Belar (1921) finds a "karyosome" in Chlamydophrys minor which breaks up and dis- appears, forming neither chromatin nor kinetic elements. If we attempt to combine these different views into a common definition we find that a karyosome may be an intranuclear body which may consist of plastin alone; or kinetic elements alone; or chromatin together with plastin; or a combination of chromatin with kinetic elements; or a combination of chromatin, plastin and kinetic ele- , 52 BIOLOGY OF THE PROTOZOA merits. Such a definition obviously would fail to specify any par- ticularly nuclear structure, and so far as its practical value is concerned the term karvosome is no more useful than the non- Fig. 25. — Dileptus gigas: A, vegetative individual in culture with nucleus in the form of scattered chromatin granules; B, individual showing the effect of treatment with beef extract on the chromatin granules. (Original.) committal term Binnenkorper or Minchin's equivalent term endo- some. I would advocate, therefore, discarding altogether the term karvosome which seemingly bears the earmarks of something definite in the cell, using in its place the general non-committal THE FUNDAMENTAL ORGANIZATION 06 expression Binnenkorper, or its equivalent term endosome, the latter as yet, at least, having no specific significance, while for the endosomes having functions characteristic of the kinetic complex a specific term may well be applied. In the present work 1 shall employ the term endosome in a general way to indicate all central intranuclear structures including those of kinetic function, while for those which are known to be of the nature of kinetic elements I shall use the term endobasal body. Fig. 26. — Division of amebae. A to /, successive stages in division (promitosis) of Vahlkampfia Umax; J to L, mitosis in Endamoeba coli. (Original.) The endosome-bearing vesicular nuclei present manifold variations in the arrangement of chromatin. In some the entire chromatin content is confined to the endosome which seems to rest in the center of a colorless enchylema traversed by strands of linin radiating from the endosome to the nuclear membrane (Arcella vulgaris, Cochlio- podium bilimbosum and rhizopods generally, as well as in many 54 BIOLOGY OF THE PROTOZOA Coccidia and Gregarinida). In other cases the endosome retains only a little of the chromatin, the bulk of which is present as a dense network in the zone between endosome and membrane (Endavioeba intestinalis, A. crystalligera, etc.). In still other cases the chromomeres are distributed more or less uniformly throughout the nuclear reticulum (Euglypha alveolata, etc.). In vesicular nuclei with endobasal bodies the chromatin may be in the form of more or less regular chromomeres uniformly dis- tributed in the nuclear space (Euglejia type), or more or less com- pactly aggregated about the kinetic element (many species of Endamoeba, various flagellates, Coccidia and Myxosporidia, etc.). Or, finally, the chromatin may be in the form of relatively large granules collected in a zone just within the nuclear membrane (e. g., Pelomyxa), or in fine granular form may make up the chief part of the nuclear membrane (Vahlkampfia Umax, Fig. 26). 1. Chromatin. — Chromatin has been more a conception than a specific thing, the term being used to designate substances which appear under different forms at different phases of cell life. It appears normally in the form of minute granules or chromomeres (chromidiosomes of Minchin) in the resting nucleus, but during division of the nucleus these granules are massed together usually to form characteristic solid and individualized structures, the chromosomes. On a 'priori grounds chromosomes were early regarded as intimately associated with the phenomena of inheritance (Roux, Weismann, Boveri) and the more recent experimental work in genetics has given substantial evidence of the soundness of this early conclusion. Our conception of chromatin is based largely upon investigations upon the nuclear substances of Metazoa and the higher plants. In ordinary descriptions, however, the term is often used in a vague sense to include any substance or body which stains with the so- called nuclear stains, i. e., the basic anilin dyes, while direct chem- ical tests to determine the exact chemical composition of chromatin have been made in very few cases. The best of these show it to be composed mainly of nuclein, one of the most complex of protein substances and rich in phosphorus. 1 Vague as is the conception of chromatin in Metazoa it is even more so in connection with the Protozoa, where little has been done in a concrete way to throw light on the subject, although much has been written about it. Many of the granules found in the cell body of a protozoon as well as those within the nucleus, stain with the usual nuclear dyes and their identification as chromatin is a matter requiring knowledge of their history and fate in the cell. It is only within recent years 1 For :i critical discussion of chromatin, see Wilson, 1925. THE FUNDAMENTAL ORGANIZATION 55 that an effort has been made to discriminate between the various granules in the Protozoa which stain intensely with the basic stains, and to distinguish the chromatin granules which enter into the make up of chromosomes from other chromatoid granules which are distributed throughout the cell, particularly the chromidia and the volutin grains. This is the more difficult in Protozoa because chromatin granules are not necessarily confined to the nucleus. Even in Metazoa and plants there are times during division when the chromatin is not confined within a nuclear membrane. In the Protozoa such a condition is permanent in many cases (e. g., in some flagellates; in Dileptus gigas, Holosticha, etc.). In other cases the nuclear chromatin, by transfusion or by nuclear fragmen- tation, spreads more or less widely throughout the cell protoplasm (rhizopods, Actinosphaerium eichhornii, etc.). Here in different species, the fate of the distributed chromatin varies. In some cases this diffusion of chromatin indicates a degenerative change, the chromatin ultimately losing its characteristic reactions. Thus in Actinosphaerium eichhornii, Hertwig has shown that, under adverse conditions such as starvation, or overfeeding, or during periods of depression, such distribution of the nuclear chromatin occurs, the granules ultimately becoming transformed into a characteristic pigment of the cell. In other cases the distributed granules retain their chromatin nature and according to numerous observers are ultimately aggregated into minute secondary nuclei which become the nuclei of conjugating gametes (see p. 69). In these instances, other chromatin which is retained in the "primary nucleus" takes no part in the germinal activities but degenerates and disappears after the gametes are liberated. It must not be inferred that germinal chromatin is thus distributed in the cyto- plasm in all cases; on the contrary in the majority of Protozoa the gamete nuclei are derived by division of the morphological nucleus with its contained chromatin, and some authorities, notably Kofoid (1921) deny in toto the origin of gamete nuclei from chromidia. While chromatin thus has a definite germinal function there is equally little doubt of the important participation of the nucleus and presumably of chromatin in the ordinary metabolic activities of the cell. Thus, if an Amoeba proteus or the ciliate Uronychia transfuga (see Fig. 135, p. 262), be cut into two portions one of which contains the nucleus while the other is enucleate, the former portion only will digest and assimilate food, grow and regenerate the lost part, while the enucleate portion will continue to move and manifest various activities characteristic of destructive metab- olism, but it will not take in food, nor digest what food may have been taken in before cutting, and in the course of a week or ten days it dies (Hofer, Verworn, Balbiani and many others). It is evident that chromatin is directly associated with all of 50 BIOLOGY OF THE PROTOZOA the important vital activities including reproduction, and the view has been repeatedly advanced that, for these varied activities at least, two different kinds of chromatin are responsible. One kind, the so-called vegetative or trophochromatin, is active in the ordi- nary metabolic functions of the cell, while the other, the germinal or idiochromatin, has to do solely with perpetuation of the race. While this view of the dual nature of chromatin would seem to be sustained by the phenomena in rhizopods, gregarines, and by the dimorphic nuclei in the ciliates, it is by no means assured that this duality represents a fundamental difference in chromatins. On the contrary it is much more probable, as Hertwig has maintained, that there is only one chromatin and that its functional activity depends upon different factors and conditions which may arise during the life cycle; germinal chromatin in one cell-generation may become vegetative chromatin in the next and vice versa. This is particularly clear in the case of the ciliates where the macronucleus, a distinctly vegetative nucleus, and the reproductive micronucleus, arise as subdivisions of a fertilization nucleus after conjugation or its equiva- lent parthenogenesis. The importance of chromatin for life of the cell is indirectly indi- cated by the extreme precision with which it is distributed to daughter cells at the time of division. Like other granules of the cell each chromomere grows and reproduces its exact duplicate by division. Chemically it probably represents the pinnacle of complex structures formed as a result of the activities of constructive meta- bolism while its derivatives, likewise granular in form and difficult to distinguish as chromatin, give rise to many more or less permanent or temporary structures in the cell body, each of which may per- form some cellular activity in its passage through the various stages of chemical breakdown. Few investigations of a purely chemical nature have been made on protozoan chromatin. The usual procedure is to designate as chromatin all structures of the nucleus which stain with the so-called nuclear dyes, or to interpret chromatin mainly on a morphological basis. Micro-chemical tests of all protoplasmic substances are made primarily on the basis of solubility or insolubility with acids, alka- lies, salts, etc., and the conclusion that certain structures are made up of certain substances follows from the microscopic picture pre- sented after such treatment. Such tests do not prove that a given structure is composed of a definite substance and is not a mixture of substances. Kossel, Miescher and others have shown that the chromatin bodies composed mainly of the chemical substance nuclein are not dissolved under the action of artificial gastric juice (pepsin and trypsin in appropriate acid and alkaline media) while other portions of the nucleus such as nucleoli and reticulum are entirely dissolved. Chromatin bodies on the other hand are dis- THE FUNDAMENTAL ORGANIZATION 57 solved in strong acids, dilute alkalies, calcium carbonate and sodium phosphate. There has been a tendency to regard chromatin as the most important substance of the living cell, and the chromosome as the most important nuclear structure. Important they doubtless are, but in many cases chromatin is known as such only in the form of chromosomes which belong to the derived and not to the funda- mental organization (see p. 88). In other words, chromatin is manufactured in the nucleus and the substances or substance from which it is made are still more fundamental. There appears to be little justification for Heidenhain's view of two kinds of chromatin, one— oxy chromatin— unstainable with basic dyes, the other— basi- chromatin — staining readily. A substance in the nucleus is either chromatin or it is something else. With the growing use of the Feulgen nucleal reaction there is reason to believe that a more precise definition of chromatin will be developed. This reaction finds its explanation in Steudel's (1912) analysis of thymonucleic acid of which the empirical formula is: C43H65P4X15O34. 1 Under moderate hydrolysis with HC1 the purin bodies are split off the molecule of thymonucleic acid and reducing groups are freed. These behave like aldehydes and give the charac- teristic red-violet color with Schiff's test (Magenta in the presence of sulphuric acid). The nuclei of various groups of Protozoa give positive chromatin reactions with this test, and it is a useful method in tracing the development of chromatin in ex-conjugants or in the chromosomes of the maturation divisions. (See Feulgen and Rossenbeck, 1924; Bresslau and Scremin, 1924; Robertson, 1927; Zuelzer, 1927; Jirovec, 1927; Reichenow, 1928, and infra pp. 93 and 315.) 2. Other Substances of the Nucleus. — Belaf (1926) makes this state- ment concerning nuclei of the Protozoa : " For the most part chro- matin of the resting nucleus cannot be distinguished from the ground substance of the nucleus (loc. cit., p. 241)." This refers to the conditions of the living nucleus and not to fixed and stained material. In the latter chromatin in the form of granules can be 1 This may be written: (H 2 0)2— P— CsHioOs— C 5 H 4 N 6 (adenine) / \ O O \ / P— C 6 H I0 O5— C6H4N5O (guanine) / \ O O \ / P— CeHioOs— C5H5N2O2 (thymine) / \ O O \ / (H20)2— P— C 6 Hio0 5 — C4H4N3O (cystocine) 58 BIOLOGY OF THE PROTOZOA distinguished from other substances of the resting nucleus by their color reactions to basic and acidic dyes. Sometimes the chromo- meres or chromioles are apparently suspended in a more or less definite "linin" reticulum which is recognized as being a coagulation product of the colloidal ground substance or karyolymph. In other Fig. 27. — Origin of macronucleus after conjugation in Uroleptus mobilis. (1) First metagamic mitosis of the amphinucleus; (2) one of the progeny of this division dividing again; (3), (4), (5) telophase stages of second division of the amphinucleus resulting in a new macronucleus (above) , and a degenerating nucleus (below) ; (6 to 10), stages in differentiation of the young macronucleus and disintegration and absorption of the old macronucleus; in (10) two new micronuclei are in mitosis preparatory to the first division of the ex-conjugant. (At) new macronucleus; (m) new micronuclei; (d) degenerating old macronuclei. (After Calkins.) cases they are combined with the substance "plastin" to form a clearly-defined endosome (karyosome) which, depending apparently on the relative proportions of plastin and chromatin, may or may not be visible in life. Plastin appears to be a well-defined nuclear substance and writers generally speak of it with familiar ease, THE FUNDAMENTAL ORGANIZATION 59 despite the fact that very little definite information is at hand con- cerning it. In pure form it is the nucleolus of tissue cells and stains intensely with acid dyes. Such nucleoli are rare in Protozoa, but the combination of plastin with chromatin in some degree is char- acteristic of Protozoa, and the staining reaction with basic or acidic dyes varies with the preponderance of one or the other. The ground-substance of the nucleus or karyolymph (Lundegardh) is difficult to define, a difficulty which Belar (1926) recognizes by the statement: ". . . at best it can be defined as that part of the nuclear space which is neither chromatin nor plastin" (loc. cit., p. 242). From this negative definition and from the fact that it cannot be demonstrated by specific staining reactions or character- ized by definite structures, it might seem that karyolymph is a negligible part of the nuclear make-up. Such a conclusion, how- ever, would be a mistake for some of the most important structures of the active nucleus take their origin from this ground substance (see pp. 88, 200). Fig. 28. — Vahlkampfia Umax; chromatin forming the nuclear membrane and giving rise to chromidia. (After Calkins.) Membrane. Like other constituent parts of the protozoon nuclei, the membranes are highly variable, sometimes presenting in optical section only one contour on the outer side (e. g., Actinosphaerium) ; sometimes showing contours both outside and inside (Amoeba pro- teus) . In the former case the inner zone adjacent to the membrane shows a decreasing density inwards, until the linin merges insen- sibly into the intranuclear reticulum. In free-nuclei formation, antecedent to gamete formation described above, the nuclear mem- branes are probably formed from the cytoplasmic reticulum in which the chromidiosomes are lying. Chromomeres also take part in the formation of nuclear membranes in some cases, e. g., in Vahlkampfia Umax, where the linin membrane is too delicate to be seen, although the definite limitation of the chromomeres indicates its presence (Fig. 28). One peculiarity of the nuclear membranes of Protozoa which dis- tinguishes them from nuclear membranes of tissue nuclei, is that in the majority of cases they remain intact during all phases of cellular activity and only rarely disappear, or disappear in part only, during division processes of the cell. (For description of chromatin, mem- branes, etc., during division, see p. 209.) 60 BIOLOGY OF THE PROTOZOA Intranuclear Kinetic Elements. The kinetic elements, some of which are intranuclear and a part of the fundamental organization, are those structures of the cell which are closely connected with the visible expression of the transformation of energy resulting from destructive metabolism. Such expression may be in the form of movement due to the activity of specific motile organs formed as a rule from the substance of kinetic elements, or it may be in the form of intracellular activities as indicated by the transformation and movements of internal attraction centers, center of radiation, of nuclear division, etc. The kinetic elements are justly regarded by many observers as the most elusive and perplexing, but at the same time amongst the most fascinating of all the organoids of Protozoa. Kinetic elements appear in Protozoa in a multitude of structures, sometimes intranuclear, sometimes cytoplasmic, and often both inside and outside the nucleus. Whether or not they are permanent organoids of the cell is subject to the same arguments pro and con which have been raised for and against the permanency of the cen- trosome in Metazoa. There is strong evidence, as the following pages will show, that not only are many types of cytoplasmic kinetic elements derived from the nucleus, but also that chromatin and intranuclear endobasal bodies are closely related, while some types that are confined to the cytoplasm are composed in part, or entirely, of a substance which closely resembles chromatin (parabasal bodies). Little is known of the chemical composition of the latter, but both intranuclear and cytoplasmic kinetic elements stain intensely with some of the nuclear dyes and divide by simple constriction at periods of cell division. In many cases it is impossible to tell from observations on ordi- nary vegetative individuals, whether a given structure belongs to the kinetic elements or to some other group of the many types of protoplasmic granules. This is particularly true of the intranuclear forms where incomplete extraction of a stain may give the appear- ance of a granule in some chromatin or plastin mass. In such cases the identity of the structure can be determined only by its history during nuclear division. Cytoplasmic forms can be more easily detected by reason of their relation to motile organs or to more or less complex fibrillar structures. («) Endobasal Bodies. — Pmdobasal bodies in nuclei of different Protozoa are highly variable and no general description is possible. In some cases they stain intensely with nuclear dyes, especially with iron hematoxylin; in other cases they stain feebly or not at all with the same dyes that color the chromatin (e. g., Chilodon). In some cases they are large and appear homogeneous throughout; in other cases there is a definite, deeply-staining central granule embedded in a more faintly staining plastin (?) matrix, or such a granule may be present without the accompanying matrix; or, THE FUNDAMENTAL ORGANIZATION 61 finally, there is no evidence at all of kinetic elements in resting nuclei, but collections of homogeneous substance (karyolymph) are present at the poles of the nucleus during division (pole plates). 1. Large Homogeneous Endobasal Bodies. — In this type the endo- basal body is conspicuous by its large size and homogeneous struc- ture. It was first described by Kenten (1895) in Euglena viridis and was early recognized as a kinetic element connected with nuclear division as attested by the names intranuclear centrosome, Fig. 29. — Bodo ovatus Stein (edax, Belaf). (1) Vegetative individual with two flagella; blepharoplast (bl) and nucleus with endosome. (2 to 6) Division of the basal bodies, blepharoplast and nucleus; (7 to 10) completion of nuclear division and division of cell body. (After Belaf, from Doflein.) division center, etc., applied to it, while nuclei containing it were included by Boveri in his "centronucleus" type. In Euglena viridis and euglenoids generally, this endobasal body according to earlier descriptions of Keuten, Tschenzoff (1916) and others is the most conspicuous structure of the nucleus, where, in the resting nucleus, it appears as a spherical or elongated ellipsoidal body with chromatin granules of limited number suspended between it and the nuclear membrane. It divides prior to division of the chromatin, first elongating with a concentration of its material at the poles. The G2 BIOLOGY OF THE PROTOZOA m o. e n d. elongation continues until a thin fibril, called a centrodesmose, alone connects the two halves. The centrodesmose ultimately breaks and its substance is ab- sorbed by the two daughter ele- ments. [See also Baker, and Hall (1923).] In the rhizopod Chlamy- drophrys stercorea, as well as in the flagellate Bodo ovatiis, the endobasal body which is quite similar to that of Euglena, divides subsequently to division of the chromatin (Schaudinn, Belaf, Fig. 29), while in Amoeba crystal- ligera (Schaudinn) there is no centrodesmose formed during division, a condition nof un- common in the rhizopods (e. g., Arcella vulgaris according to Swarczewsky ; Vahlkampfia Umax [Fig. 28], and many species of Endameba). Not only is this simple type of endobasal body found in rhizopods and flagel- lates, but also in some cases in the more complex ciliates, where, in Chilodon cucullus, for example, the macronucleus contains a definite endosome which behaves exactly like that of Euglena. It is highly Fig. 30. — Chilodon sp. Macronucleus with endosome and endobasal body (end) . (mo) Mouth surrounded by pharyngeal basket. (Original.) B Fig. 31. — Endamoeba dysenteriae (Councilman and Lafleur). Two stages in the metamorphosis of endosome and endobasal body. (After Hartmann.) probable that in all of these cases the endobasal body is em- bedded in a core of plastin. THE FUNDAMENTAL ORGANIZATION 63 2. Endobasal Bodies With Centrioles. — Centrioles are kinetic ele- ments in the form of minute granules, which in Metazoa and in some types of Protozoa, form the focal points of the mitotic spindle. In many Protozoa minute granules may be embedded in a matrix of chromatin or plastin, or in a combination of both. These in some cases form the poles of typical spindles, but in the majority of cases, apart from the polar granules and the connecting centrodesmose, there is little evidence of a typical spindle. In some forms this type of endosome undergoes changes in appear- ance which Hartmann (1911) and his followers have interpreted as periodic or cyclical in nature. Such variations have to do with the concentration of the chromatin substance about the endobasal body or centriole, being massive and dense in certain phases and distributed in others. In Endamoeba dysenteriae the centriole in the latter phase is distinct and definite but in the former phase it is hidden by the dense chromatin (Fig. 31). From such conditions Hartmann infers that all massive types contain hidden centrioles, a conception applied by Naegler to all of the smaller amebae and endamebae, but, according to Glaser, it is limited to comparatively few types. Typical endobasal bodies in the form of centrioles are contained in the first maturation nuclei of Vroleptus mobilis. Here each massive micronucleus fragments into chromatin granules which remain in a dense reticulum at one pole of the enlarging nucleus until the chromosomes are formed. A centriole, hidden in this mass, divides and one-half traverses the nucleus to form the first pole of the maturation spindle but remains connected by a centrodesmose with the other centriole which, in turn, forms the other pole of the spindle (Fig. 32, b-g). Similar centrioles are found in widely separated groups of Protozoa. In Coccidivm schubergi, according to Schaudinn (1900), the endobasal body divides with a long con- necting centrodesmose. Here, however, part of the material of the centrodesmose collects into two granules with a more densely stained connecting thread, thus producing a structure which Doflein interprets as analogous to the mid-body (Zwischenkorper) of Metazoa and plant cells. The fate of the centrioles after division differs in different cases. In some, e. g., Bodo lacertae (Belaf, 1921, Figs. 33, 34), they come from the nucleus and re-enter the daughter nuclei; 1 in others they arise from basal bodies and become basal bodies of the flagella after division (e. g., Chilomastix aulostomi, Belaf, 1921; Spongomonas, Hartmann, etc.). While the embedding matrix in most of the above cases is similar to chromatin in its reaction, and forms an important part of the endobasal body, there are other types (e. g., My.vobolus pfeifferi, 1 See, however, the earlier contradictory accounts of Prowazek (1904), Alexeieff (1914), and Kuczynski (1918). 64 BIOLOGY OF THE PROTOZOA Fig 32.-Uroleptus mobilis Eng. First and second meiotic divisions during con- jugation. (A) Two conjugating individuals; (B to G) formation of the first spind e pole by division of the endobasal body (with centrodesmose) ; (H to M) first meiotic nuclear division; (.V to Q) second meiotic division. (After Calkins.) THE FUNDAMENTAL ORGANIZATION 65 one of the Myxosporidia) in which the centriole emerges from an enveloping plastin-like matrix, which, like a nucleolus, then degen- erates and disappears. 3. Nuclei With Pole Plates and Without Endobasal Bodies. — This type of nucleus is characterized by the entire absence of endobasal bodies. A hyaline mass, which stains with difficulty, may, however, be present at the spindle poles during nuclear division, but in many cases it cannot be detected in the resting nucleus. During division it occurs in characteristic forms known as pole plates. Fig. 33. — Bodo lacertae Grassi. Early stages of division of the basal bodies, (l/b); blepharoplast ring (bl); nucleus and parabasal body (p). (After Belaf.) In the micronuclei of Paramecium caudatum such a mass forms a hyaline cap at one pole of the otherwise chromatin-filled resting nucleus. Observations are entirely lacking in regard to division of this mass during reproduction, but similar aggregates of non- staining substance are present at the distal ends of the daughter nuclei during stages of division (Fig. 35). Similar pole plates appear as broad, flat and hyaline ends of the spindles of Actinosphaerium eichhornii according to Hertwig (1898), in the spindle of Tricho- syhaeri urn sieboldi according to Schaudinn (1899), or in the macro- 5 66 BIOLOGY OF THE PROTOZOA nucleus of Spirochona gemmipara (Hertwig). In this group, also, we would include the peculiar hyaline globular bodies at the poles of the nuclear spindles of Euglypha alveolata as described bv Schewiakoff (1888). It is quite possible, although direct evidence is lacking, that none of these peculiar pole plate structures belongs to the group of Fig. 34. — Bodo laccrtae Grassi; division stages continued. (E) Origin of centrioles in the nucleus, and their retention in the daughter nuclei (F to G); (bb) basal bodies, (c) centriole. (After Belar.) kinetic elements. Indirect evidence favoring this possibility is furnished by the entire absence of observations on the division of a definite body, the substance of which forms the pole plates. Hertwig (1898) and Doflein (1916) assume that they are formed from the "limn" substance of the nucleus. On this assumption the pole plates might be interpreted as hyaline aggregates of the ground substance of the nucleus, indeed, the hyaline and homogeneous appearance of THE FUNDAMENTAL ORGANIZATION 67 the pole plates is suggestive of ameba ectoplasm. With our present knowledge I am inclined to agree with this interpretation of pole plates and to regard Paramecium caudatum, with other species of this genus, Actinosphaerium eichhornii and the other forms men- c-.e.tv. C.St Fig. 35.— Paramecium caudatum. Section of a dividing individual; c. st., con- necting strand of dividing micronuclei; e. tr., extruded trichocysts; a. v., gastric vacuole; .1/, dividing macronucleus; m, m, divided micronuclei;^?-., trichocysts. (Original.) 68 BIOLOGY OF THE PROTOZOA tioned above, as containing no permanent intranuclear kinetic elements. To such a group we would also assign forms like Aulo- cantha scolymantha and Chilomonas paramedian, in which according to observations of Borgert (1909) and Alexeieff (1911), not only intranuclear kinetic elements but pole plates as well are entirely absent. On the whole I would interpret the intranuclear kinetic elements of Protozoa as originating by condensation of the ground substance or karyolymph of the nucleus. In Paramecium caudatum (Figs. 35, 147) both in vegetative and meiotic divisions, the ground substance forming the pole plates shows but little condensation (Fig. 57), but in the first meiotic division of Uroleptus halseyi the karyolymph forms two irregular masses which condense to form the spindle fibers and the two spindle poles which are more like pole plates than like centrioles (Figs. 151, 153). In a similar stage of Uroleptus mobilis, however, condensation results in the formation of a definite centriole which divides with a connecting centrodesmose (Fig. 32). In the flagellate type the endobasal body may well be a permanent condition of such condensation. Whether or not such condensations leading to endobasal body formation involve a specific chemical make up, different from that of the karyolymph and from chroma- tin, is an unsolved problem. The diffuse forms such as may be seen in pole plates do not stain with iron hematoxylin or other nuclear dyes nor do they give a positive Feulgen reaction. The centrioles and permanent endobasal bodies stain with iron hematoxylin but the Feulgen reaction is negative. II. CYTOPLASMIC ELEMENTS OF THE FUNDAMENTAL ORGANIZATION. Very little work has been done on the finer structures of encysted Protozoa, and we are relatively ignorant of the make-up of the fundamental organization of the cytoplasm. It is difficult, and often impossible, to distinguish between those elements which are essential parts of the germinal protoplasm and those which are formed as a result of metabolic activities. The latter, obviously, would belong to the structures of the derived organization. The great majority of the structural elements of the cytoplasm are known only in the adult organism. Many of these are undoubt- edly derived structures of the developing individual but some may be essential parts of the germinal protoplasm. Until further knowl- edge of the origin of such questionable elements is available we may regard them tentatively as parts of the fundamental organiza- tion and describe them as such. In most cases they are present in the adult organism in the form of granules which, morphologically, are almost indistinguishable from one another but which react THE FUNDAMENTAL ORGANIZATION 69 characteristically with specific staining methods, thereby indi- cating differences in their chemical composition. Amongst such characteristic granular elements of the cytoplasm are (1) Chromidia, found mainly in Sarcodina and Sporozoa; (2) Volutin grains, found mainly in flagellates, but also present in Sarcodina and Sporozoa; (3) Mitochondria, characteristic of all types; (4) Golgi apparatus, probably universal; (5) Silver Line Si/stem of the Infusoria; (6) Kinetic elements (for the latter see pages 88 and 104). 1. Chromidia.— The nature and the functions of chromidia have been and still are matters of controversy in which there are wide differences of opinion. Hertwig (1879) early called attention to extra-nuclear chromatin in Radiolaria and later (1899) described the zone of cytoplasmic, deeply staining substance which extends from one nucleus to the other and characterizes the dorsal region of Arcella vulgaris and related forms. Hertwig called this the chrom- idial net and homologized it w ith the extranuclear chromatin which he had found in Radiolaria. At about the same time (1898, 1902) Hertwig described the breakdown of nuclei and the distribution of chromatin into the cytoplasm of Actinosphaerium eichhornii. To such chromatin granules in the cytoplasm he gave the name "Chro- midien" and their appearance was regarded as a sure indication of the approaching death of the organism. These observations mark the commencement of a long controversy over the question of chromidia duality which, so far as the Protozoa are concerned, was first clearly announced by Schaudinn in connec- tion with the life histories of the testate rhizopod Centropyxis aculeata, the foraminiferon Polystomellina crispa, and some of the endamoebidae. The chromidia! net of Centropyxis is similar to that of Arcella and according to Schaudinn is the seat of the formation of second- ary nuclei by origin de novo from the chromatin of the chromidial net. These secondary nuclei become the nuclei of gametes while the primary nucleus degenerates. Similarly in Polystomellina, al- though there is no chromidial net, the cytoplasm of mature indi- viduals of the asexual generation becomes filled with minute chro- matin granules— chromidia which arise by fragmentation of the primary nuclei and ultimately become the nuclei of gametes (Fig. 123, p. 235). These findings by Schaudinn were subsequently confirmed by Lister (1905) for Polystomellina crispa; by Elpatiewsky (1907) and Swarczewsky (1908) for Arcella vulgaris; by Goldschmidt (1905) for Mastigina and Mastigella belonging to the flagellate family Rhizomastigidae; by Winter (1907) for Peneroplis pertusus, a fora- miniferon; by Goette (1917) for Difflugia lobostoma. Similar obser- vations were made in connection with Sporozoa of different kinds by Leger and Duboscq for the gregarine Nina gracilis; by Swarc- 70 BIOLOGY OF THE PROTOZOA zewsky (1910) for a species of Lankesteria a hemosporidian ; by Kuschakewitsch (1907) for Gregarina cuneata; by Lebedew (1909) for the ciliate Trachelocerca phoenicopterus. The findings and con- clusions of these different observers have been criticized by Doflein (Lehrbuch, Fourth Edition), by Kofoid (1921) and by others, as unconvincing and not, as yet, adequately confirmed, while the suggestion is repeatedly made that the "secondary" nuclei arising thus de novo from chromidia may be intracellular parasites. So far as the dualism of chromidia is concerned Schaudinn (1903) was the first to suggest the idea by the term "somatochromidia" for chromidia which are vegetative in function or the result, as in Actinosphaerium, of degeneration, and by the term "gametochro- midia" for chromidia which give rise to gamete nuclei. These terms were turned into "trophochromidia" and "idiochromidia" respectively by Mesnil (1905) with a slight difference in interpre- tation of the former. Goldschmidt (1905) likewise indicated the same interpretation by the terms "chromidia" and "sporetia" respectively. Before accepting interpretations . as above, particularly in con- nection with chromidia of the testate rhizopods, it is necessary to determine whether or not the granules in question are really chro- matin. Khainsky (1910) came to the conclusion that the chromidial net of Arcella has an active part to play in nourishment of the organism, and Zuelzer (1904) maintained that the chromidial net of Difflugia is the seat of formation of a carbohydrate nutritive substance of the nature of glycogen. If these suggestions prove to be correct it would indicate a different chemical make-up for chro- midia and intranuclear chromatin, and a difference which should be detectable by microchemical tests. In this field, however, observations are few and results are discordant. The chromidial net of Arcella vulgaris stains black with iron hematoxylin, green with the Borrel mixture and, usually, gives a negative reaction with the usual Feulgen treatment. These results confirm Hart- mann's experiment with pepsin under the action of which the chromidial net of Arcella is dissolved out while the secondary nuclei are conspicuous after subsequent staining. Belar (1926) and others apparently believe that Hartmann's experiment gives a final answer in the negative to the question of the chromatin nature of chromidia. This conclusion, however, is somewhat premature for recent experiments with the Feulgen reac- tion indicate that nucleic acid is certainly present at some stages. With hydrolysis by strong hydrochloric acid at 60° F. followed by the usual staining method the result is invariably negative, while the primary nuclei show only a faint reaction. If, however, the first part of the operation involving strong hydrolysis is omitted and the Arcella material placed directly in the staining solution for THE FUNDAMENTAL ORGANIZATION 71 from eight to fourteen hours, a positive reaction is obtained in all forms in which the secondary nuclei are present (Fig. 3(3). Here the nuclei and the embedding matrix of chromidia are intensely stained. ( 'hromidia at other stages give varying shades of purple depending apparently upon the condition of the organism. Nucleic acid which is formed in the chromidia becomes concentrated in the secondary nuclei; these obviously would resist the pepsin digestion while the residue is dissolved. Fig. 36. -Arcella vulgaris. Growth of nucleic acid bodies in the chromidia! net. (Original, X 500 and X 1000.) The problem of extranuclear chromatin, or chromidia, assumed a novel theoretical significance with the development of Hartmann's so-called polyenergid theory. Hartmann (1909) suggested a mor- phological interpretation of Sachs "energid" or nucleus with its sphere of influence, by suggesting an energid as a nucleus consisting of two components, one the chromatin or idiogenerative component, the other a centrosome or homologous structure (kinetic or loco- motor component). In 1911 he distinguished three main types of nuclei of Protozoa, viz., monoenergid, meroenergid and polyenergid types. Monoenergid types are in Protozoa having one kind of cell division as in most flagellated Protozoa. Meroenergid types are forms, originally with two nuclei, one of which has lost the idio- 72 BIOLOGY OF THE PROTOZOA generative component (as in Heliozoa with central granule, or Trypanosomes with "kinetonucleus"). Polyenergid types, finally, involve nuclei containing an aggregate of monoenergid nuclei. Since a monoenergid has but one kind of division Hartmann assumes that this division may take place while in the aggregated condition; or that the monoenergids are freed by rupture of the membrane after which they may divide as monoenergids in the cytoplasm. In all cases the monoenergids become the nuclei of gametes (as in Radiolaria, Foraminifera and gregarines). The conception is inter- esting, but apart from adding other somewhat unenlightening terms meroenergid and polyenergid it leaves us practically where we were before on the chromidia problem, and separates, without sufficient justification, the chromidial net type from the gamete nuclei type. In all probability the two types are not widely different. The monoenergids which come from a polyenergid nucleus represent chromatin which is formed in the nucleus (see p. 87) ; the gamete nuclei which arise from the chromidial net represent chromatin which is manufactured by a cytoplasmic substance of the same nature as the karyolymph and a substance which, possibly, may be derived from the nucleus. 2. Volutin Grains.— These are widely distributed in Protozoa with the exception of the Infusoria, and are not difficult to distinguish from chromidiosomes. They are usually spherical in form but may be angular and irregular and stain intensely with the basic dyes, retaining the stain even after the chromatin granules are completely extracted. They were discovered by a pupil of A. Meyer in the cells of Spirillum volutans from which the peculiar name is derived, and, according to Guilliermond, they are identical with the "meta- chromatic bodies" of Babes, and with the "red granules" discovered by Biitschli. They take a yellow stain with iodine and a blue stain with methylene blue and 1 per cent solution of sulphuric acid, while their reaction to the usual chromatin stains makes them difficult to distinguish from chromidia. They do not give a reaction with the Feulgen method as usually employed, but Reichenow (1928) found that if the preliminary acid hydrolysis is omitted a typical Feulgen reaction follows upon treatment with the fuchsin-sulphuric acid component alone. He infers from this that volutin substances give a typical Feulgen reaction, which is much more rapid than that of nuclear chromatin, and concludes that volutin consists of free nucleic acid. The same conclusion was reached by Schumacher (1926) on the basis of volutin reactions to his methylene blue phos- phin method. Meyer himself regarded them as composed largely of nucleic acid, a conclusion supported by the experiments of Reichenow (1909) on Hematococcus in which it was shown that volutin grains disappear in a medium free from phosphorus and that, during the phases of active chromatin increase in the nucleus, THE FUNDAMENTAL ORGANIZATION 73 they diminish perceptibly in size and increase in size when the chromatin content becomes stationary. From these results, con- firmed by van Herwerden (1917) on yeast cells, Reichenow con- cluded that volutin grains play a most important part in the vital activities of the cell and he regarded them as a reserve store of nucleo-proteins for the purpose of chromatin growth in the nucleus. They appear to be formed in the cytoplasm and, if these observa- tions are well founded, are entirely different in origin and in function from the other minute granules which they closely resemble. The importance of these conclusions in problems connected with biology of the cell warrants the demand for further and more complete observations and experiments. 3. Mitochondria. — The chondriome of a cell consists of the aggre- gate of cytoplasmic substances of lipoidal nature appearing in the form of minute granules termed mitochondria, as strings of granules termed chondriomites, or as smooth filaments termed chondrioconts according to the terminology of Benda (1903) and Meves (1907). The lipoidal make-up is shared with the Golgi apparatus, another group of cytoplasmic substances which are equally well distributed and similar in form and in reactions to mitochondria, but which are regarded as distinct from the chondriome and with different functions in the cell. Some of the lipoidal substances making up the chondriome are evidently autonomous bodies in the cell, while others, more transi- tory in nature, probably result from metabolic activities. It is quite probable, as Alexeieff suggests (1928), that different states or stages of a common type of substance are represented in different organisms and the terms mitochondria chondriomite, chondriocont, etc., have merely a morphological significance. Of these the mito- chondria appear to be the original neutral and most widely dis- tributed of the lipoidal substances, and as such they belong to the fundamental organization. Mitochondria are minute inclusions in the cytoplasm, varying in size from 0.5 ju to 1.5 ju. They may be spherical granules or rod-shaped, resembling bacteria, or crescentic or sickle form. (Fig. 37.) They have been identified in so many different types of Protozoa that their universal distribution may be assumed with assurance. Except in a very general way the chemical make-up of mitochon- dria is unknown. They become reduced in size or disappear after treatment with alcohol or acetic acid, but there are wide differences in the times required to bring this about. They blacken with osmic acid, turn blue green with Janus green B, or red with Janus red (Horning, 1926). Faure-Fremiet (1910) who was the first to recognize mitochondria in Protozoa regarded them as a combination of albumin and phosphates of fatty acids. Today there is no 74 BIOLOGY OF THE PROTOZOA great advance beyond this original interpretation, the accepted view being that mitochondria are combinations of a fat-like body (lipoid) and protein, the variations in staining, in solubility, etc., depending upon the relative amounts of protein in the combination, a small proportion making them highly unstable, a large proportion making them more resistant to heat, alcohol and fat solvents in general. Fig. 37. — Urole-ptus halseyi. Difference in mitochondrial content of a cultural individual (left) and an ex-conjugant (right). X 700. (After Calkins, Arch. f. Protistenkunde, courtesy of G. Fischer.) Opinions differ in regard to the autonomy and self-perpetuation of mitochondria. Observations on the living protozoon cell con- vinced Faure-Fremiet (1910) that the granules reproduce by spon- taneous division and this observation has been confirmed by others upon living and fixed material. Richardson and Horning (1931) in particular, after a slight modification of the pH of the milieu, obtained preparations of Oyalina showing practically every mito- chondrial granule in division (Fig. 38). In other cases, however, THE FUNDAMENTAL ORGANIZATION 75 particularly in the early sporozoites of Monocystis, Horning was unable to demonstrate the presence of mitochondria and concluded that they are absent in young forms but make their appearance in the process of development. This was interpreted as evidence of their origin de novo in the cytoplasm (Horning, 1929). Suspicions have been aroused from time to time as to the nuclear origin of mitochondria, although little positive evidence has been forthcoming. Some has been obtained recently, however, in con- nection with observations on the reorganization processes following conjugation of Uroleptus halseyi (Calkins, 1930). Here the old macronuclei, eight in number, break up, each into a group of minute spherules. These spherules, at first, have a deeply staining cortex * ft ." ■■ - » ft IT: m • &f^ii *^r,*f f. #•--* V- • A» ^>« 51 ' *V.**YAk a' *« * *** •* Fig. 38. — Dividing mitochondria in Opalina. (After Richardson and Horning, Jour. Morph., courtesy of Wistar Institute.) (with iron hematoxylin) and a more feebly staining medullary por- tion, thus giving the appearance of black rings in optical section. At a later stage the apparent rings break up into small crescents and the latter ultimately become rod-like mitochondria filling the cell of the ex-con jugant (Fig. 37) . Opinions are equally divergent regarding the functions of mito- chondria in the cell. The earliest suggestion was that of Faure- Fremiet (1910), who believed that they play some part in connection with the preparation of germ cells, and who was influenced no doubt, by their conspicuous presence in germ cells of Metazoa. Confirmation of this suggestion is furnished in part by observations of Zweibaum (1922), who observed an increase in the fatty acid content of Paramecium when ready to conjugate; and confirmed, 76 BIOLOGY OF THE PROTOZOA in part, by the observations of Joyet-Lavergne (1927) on the differ- ences in number, size, and staining capacity of the mitochondria in the two individuals forming a syzygy in gregarines, thus indicating what he interprets as male and female differentiation. Numerous observers have maintained that mitochondria are responsible for digestive processes in the cell. The best evidence in support of this suggestion has been furnished by Horning (1928), who, using dark-field illumination, observed mitochondria of hetero- trich ciliates adhere to food particles which had been recently ingested; the mitochondria were included in the gastric vacuoles, where they disappeared pari passu with the breakdown of the food substances. Horning concludes that, among other possible func- tions, mitochondria are direct agents in food hydrolysis, playing the part of zymogen granules in the preparation of proteolytic digestive ferments. Causey (1925-1926, etc.) likewise associates mitochondria with food digestion, but he distinguishes between spherical and rod-like forms, the latter being found clustered about the gastric vacuoles (Endomoeba gingivalis) while the former are distributed about the cell, where they act as centers of katabolic activity (?). The difficulty of distinguishing between mitochondria and bacteria is obvious, particularly when inside a gastric vacuole, and this has been the main criticism directed against Homing's interpretation, who meets it by describing the stain used which was specific for bacteria and did not stain the mitochondria. Still other interpretations of the functions of mitochondria have been advocated more or less vigorously by different observers. As active centers they have been associated with the formation of plastids (e. g., leucoplasts, pyrenoids, etc.) filamentous structures of various kinds and with practically all of the cytoplasmic elements of the derived organization. Cowdry (1924) states that more than eighty substances have been claimed to come from mitochondria (see especially Causey, 1926). Not only in cell activities have they been regarded as direct causes, but also as latent or static centers they have been interpreted as cytoplasmic transmitting agents in heredity. None of the suggested interpretations mentioned above seems to be adequate to explain the purpose of mitochondria. Their universal distribution in Protozoa and in Metazoa indicates some important, possibly fundamental activity which is closely bound up with life of the cell or protoplasm in action. Kingsbury (1912) long since suggested that mitochondria might be associated with cell respira- tion, a suggestion adopted and enlarged by Joyet-Lavergne (1927) mainly from study of gregarines and coccidia. According to this observer there is a close connection between mitochondria in coccidia and the catalyst glutathion which is a powerful oxidase. (See Needham and Needham, 1926; Tunnicliffe, 1926; etc.) He THE FUNDAMENTAL ORGANIZATION 77 noted that glutathion is abundant where mitochondria are abun- dant and vice versa. He has also shown that the oxidation-reduction potential (indicated by the expression rH) varies with the distri- bution of glutathion, low when glutathion is abundant, and high when it is scarce. While there is considerable evidence to indicate an association between mitochondria and protoplasmic respiration, Joyet-Lavergne himself finds that the association is not always demonstrable and in some cases is highly improbable, and admits that there are probably other functions of the mitochondria. On the whole we are still in the air as regards the function or func- tions of mitochondria. The variety of interpretations that have been advanced, and often upon good evidence, suggests that we may have to do here with cellular elements which have a general enzymatic significance and functional both in constructive and in destructive activities. As synthesizing enzymes they may be agents in the selection of different materials from the cytoplasm and in fashioning them into proteins, starch, fats, essential oils, etc. (Cowdry, 1924, 1926; Regaud, 1909, etc.), or by metamorphosis they may give rise directly to plastids of different kinds in the cell (Guilliermond, et al.) ; or by degeneration giving rise to substances like chromidia which Gatenby regards as badly damaged mito- chondria. As catalytic enzymes they may act as oxidases in respira- tion, or as hydrolyzing agents in protein and carbohydrate digestion. It would seem that we are either demanding too much of one type of protoplasmic substance or that the term mitochondria embraces a large number of substances having different functions, but with a common lipoidal composition in which the protein com- ponent is the chief variable. Furthermore, it is not improbable that the Golgi apparatus of the cell represents an extreme variation of this type of substance. 4. Golgi Apparatus.— Another cytoplasmic substance which had been identified as a phospholipin (Faure-Fremiet) or lipoproteid (Bouin, Bowen, Hirschler, King, Horning, Joyet-Lavergne, etc.), and known as the Golgi apparatus, Golgi bodies or (in part) dictyo- somes, is also widely distributed in different groups of Protozoa. There are many points of resemblance between this substance and that of mitochondria, particularly in their lipoid composition and consequently in their reactions to special stains. In many types the Golgi bodies— dictyosomes— and mitochondria are apparently indistinguishable (e. g., Gregarina blattarum, Spirostomum ambiguum and Opalina ranarum according to Hirschler, 1924), but in cases where, on morphological grounds, they are unmistakable, they differ from mitochondria in their larger size and in their tendency to clump together in masses, or to form a definite reticulum or net- work (Metazoa) in the vicinity of the nucleus. 78 BIOLOGY OF THE PROTOZOA In Metazoa the Golgi apparatus appears under two main aspects, one diffused, the other localized. These may be converted one into the other in different stages of cell activity and they should be regarded as variations of the same substance in the cell or of the same structural element. The localized phase was termed by Golgi (1898) the "internal reticular apparatus" from the characteristic net-like structure which it assumes in nerve cells. The granular phase is derived, apparently, from the fragmentation of the fibrils which make up the net structure. In Protozoa the Golgi apparatus rarely appears in the form of a network, although aggregates of lipoprotein^ which are found in some cases are regarded as the equivalent of the localized phase typical of metazoan cells. The granular phase, however, is widely distributed in the form of spherules which are larger in size than Fig. 39. — Golgi apparatus in Amoeba proteus. (After Brown, Biological Bulletin, courtesy of the Marine Biological Laboratory.) mitochondria and have an osmium blackening lipoidal cortex (osmi- ophilic portion) and a gray-staining medullary part (osmiophobic portion). This gives them the appearance of black rings or, if imperfectly stained, of crescents or even of rods. In the latter condition they are easily mistaken for mitochondria (Fig. 39). Golgi bodies as distinct from mitochondria, were first recorded by Hirschler (1914) in Monocystis ascidiae a gregarine and similar parasitic forms seem to have been the favorite material for their study. King and Gatenby (1923) and Joyet-Lavergne (1923) described them again in Sporozoa. Since this time, however, descrip- tions of Golgi bodies from many forms, including representatives from all groups of Protozoa, have been published and various attempts have been made to attach some specific function in the cell to them. Following the course of development of the subject in Metazoa, THE FUNDAMENTAL ORGANIZATION 79 the function of Golgi bodies in Protozoa is generally associated with the secretory activities of the cell. These activities, in turn, fall into different categories but mainly in the group of enzymatic functions. Thus Joyet-Lavergne describes a structure near the tips of young forms (agametes, sporozoites) of coccidia, i. e., that portion which first penetrates an epithelial cell, which he compares with the acrosome of metazoon sperm cells (Fig. 40), the substance of the Golgi body being the source of the cytolyzing agent. There is some evidence also that the so-called parabasal bodies of the Polymastigida and the Hypermastigida are made up of varying proportions of lipoid and of proteid substances and have many of the morphological attributes of Golgi bodies (Duboscq and Grasse, 1925). Duboscq and Grasse hold that the parabasals here have a secretory function in connection with the transformation of energy underlying flagellar movements. This, however, has not Fig. 40. — Golgi apparatus in reproductive cells. 1, 2 and 3, merozoites of Aggrc- gata eberthi; 4, sporozoite of same; 5, microgametes of same; 6 and 7, sporozoites of Gregarina polymor.pha. In all, the Golgi apparatus at anterior end recalls the acro- some of spermatozoa. X 1000 U and 5), X 2000 (1, 2, 8, 6 and 7). (After Joyet- Lavergne, Arch, d'anatomie microscopique, courtesy of Masson et Cie.) been confirmed by later workers and there is high probability that all of the structures which have been called parabasal bodies are not identical in chemical composition (see Hall, 1931; V. E. Brown et al, 1930.). Another type of secretory activity of Golgi bodies in Protozoa is described by Nassonow in connection with the lipoidal membranes, homologized as Golgi apparatus, about the contractile vacuoles and canals of flagellates and ciliates. Nassonow sees in this a special apparatus for the secretion of nitrogenous waste into the vacuole whence it is excreted (see below, p. 170). There is no satisfactory evidence of the origin of the Golgi bodies in Protozoa. If the parabasals are to be included in this group of substances and there is equal evidence for regarding them as chroma- toid substances, then there is evidence that in some cases they arise from the blepharoplast and the latter from the endobasal body of the nucleus. Causey (1925), upon rather hazy evidence, concludes that the Golgi bodies of Endamoeba gingivalis arise as thickenings of the walls of gastric vacuoles. so BIOLOGY OF THE PROTOZOA Further work on these different types of lipoidal elements of the cytoplasm of Protozoa is much needed and a more critical classifi- cation of the formed structures of the cell is greatly to be desired, particularly in connection with chromidia, parabasals, mitochondria and Golgi bodies. 5. Silver Line System.— Recent technical developments have led to the discovery of a complex system of fibrils in the cortex of ciliates. The way was paved for this by observations of Bresslau (1921) who m jMftf!! ; Fig. 41. Fig. 42. Fig. 41. — The silver line system of Discomorpha pectinata. Right side. (After Klein, Arch. f. Protistenkunde, courtesy of G. Fischer.) Fig. 42. — The silver line system of Discomorpha pectinata. Left side. (After Klein, Arch. f. Protistenkunde, courtesy of G. Fischer.) endeavored to find some chemical (stain) which would cause imme- diate coagulation of the colloidal structures, especially of the cortex. He used a mixture of equal parts of a 10 per cent opal blue stain and of 6.5 per cent phloxin-rhodamin stain. Ciliates were allowed to dry in this mixture and were then mounted in balsam. Success- ful preparations made in this way revealed specific types of cortical markings of rectangular or rhomboidal shape. Here areas of coagulation gave evidence of more or less definite boundaries. B. Klein (1926) also used the method of drying, but drying with- out coagulation. He argued that small organisms may lose their THE FUNDAMENTAL ORGANIZATION 81 water without loss of organization and may re-establish vitality by subsequent hydration (e. g., as in dried rotifers or protozoan cysts). He maintained that normal structures are not disturbed by such desiccation provided the latter process is correctly carried out. Dried forms obtained in this way were treated with a 2 to 3 per cent solution of silver nitrate, which was allowed to act for from eight to ten minutes. The organisms were then submerged in distilled water and exposed to sunlight. Blepharoplasts or basal bodies of the cortex are apparently composed of a substance which ' ■ c >s '.-.'.* ..... ■ .\ • ■ Fig. 43. — Podophrya fixa. Silver line system at the time of budding. A, budding region of tentacle-bearing parent organism, aggregation and divisions of primary blepharoplasts; B, later stage with final divisions of blepharoplasts; C, bud in which the blepharoplasts have "satellites" which form the cilia. (After Chatton, Lwoff and Tellier, Compt. rend. Soc. d. biol., 1929, courtesy of Masson et Cie.) has an affinity for silver (argentophile substances). The silver is reduced in sunlight and the basal bodies, their connectives and associations are revealed in jet black lines and grannies against a yellow background. Klein termed these structures the silver line system and has shown that, specific systems characterize each species of ciliate (Figs. 41 and 42). Chatton and Lwoff (1929) have extended the silver nitrate method for fixed material, thus avoiding the somewhat brutal desiccation. Their results in general confirm Klein's. 6 82 BIOLOGY OF THE PROTOZOA The silver line systems then are definite aggregates of granules and fibrils which, in some pattern or other, form a part of the cortex of every ciliate. It is present over large stretches of the cell body, even where cilia are absent; for example, throughout the surface of a Vorticella. In Suctoria and in some ciliates (e. ). Or the function may be of a metabolic nature in one type and reproductive in the other, as in the Infusoria, where the two types show great differences in form and size. Here the nucleus having to do with metabolism makes up a large part of the volume of a cell and is usually of relatively large size, hence is called the macronucleus, while nuclei having to do with reproduction and fertilization are always minute and are called micronuclei (Fig. 44). Usually the micronucleus is closely attached to the macronucleus and, in some cases, may be partially hidden in a depression or pit in the macro- nucleus, or it may be entirely independent of the larger nucleus and lie freely in the cytoplasm. A typical example of dimorphic- nuclei is shown by Paramecium caudatum (Fig. 23, p. 50). The derived forms assumed by macronuclei and the number in a single cell vary within wide limits. The most generalized condi- tion is a simple, spherical form; but ellipsoidal, rod-like, horse-shoe- shape, beaded and branched macronuclei are not uncommon. The beaded forms frequently appear like several separated nuclei but the segments are usually enclosed in a common membrane con- tracted at the nodal points, the entire aggregate forming a single nucleus (Spirostomum, Stentor, Amphileptus, Uronychia, etc.). The size of the macronucleus bears no constant relation to the size of the organism (Fig. 44). Micronuclei do not differ much in form but vary in structure from typical vesicular to compact massive types. Their number in the cell likewise varies from 1 to as many as 80 or more (Stentor). They are never connected with one another, but are quite indepen- dent and distributed at intervals along the sides of the macronuclei. There is little or no evidence of the phylogenetic origin of these dimorphic nuclei which are distinctive of the Infusoria. In onto- genetic origin the nuclei are invariably derived after conjugation from division products of the fertilization nucleus, the latter being formed by the union of two micronuclear elements. Hence the statement is usually made that macronuclei arise from micronuclei, a statement which is not strictly accurate, since the fertilization nucleus is neither one nor the other, but merely a cell nucleus of a fundamental organization. In some cases macronuclei and micro- 86 BIOLOGY OF THE PROTOZOA A L -m o. --M C. V. M-— ! Fig 44 —Illustrating volume relations of macronuclei and cell body. A, in Spiro- slomum amUguum; B, in Spirostomum teres; and C, Lionotus procerus; (A) anal pore ; (CV) contractile vacuole; (M) macronucleus ; (mo.) mouth. In Lionotus the mouth is a long slit, in Spirostomum a circular opening at the posterior end of the peristome. (A and B, after Stein; C, original.) DERIVED ORGANIZATION 87 nuclei are not differentiated until the third division of the fertiliza- tion nucleus (e. g., in Cryptochilum nigricans, Paramecium caudatum, Par. putrinum, Bursaria truncatella, Carchesium polypinum, Oper- cularia coarctata, Ophrydium versatile, Vorticella monilata, V. nebu- Ufera, etc.); in other cases differentiation occurs after the second divisions (e. g., in Anoplophrya branchiarum, Colpidium colpoda, Di ~5& x< ."*.'■ ■*. i; »■*»" „ ■•---*,"« V — Fig. 50. — Nuclear division and budding in Heliozoa. A, Vegetative cell of Spfuu r- astrum with axial filaments focussed in a central granule (centroblepharoplast) ; B, C, D, division of central granule and spindle formation in Acanthocystis aculeata; E, F, formation of buds of same; G, exit of central granule from the nucleus of young cells. (After Schaudinn.) £ 96 BIOLOGY OF THE PROTOZOA for division, the endosome fragments into minute chromomeres, which may be strung out in lines through the nucleus, these strings being divided transversely at division. Or the chromomeres may be aggregated in a fairly homogeneous transverse plate in the center of the dividing nucleus (Fig. 51). The former condition is illustrated by the nucleus during vegetative division of Actinosphaerium eich- hornii according to Hertwig, the latter condition by Sphaerastnnu and Acanthocystis (Fig. 50), Collodictyum (Fig. 51), Paramoeba chaetognathi, or the myxomycete Comatricha obtusata according to Lister. Fig. 51. — Nuclear division in Collodictyum tried latum. (After Belaf.) A slight modification of this type is shown by nuclei containing multiple endosomes as in Pelomyxa binucleata which fragment at periods of division, giving rise to a granular nuclear plate (?) which presumably divides to form the daughter plates as shown in Schau- dinn's well-known figure or to division figures like that of Centwpyzis aculeata. Another widely distributed type of division figure is derived from vesicular nuclei in which the chromatin is not contained in one or more endosomes but is distributed peripherally about the nucleus where it usually forms a distinct chromatin reticulum. Such nuclei usually contain an endosome which may be the most conspicuous structure of the nucleus. In . 1 moeba crystalligera the peripheral chromatin appears to be passively divided without any appreciable change in its make up. In Amoeba vespertilio the peripheral chro- DERIVED ORGANIZATION 97 matin is similarly divided and distributed but the endosome appar- ently contains some chromatin in addition for a complete division figure is formed from its substances, chromatin-like granules form- ing a nuclear plate (Fig. 52). In other cases, as for example End- amoeba intestinalis and E. cobayae, the peripheral chromatin is broken up into chromomeres, which collect in the center of a spindle ife: '■•.n't. Fig. 52. — Amoeba vespertilio Dof. Origin of the spindle within the nucleus (1, 2), nuclear division (5, 6, 7), and reconstruction of nuclei after division (3, 4, 8, 9). (After Doflein.) from the linin of the nucleus and with centrioles at the poles. In Chlamydophrys the endosome apparently divides before it disap- pears, the chromosomes being formed from the peripheral chromatin. In still another general type, derived also from vesicular nuclei, the chromatin in the form of chromomeres is suspended in a loose reticulum. In Opalina chromatin appears to be aggregated in a few larger granules, which divide where they happen to be without 7 98 BIOLOGY OF THE PROTOZOA further formality, the nucleus meantime assuming an indefinite division figure. More frequently, however, the chromomeres are suspended between an endosome and the nuclear membrane, as in Eimeria schubergi, or various species of Trypanosoma. In some of these, at division the chromomeres appear to form a nuclear plate, and are distributed in equal groups to the daughter nuclei (Fig. 51). In a final group of types of nuclear division figures either from massive or vesicular nuclei, the chromomeres are derived from the fragmentation of endosomes or from a chromatin reticulum. The common feature in this large group is the fact that these chromo- meres unite secondarily to form definite chromatin bodies which satisfy, in part at least, the definition of chromosomes as given above. These chromosomes are divided equally, one-half going to each pole of the division figure. In some cases it is obvious that their '^^■W~'^M Fig. 53. — Metaphase and anaphase of nuclear division in the radiolarian Aula- cantha scolymantha. X 300. (After Borgert, Zoolog. Jahrbucher, courtesy of G. Fischer.)] division is longitudinal, but in the majority of cases it cannot be ascertained with assurance whether their division is longitudinal or transverse. Nuclear figures of this general type may be divided into two groups, in one of which the chromosomes are too numerous to permit of decision as to their constant number, and the second comprising forms in which the chromosomes are constant in number and in some of which this number is reduced to one-half at meiosis. In the first of these groups we would include types like Euglypha alveolata, the various species of Paramecium and some Radiolaria (Fig. 53). In the second group we would place such forms as Actinophrys sol, Aggregata eberthi, Trichomonas and allied flagellates, Trichonympha and related forms, and the majority of filiates in which the maturation processes are known. In Euglypha alveolata the chromatin of the vesicular nucleus is distributed throughout the resting nucleus. During the early divi- DERIVED ORGANIZATION 99 sion stages the chromomeres are rearranged in rods or fibrils which form a more or less definite skein within the nucleus; this skein fragments into a large number of chromosomes which, according to Schewiakoff, are longitudinally divided. A more aberrant history is followed by the chromatin of the nuclei of various species of Paramecium. In Paramecium caudatum the micronucleus belongs to the massive type, and there is no satisfactory account of the origin of chromosomes in vegetative division (Fig. 35, p. G7), but the number is much smaller than in the meiotic divisions (see Fig. 147, p. 297). A more definite metazoan type of chromosome formation is shown by the organisms with a definite number of chromosomes which is reduced to one-half at meiosis. Here the number of. chromosomes is usually smaller and their individual history during nuclear divi- sion is less difficult to make out. A good example, typical of the more complex flagellates, is Trichonympha campanula, as described by Kofoid and Swezy. Here the resting nucleus contains a large granular endosome. In the prophase of division the granules of this endosome give off chromatin along the walls of the linin reticulum until a definite skein stage results (Fig. 54). Double chromosomes, 2(1 in number, and formed by the splitting of the spireme segments, make up a definite nuclear plate. They are attached by intranuclear fibers to the daughter blepharoplasts and are divided longitudinally with the division of the nucleus. The original connecting fibrils between the separating halves of the blepharoplast ('' centroblepharoplast ") remain at all times outside the nuclear membrane, hence it is called a paradesmose by Kofoid and Swezy. One of the chromosomes appears to be different from the others, both in resting and division stages, and is called the heterochromosome, although its function or significance is quite unknown. Similar odd chromosomes are known in some Gregar- inidae and Coccidiida where the vegetative stages are haploid, as well as in other polymastigote flagellates. Except for the complica- tions brought in by the extensive neuromotor apparatus of Trich- onympha campanula, the division figures of other related flagellates are quite similar, although the number of chromosomes is usually smaller. Thus Kofoid and his collaborators found about 24 in Leidyopsis sphaerica, 12 in- Trichomitus termitidis and 4 in Giardia maris (Fig. 54, p. 100). A smaller number of chromosomes is likewise found in a number of the Gregarinida, and their history in division approaches that of metazoan chromosomes. Thus in the case of Monocystis rostrata Mulsow describes 8 definite chromosomes formed from a portion of the nuclear chromatin, the number being reduced to 4 in the gamete- forming divisions (Fig. 55). Shellack and Leger, also, have described similar chromosomes in Monocystis ovata and in Stylorhynchus longi- 100 BIOLOGY OF THE PROTOZOA roll is. In the latter case, also, there is a peculiar lagging hetero- chromosome ("axial chromosome") of unknown significance. (6) Origin of Fertilization (Meiotic) Chromosomes. — In practically all Protozoa the sequence of stages leading to formation of chromo- somes which enter into pronuclei is quite different from that of the division nuclei. This phenomenon is one of the final acts of develop- ment and in Protozoa represents a last stage of differentiation of A Fig. 54. — Triehonympha campanula in division. A, and B, prophase and anaphase of nuclear division; the divided centroblepharoplast forms the poles of the spindle and are connected by a paradesmose. C and D, breaking up of chromosome spireme into chromosomes which show a tendency to unite in pairs. (After Kofoid and Swezy.) the derived organization of the nucleus. Here, as in Metazoa, there are at least two maturation divisions, while in ciliates the number is increased to three. As in Metazoa, one or the other of the matura- tion divisions is a reducing division or reduction may be parcelled out in both divisions, the end-result being that the number of chromosomes is reduced by one-half, i. e., from the diploid to the haploid number. As in Metazoa, the first of the meiotic divisions DERIVED ORGANIZATION 101 is usually preceded by activity in the nucleus resulting in a skein- like arrangement of the chromatin (spireme) from which definite chromosomes emerge. This spireme, in Metazoa, is the stage of pairing of homologous chromosomes, i. c, chromosomes representing the same characteristics in the two parents. By such association D c Fig. 55. — Monocystis rostrata; chromosome reduction. ^4, Formation of spindle in pseudo-conjugant; B, C, nuclear plates of progamous divisions, 8 chromosomes; D, anaphase of same; E, anaphase of last progamous division, the number of chromo- somes is here reduced from 8 to 4. (After Mulsow.) the chromosomes when fully formed are apparently reduced to the haploid number, but each is double, and the actual reduction occurs in the ensuing divisions. In Protozoa the antecedent or prophase stages of the first meiotic division rarely conform to the metazoan scheme, but in most cases 102 BIOLOGY OF THE PROTOZOA there are stages which have some resemblance, at least, to spireme formation of the metazoan type. For Actinophrys sol, Belaf (1922) has described in great detail the transformations of the chromatin of the vesicular nucleus in the first maturation division. A spireme, bi> d cf B d ri- ff G si X %W C H E Fig. 56. — Chromosomes 1 if Aggregata eberthi. Letters a to /, or a' to /', "^designate the haploid groups. .1, prophase of the first division (male); B, nuclearjplate of same; C, anaphase groups at first division; E, chromosomes in macrogamete nucleus before fertilization; F, chromosomes in zygote nucleus (diploid); G, paired chromo- somes in nuclear plate of first zygote division; H, early anaphase groups of first zygote division, and separation of homologous haploid groups. (After Dobell and Jameson.) passing through bouquet, pachytene, strepsineme and synapsis stages, into double chromosomes of the metaphase nuclear plate, are strikingly similar to analogous stages in metazoan meiosis (Fig. 157, p. 309). Here there is very little to suggest individuality DERIVED ORGANIZATION 103 of the chromosomes, but in the coccidian Aggregate, eberthi where reduction is zygotic (the vegetative stages being haploid) the twelve chromosomes unite in six pairs of homologous chromosomes (Dobell) (Fig. 56) and a modified spireme occurs in the progamous divisions. Similar but less definite conditions are shown in the gregarine Diplocystis schieideri as described by Jameson (1920) (Fig. 158, p. 310). A somewhat simplified history of the chromatin was given by Mulsow (1911) for the progamete nucleus of the gregarine Monocystis rostrata (Fig. 55). Here, differing from Diplocystis, re- duction is gametic and the vegetative stages are diploid. The resting nucleus is vesicular and the chromatin granules join chain- wise to form eight chromosomes. These split lengthwise in the metaphase stage, a preliminary spireme stage, apparently, being absent. Fig. 57. — Micronucleus of Paramecium caudatum in the prophases of the first meiotic division. .4, Early stage in the formation of chromosomes; B, elongation of the nucleus prior to crescent formation ; C, metaphase of the first division. Dehorne describes the entire chromatin aggregate as forming one highly convoluted chromo- some. (After Dehorne.) In the hypermastigida (Trichonympha, Dinenympha, Stauro- joenina, etc.) flagellates, fertilization is unknown, but ordinary nuclear division is preceded by formation of long chromosomes which give the appearance of a spireme. Quite a divergent type of spireme formation is found in the ciliates where the chromatin is massed in homogeneous micronuclei. In Paramecium caudatum the micronucleus elongates to form a bar nearly equal in length to the macro nucleus (Fig. 57). The massed chromatin becomes granular, and the granules stretch out in an elongate network which, in the following crescent phase, breaks up into a multitude of double chromosomes. 104 BIOLOGY OF THE PROTOZOA In other ciliates the massive micro-nucleus gives rise to a group of chromatin granules which form an umbrella shape mass at one pole of the nucleus (Didinium, Oxytricha, Euplotes, Uroleptus, etc.). This has been described as the "candelabra" stage by Collin (Ano- plophrya) or the "parachute" stage by Calkins {Uroleptus). The number of granules is much larger than the number of chromosomes of the later reducing division, but this large number is halved at the first meiotic division (Fig. 32, p. 64). With the second division the remaining granules usually fuse to form the diploid number of chromosomes and this number of chromosomes is finally reduced to one-half. At the third division these resulting haploid chromo- somes become granular and are divided transversely. B. Derived Organization; Cytoplasmic Changes. — 1. Cytoplasmic Chromatin. — During the metabolic activities of the cell, substances which are undoubtedly derived from the nucleus are cast off into the cytoplasm. The majority of these are not represented by demonstrable structures of the cytological organization. Thus in Uroleptus (mobilis and halseyi) fully one-third of the macronuclear chromatin is shed into the cytoplasm at each division and disap- pears as chromatin, while in ciliates generally the entire substance of the macronuclei and a variable proportion of micronuclear substance (fifteen-sixteenths in Uroleptus mobilis) is absorbed in the cytoplasm at periods of conjugation. In the latter case, again, this nuclear substance cannot be definitely traced into cytoplasmic structures (see, however, the described origin of mitochondria in Uroleptus halseyi, p. 75). Secondary nuclei which are formed in the cytoplasm of Foramini- fera, Radiolaria and some ameboid forms are traced directly back to nuclear chromatin. Thus in Polystomellina crispa, Peneroplis and other foraminifera the nuclei fragment distributing quantities of chromatin granules (chromidia) in the cytoplasm. These granules in groups of two or three form minute secondary nuclei, one such nucleus in each swarm spore (amebula) which then develops into a megalospheric generation with hundreds of small nuclei formed by division (see p. 69). When mature the protoplasm breaks up into swarms of flagellated gametes, each with one of these minute nuclei (Schaudinn, Lister, Winter et at.). The testate rhizopods secondary nuclei develop from chromidia which form the nuclei of ameboid swarmers {Centropyxis Schaudinn, Arcella). Similarly in pseudopodia-forming flagellates (Rhizomas- tigidae) Goldschmidt (1905) describes the formation of secondarj' nuclei in Mastigella and Mastigina (Fig. 45, p. 88) from the cyto- plasmic chromidia. 2. Cytoplasmic Kinetic Elements. — It is in the cytoplasm that kinetic elements are most highly differentiated, and the often perplexing structures which appear in different types of Protozoa DERIVED ORGANIZATION 105 have led to much confusion in terminology as well as in interpreta- tion. Indeed the type of development of the kinetic elements in flagellates is entirely different from that in ciliates and at the present time, at least, they cannot be homologized. Any attempt, therefore, to present a clear picture of the diverse elements and to distinguish one type from another inevitably leads to contradictions in interpretation. The facts may be marshalled, however, into fairly logical series indicating increasing complexity in the organiza- tion of the cell. Such series are presented in the following pages with the understanding that they involve no claim of finality, nor do they indicate phylogenetic relationships. The kinetic structures most frequently found in the cytoplasm of Protozoa are relatively simple, the more complex types which have been revealed being found in comparatively few cases. In considering Protozoa as a group, therefore, too much weight should not be attributed to these more complicated forms. For purely descriptive purposes they may be considered in the following order: (1) Kinetic elements, which are morphologically and functionally equivalent to intranuclear centrioles forming parts of endobasal bodies and usually derived from them ; (2) blepharoplasts equivalent to basal bodies, or independent of basal bodies, which lie at or near the bases of motile organoids and give rise to the kinetic structures in them ; (3) basal bodies derived from and independent of blepharo- plasts; (4) parabasal bodies which are closely connected with the blepharoplasts and probably derived from them ; (5) centrodesmoses and paradesmoses, or connecting fibrils between kinetic elements at the spindle poles; ((>) rhizoplasts, or fibrils originating as outgrowths from the substance of specific kinetic elements and connecting two such elements or ending blindly in the vicinity of the nucleus; (7) astrospheres and centrosomes, similar to analogous structures in the cells of Metazoa; (8) miscellaneous kinetic elements such as centroblepharoplasts, axostyles, parastyles and the neuromotor apparatus of flagellates. An entirely different series involves the motorium, conductile fibrils, and myonemes of Infusoria together with the silver line systems of the ciliates which we have included in the structures of the fundamental organization (see p. 80). Since many of these are characterized by their functional activi- ties as well as by their specific structures, it is not illogical to find that the same organoid performs generalized functions. Thus a blepharoplast may be the same as a centriole, or as a basal body; rhizoplasts may arise as a broken centrodesmose or paradesmose; a myoneme as a conductile element, etc. The complexities of organi- zation arise from the simultaneous presence of many of these differ- ent kinetic elements in the cell where they may form a coordinating system of organoids which Sharp and Kofoid have aptly designated the neuromotor system. 10(1 BIOLOGY OF THE PROTOZOA D ■9 Ci.r'^g,, — w . .:-s^.. w ■- -'■;»#' ^ G Fig 58.-Hartmannella klitzkei Arndt. Centrosome and centnole in a testate rhizopod A, Animal with watch-glass-like shell; B to F origin of the centrosome In the cytoplasm, its division, and position on the spindle; G, anaphase stage of nuclear division. (After Arndt.) DERIVED ORGANIZATION 107 1. Blepharoplast, Basal Body and Centriole.— In many of the comparatively simple Protozoa which have no specialized motile organoids, the cytoplasm apparently lacks all traces of specific kinetic elements. Thus in the entire group of Sporozoa, in the simpler Gymnamebida and in testate forms of rhizopods, kinetic elements, if present at all, are in the form of endobasal bodies within the nucleus or as centrosomes close to it. Arndt (1924), however, described a centrosome, with centriole, which divides and forms the poles of the mitotic figure in Hartmannella Mitzkei, a testate rhizopod (Fig. 58). In some of the relatively simple rhizopods, however, especially those belonging to the family which Doflein has called the Bistadiidae, from the fact that two distinct phases an ameboid and a flagellate phase— are interchangeable, we find organisms which throw light on the origin of cytoplasmic- kinetic elements. Such dimorphic types of rhizopods have been repeatedly observed since Dujardin first called attention to them, but details concerning the origin of kinetic elements and the flagellum have been made out only through use of modern cytological methods. In some Protozoa, e. g., Codosiga botrytis, the kinetic elements of the flagellum grow directly out of an endobasal body of the nucleus, indicating their origin from an intranuclear kinetic element (Fig. 59,^4), in other simple forms the flagellum arises from a kinetic element situated in the cytoplasm but connected with the intra- nuclear kinetic element by a rhizoplast at some stage (Fig. 59, B). In the phytoflagellate Polytoma uvella, according to Geza Entz (1918), the relation between intranuclear and cytoplasmic kinetic elements varies with the age of the cell. The usual condition in adult cells is two basal bodies, one at the base of each flagellum, and neither of them is connected by a rhizoplast with the nucleus. In young individuals, however, the original single blepharoplast (= basal body) is connected by a rhizoplast with an intranuclear endobasal body, or a larger rhizoplast from the blepharoplast may break up into a calyx of fibrils which enter the nucleus at different points. The inference might be drawn in all such cases that the cytoplasmic body represents one of the daughter halves formed by division of the nuclear endobasal body, while the connecting fibril represents the rhizoplast formed during such division. Such stages are well illustrated by the dimorphic forms of rhizopods during the transition from the ameboid to the flagellated phase. Thus Whit- more described a cytoplasmic kinetic element functioning as a basal body which is connected by a fibril with the nucleus and which lies at the base of the flagella in Trimastig amoeba philipijinensis , and Puschkarew described a similar condition in Dimasiigamoeha bista- dialis (Fig. 59, C). The most complete observations, however, were made by Charlie Wilson in connection with the transition from ame- boid to flagellated stage in a closely-related form, Dimastig amoeba Fig. 59.— Flagellum insertion. A, Codosiga botrytis, with flagellum arising from the nucleus. B, Dimastigamoeba bistadialis Pusch. with blepharoplast connected by rhizoplasts with the nucleus, and with independent basal bodies. C, Dimastigamoeba gruberi and origin of the blepharoplast from the endosome in the nucleus; (b) bleph- aroplast; (w) nucleus; (r) rhizoplast. {A and B from Doflein, C from Wilson.) (108) DERIVED ORGANIZATION 109 gniberi, one of the soil amebae. She describes the nucleus of this organism as containing a typical endosome within which an endobasal body is embedded. At the period of flagellation this endobasal body divides and one daughter element migrates through the substance of the endosome and through the nucleus to the cytoplasm, retaining its connection throughout with the intranuclear kinetic element (Fig. 59, C). In the cytoplasm it becomes a basal body which gives rise to the kinetic elements of the flagella. In these cases the ex- truded kinetic element combines the functional characteristics of a blepharoplast and a basal body or group of basal bodies. In this dual capacity it may be regarded as a blepharoplast— basal body. In Dimasiigamoeba bistadialis according to Puschkarew it divides, one part remaining as a blepharoplast, the other becoming a basal body; the two parts, however, are connected by a rhizoplast and rhizoplasts connect the blepharoplast with the endobasal body (Fig. 59, B). In Bodo lacertae according" to Belaf the centrioles after division are taken into the daughter nuclei. Here the kinetic elements, although originating from an endobasal bod} 7 , are different in func- tion from those described in the preceding paragraph. Forming the poles of the mitotic spindle they are correctly described as centrioles, but apparently they again become endobasal bodies (Figs. 33, 34, p. 65). While the flagella appear to emerge directly from the nucleus in some cases, e. g., in Mastigamoeba invertens according to Prowazek, or Codosiga botryiis according to Doflein, in many cases they take their origin actually from kinetic elements in the form of centrioles which lie on the outside of the nuclear membranes, as in Mastigina setosa, Phialonema cyclostoma, Cercomonas longicauda, Oicomonas termo, or Chilomastix gallinarum (Fig. 60). In such cases, illustrated by Chilomastix aulostomi according to Belaf (1921), centrioles, become the basal bodies, and the latter become centrioles. In such cases the basal bodies are unquestionably blepharoplasts. In other cases the blepharoplast does not remain connected with the nucleus by any fibrillar process, but as an entirely separated and independent kinetic element gives rise to the flagella at or near the anterior end of the cell (Leptomonas jaculum) or Herpetomonas gerridis (Fig. 169, p. 366). In Chilomastix mesnili Kofoid and Swezy (1920) describe three blepharoplasts, one of which gives rise to two flagella, another gives rise to one flagellum and the parastyle, the third to the parabasal, peristomial fibril and the cytostomal flagellum (Fig. 60, B). Boeck (1921) has confirmed these findings. Or, the blepharoplast may migrate toward the posterior end of the cell where with or without division to form blepharoplast and basal body it gives rise to a flagellum, which becomes the vibratile margin of an undulating membrane as in the majority of trypanosomes (Fig. 110 BIOLOGY OF THE PROTOZOA 61, E). In still other cases the blepharoplast also gives rise to one endoplasmic fibril or rhizoplast, which extends deeply into the cell as in Rhizomastix (Mackinnon), or a number of such rhizoplasts may be formed as in Mastigella vitrea. In these cases the blepharo- plast divides independently of the nucleus at periods of cell division. 2. Parabasal Body and Blepharoplast.— As a centriole may be contained in an endobasal body which consists largely of chromatoid substance, so may a basal body be enclosed in chromatoid substance u.m Fig. 60. — Flagellum insertion. A, Phialonema cyclostomwm; B, Chilomastix nu snili; < ', the same, encysted, {u.m.) Margin of undulating membrane in cytostome. (A, Original; B, C, after Kofoid and Swezy.) of a blepharoplast, as shown by Goodey (1916) in the flagellate Prowazekia (Bodo) saltans, or by Kofoid and Swezy (1915) in Trichomonas augusta. Again, just as a centriole may be freed from its enclosing chromatoid substance in an endosome, so may the basal body be freed from the blepharoplast. In a similar way the blepharoplast may be contained in an embedding chromatoid mass of a cytoplasmic kinetic element, or it may be free from such a mass. We may then have in the same cell a kinetic complex consisting of one or more basal bodies, one or more blepharoplasts, DERIVED ORGANIZATION 111 and a residual kinetic element in the form of a chromatoid mass. To this residual chromatoid mass the name parabasal body is applied, the term originating with Janicki (1915). Kofoid (1916) interprets its function as a storage or feeding reservoir for the kinetic elements, its substance in turn being derived from the nucleus. /" /.;/ *%> D Fig. 61. — Relation of parabasal to nucleus. A, Crithidia euryophthalmi endosome of nucleus and parabasal connected by rhizoplast; B, origin of parabasal from endo- some of nucleus; C and D, differentiation of parabasal and rhizoplasts; E, Trypano- soma cruzi, and F, Crithidia leptocoridis, for comparison. (After MeCulloch.) It is in connection with the parabasal body that most of the difficulties have arisen concerning the interpretation of cytoplasmic kinetic elements. It is still in the stage of polemics and contro- versies continue over the chemical nature of its substance. The difficulties began with Schaudinn's work (1904) on the trypanosome 112 BIOLOGY OF THE PROTOZOA of the little owl (Glaucidium [Athene] noctuae). Schaudinn's descrip- tion and figures of the history of the kinetic elements at the base of the flagellum have been cited and copied in practically every text-book dealing with the Protozoa and have had a wide influence in theoretical protozoology. Other keen observers, however, have sought in vain for evidence corroborating this history. In the absence of such confirmation and in view of the multitude of differ- ent observers who find a simpler explanation in many different types of trypanosomes, including that of the little owl (see Minchin, Robertson, Sergent, et al.), Schaudinn's interpretation and conclu- sions can be accepted only with many reservations. The essential point in Schaudinn's description was the origin by heteropolar mitotic division of the nucleus of a recently " fertilized cell," of a larger nucleus which becomes the nucleus of the cell, and a smaller nucleus which forms the kinetic complex. This smaller nucleus divides again by mitosis, also heteropolar, the smaller portion becoming the basal granule which forms the flagellum and the "myonemes" of the undulating membrane, while the larger portion remains intact as a homogeneous deeply-staining granule. The contested points in regard to this phase of Schaudinn's work are, first, the "fertilized cell" of the trypanosome, which is now generally regarded as a stage in the life history of an entirely differ- ent parasite of the little owl (Minchin enumerates no less than five different types of protozoon parasites which may live simultaneously in the blood of this owl). A second contested point is the origin of the kinetic elements of the cytoplasm by mitosis. Other con- tested points and untenable conclusions drawn from them have to do with sex differentiation and parthenogenesis which need not be considered here. It is not at all impossible that Schaudinn may have seen the emer- gence of a kinetic element from the endosome of the nucleus as de- scribed above in the case of Dimastigamoeba gruberi, and the similar emergence of a basal granule or blepharoplast from a chromatoid mass in the cytoplasm. The interpretation of such possible stages as mitotic nuclear division, and the smaller products of such division as nuclei, has led to numerous theoretical developments which have only a narrow basis of fact. Two years after Schaudinn's paper appeared, Woodcock translated it into English and conferred the name " kinetonucleus " on the smaller body resulting from the heteropolar mitotic division and the name " trophonucleus " on the nucleus of the cell. Schaudinn himself was the first to announce this binucleate character of the trypanosome body and the hypoth- esis was taken up by his followers, Prowazek, and notably Hartmann (1907). The latter developed the conception into an elaborate view of original nuclear dualism upon the basis of which he created a special group of the Protozoa including trypanosome-like flagel- DERIVED ORGANIZATION 113 lates and hemosporidia, which he called the "Biiiucleata." 1 As Doflein points out, not only do the hemosporidia have no blepharo- plasts as do the trypanosornes, but blepharoplasts in the latter are not to be considered nuclei. In this use of the term blepharoplast Doflein includes the structure to which Woodcock gave the name kinetonucleus, but he employs the term in a special sense as a kinetic element, while German writers generally use it for structures of widely different significance. Thus Schaudinn, although con- vinced of its nuclear character, nevertheless called it a blepharo- plast. French writers, as a rule, speak of it as a centrosome (e. g., Mesnil, Laveran, etc.) as do some English observers (e. g., Moore and Breinl) ; many of the latter, however, follow the original nuclear interpretation, Bradford and Plimmer following Stassano, regarding it as a " micronucleus " and comparing it with the smaller nucleus of the ciliates, while Woodcock and Minchin considered it a "true nucleus." The essence of the problem indicated by the various usages of these familiar terms comes down to a decision as to whether the so-called kinetonucleus, by which is meant the relatively large chromatoid body in the cytoplasm and closely connected with the basal granule, is a nucleus, or a kinetic center of the cell, or neither. Woodcock's term connotes a happy combination of both nuclear and kinetic possibilities; the kinetic function evident from its relation to basal granules or blepharoplasts, while its nuclear characteristic is seen mainly in the deeply-staining chromatin-like substance of which it is composed as well as by its frequent connection with the nucleus. Some writers, notably Rosenbusch (1909), giving free play to the imagination, and under the conviction that it is a nucleus, describe it as such, with centriole, "karyosome," nuclear space which may contain chromatin granules, and a nuclear membrane. The extremely minute size of this organoid and the pranks which the Romanowsky stain or any of its modifications may play with it, as they do with structures of the actual nucleus, together with a fertile imagination, are sufficient to account for the perfect nuclear type which Rosenbusch, for example, described. Other observers, while maintaining its nuclear character, do not accept this extreme inter- pretation; Minchin, for example, describes it as a "mass of plastin impregnated with chromatin staining very deeply, rounded, oval, or even rod-like in shape" (Prot., p. 2SS). If we bear in mind the many types of granules in the cell which stain like chromatin with certain dyes, it seems unnecessary, to say the least, to make the term nucleus, which stands for a well-known and easily recognized organoid of the cell, elastic enough to embrace cytoplasmic bodies in regard to which there is so little evidence of nuclear structure or nuclear function. In well fixed and stained 1 For critiques of the Binucleata, see particularly Minchin (1912), Dobell (1911). 8 114 BIOLOGY OF THE PROTOZOA material the so-called kinetonucleus affords little evidence of nuclear make-up ; it appears as a homogeneous mass of chromatoid material which divides into equal parts prior to division of the nucleus. Such features do not make it a nucleus any more than similar features make nuclei of pyrenoids, or of other plastids of the cell. Func- tionally, and unlike the nucleus, it is not necessary for the vital activities of the organism, as shown by the experiments of Werbitski (1910), confirmed by others, in which by the use of certain chemicals (e. g., pyronine) the "kinetonucleus" of Trypanosoma brucei disap- pears without any effect upon the movements and reproduction of the trypanosome, a race being formed in which this organoid is absent. Nor can the " kinetonucleus " be regarded as a centrosome, for although closely connected with basal granules, it never behaves like an attraction center. With the exception of Schaudinn's account and the overdrawn account by Rosenbusch there is no evidence that it divides by mitosis; it never develops chromatin structures which by any stretch of the imagination can be called chromosomes. If the " kinetonucleus " is not a nucleus nor an active kinetic center of the cell, then any misleading appellation such as kinetonucleus, centrosome, or blepharoplast, which indicates co-partnership with the actual cell nucleus or other easily recognizable organoid, should be discarded together with the supplementary term trophonucleus. Among names suggested to replace the term kinetonucleus is " kine- toplast" used by Wenyon, Dobell, and Alexeieff, and "parabasal body" (Janicki) as used by Kofoid. The non-committal term parabasal body was first employed by Janicki (1915) to designate an accessory structure in the kinetic complex of Lophomonas (Fig. 105, p. 211). Analogous structures have since been found in practically all of the parasitic flagellates thus far described, although not found in free-living types generally. It is present as a globular mass of deeply-staining substance close to the blepharoplasts of types like Trypanosoma brucei, Bodo edax or Bodo lacertae (Fig. 33, p. 65) ; as an elongate mass in most of the Cryptobia species (Fig. 61, C) ; as a long basal filament in Trichomonas augusta (Fig. 77, p. 145) ; or Chilomastix mesnili; as a spirally coiled mass in Devescovina striata (Fig. 62, F), etc. It apparently differs in size and form in different phases of the same organism as in Bodo lacertae where, in addition to the globular form, it may be rod-like or partly coiled or absent altogether. In Chilomastix mesnili an homologous rod-like body, termed the parastyle, arises from a second blepharoplast (Kofoid and Swezy, 1920) (Fig. 60). The most extensive work on the parabasal body has been carried out by Kofoid and his followers who regard this structure not as a nucleus nor as a kinetic center, but as a "kinetic reservoir" or a reservoir of substances which are used by the animal in its kinetic activities under the conditions of its dense environmental medium. DERIVED ORGANIZATION 115 This substance, according to Kofoid, appears to form at the expense of the nuclear chromatin and increases or decreases— that is, the parabasal body becomes larger or smaller apparently in relation to metabolic demands. When the parabasal body is poor in chromatin the blepharoplast and nucleus may be rich and vice versa. "Our data are too incomplete to give a clear picture of the process, but as far as they go they suggest the origin of the parabasal at the expense of the chromatin of the nucleus, the movement of stain- able substance on the rhizoplast, either to or from the blepharoplast at the base of the flagella, and the wax and wane of the parabasal" (Kofoid, 1916, p. 5). This interpretation is strengthened by the positive reaction of the parabasal of some species to the Feulgen nucleal test (see p. 57). Kofoid's interesting and suggestive interpretation of the nature of the parabasal is very well sustained by the morphological rela- tions of blepharoplast, nucleus and parabasal body in widely diverg- ent types of flagellates. Morphologically, a series representing a gradually increasing complexity is illustrated by : (1) Dimastigamoeba gruberi, in which the blepharoplast arises by division of the intra- nuclear kinetic center and remains connected with it by a centro- desmose or, in this case, a cytoplasmic rhizoplast; (2) Scytmnonas subtilis in which the blepharoplast is not connected with the nucleus and gives rise only to the flagella ; (3) Bodo edax, or species of Cryp- tobia in which a large chromatoid mass, the parabasal body, is con- nected by rhizoplasts with the blepharoplast, or may be indepen- dent of it; (4) Bodo lacertae in which basal bodies (arising from the blepharoplast), blepharoplast and parabasal body are all indepen- dent; (5) Giardia augusta, in which the independent blepharoplast, basal bodies and parabasal body are all double and arranged in perfect bilateral symmetry; (6) Calonympha grassii (Fig. 63), in which nuclei, parabasal bodies, blepharoplasts and basal bodies are multiple and in which axial threads (rhizoplasts) unite to form a central axial supporting rod; (7) Trichonympha campanula, in which the blepharoplast (centroblepharoplast) acts as a centrosome in mitosis while long rhizoplasts connecting distal basal bodies with the blepharoplast form a complex radial system of astral rays (Figs. 61 to 65). In many cases the blepharoplast, which is the central element of the kinetic complex, remains connected with the nucleus by a rhizo- plast as a permanent record of the intranuclear origin of the entire complex (Fig. 62). In many cases the blepharoplast is double, as in most biflagellated forms; in others it is triple as in Trimastig- amoeba p)iilippinensis or Chilomastix mesnili (Fig. 60, B); in some it is quadruple, or contains four basal bodies as in Trichomonas; in others it is multiple, forming a ring of blepharoplasts about a bundle of flagella as in Lophomonas blattarum (Fig. 105, p. 211). 116 BIOLOGY OF THE PROTOZOA Finally in flagellates with multiple nuclei (family Calonymphidae) , in addition to a number of free blepharoplasts and parabasal bodies, each nucleus is accompanied by a blepharoplast which gives rise Fig. 62. — Types of parabasal body. A, Polymastix; B, Trypanosoma cruzi; C, Cryptobia sp.; D, Bodo lacertae; E, Prowazekia sp.; F, Devescovina striata; G, Herpeto- monas musca-dome.sticae. (b) Blepharoplast; (p) parabasal body; (n) nucleus; (x) axostyle. (A, C, D, G, after iSwezy; B, after Chagas; E and F, after Doflein.) to three uniform flagella and one longer, band-formed flagellum, by a parabasal body, and by a rhizoplast (axial strand, Fig. 63). Many of these aggregations of kinetic elements are sufficiently DERIVED ORGANIZATION 117 complex to justify the term neuromotor system of Sharp and Kofoid and appear to form a coordinated whole, as shown by the reaction after maceration when they retain their connections and remain together for some time after the supporting protoplasm has disap- peared (Trichomonas, Kofoid). The term is certainly justified in connection with the remarkable kinetic structures of flagellates belonging to the family Trichonymphidae. In Trichonympha cam- panula, Kofoid and Swezy (1919) describe the system as composed of an external coating of cilia-like motile organs, three zones of Fig. 63. — Calonympha grassii Foa. (From Doflein.) flagella with their basal bodies, rhizoplasts connecting basal bodies with a great anteriorly placed blepharoplast, and more deeply-lying myonemes which apparently are not connected with the blepharo- plast (Fig. 64). Kofoid and Swezy regard the central organoid as a kind of superblepharoplast, calling it the "centroblepharoplast" since it has the attributes of a centrosome. When it divides the entire aggregate of kinetic elements of the cortical zone divides with it, forming a mitotic figure with centrosomes, central spindle and astral rays (Fig. 54). The connecting fibrils of the centrosomes, unlike 118 BIOLOGY OF THE PROTOZOA the centrodesmose in Metazoa, remain outside of the nucleus (as it does in many other flagellates) and is called the paradesmose by Kofoid to distinguish it from the centrodesmose or central spindle. From this review of the cytoplasmic kinetic elements in the flag- ellates it is apparent that in endobasal bodies, basal bodies, and parabasal bodies we have to do with structures closely connected with the kinetic activities of the organism and closely related to each other. The chromatoid substance of which they are composed may or may not be chromatin, although the evidence adduced indi- cates that it arises from the nucleus and in some cases is similar to chromatin in its staining reactions. It does not behave like chro- ^V^' V^Vf -V-V^C" " i, J, ^ ■*.**--. A « L.vJ '/ Fig. 64. — Trichonympha campanula Kof. and Swez. (After Kofoid and Swezy.) matin during division of the cell, but like pyrenoids, or chromato- phores, where each granule reproduces its like by division; nor does it afford any evidence of constructive metabolic activities in the cell. For these reasons I believe, with Kofoid, that the term "parabasal body" expresses the relationships and functional activi- ties of the so-called "kinetonucleus" much better than does the latter term and should take its place in literature dealing with the Protozoa. The interpretation of kinetonucleus and parabasals, however, is still incomplete. In Trypanosoma, as stated above, the kinetic element known as the "kinetonucleus" (aud.) or the "para- basal" (Kofoid, Swezy, et al.) gives a positive Feulgen nucleal reaction, indicating the presence of thymonucleic acid (Bresslau and Scremin, 1924; Robertson, 1928; Jirovec, 1927; DaCunha and DERIVED ORGANIZATION 119 m- Muniz, 192S). Lwoff (1931) finds that this nucleal reaction is confined to a cortical zone of the body in question, and holds that probably in all cases the so-called kinetonucleus is composed of two quite different substances, one of which, the medullary sub- stance according to the observations of Grasse (1926), is apparently of lipoid nature. Lwoff (1931) gives a new interpretation of para- basals and kinetonuclei in the simpler parasitic flagellates such as Leptomonas ctenocephali (Fig. 65). Here the so-called "parabasal filament" (p.f.) does not originate from the blepharoplast ("mastigosome" of Lwoff = m) but from a flagellar ring (r) quite re- moved from and not connected with the blepharoplast. The latter, however, gives rise to and is connected with what he terms the "kinetonucleus," which he shows has a chromatin cortex (k). The latter gives rise to still another element which he calls the "paranuclear" body (c.Bin). In this case the "parabasal" is not derived from the blepharoplast, but is of entirely differ- ent origin from parabasals of other forms. What Lwoff calls the "kinetonucleus" has the same relation to the blepharoplast as do the majority of parabasals (e. g., Crithidia, Trypanosoma cruzi, etc., Fig. 61). Further study of these perplexing fibrils in flagellates and particularly in the hyper- mastigida, must be made before the puzzle of exact homologies can be solved. 3. Other Cytoplasmic Kinetic Elements.— A unique cytoplasmic kinetic element, ap- parently homologous with the centrobleph- aroplast of certain flagellates, is found in some types of Heliozoa. The non-com- mittal name central granule (Centralkorn) was given to this structure by Grenadier (1869), who was the first to observe it. In some types it lies in the geometrical center of the cell (Acanthocystis aculeata, Sphaerastrumfockei, Raphi- diophrys pallida, etc.) ; in other types it is ex centric (Dimorpha m utans, Wagnerella borealis) or absent altogether (Actinophrys sol, Actino- sphaerium eichhornii, Camptonema nutans, etc.). In the ordinary vegetative activities of the cell, radiating fibers starting from the central grain extend through the protoplasm to the periphery, where they form the axial filaments of the pseudopodia (Fig. 66) . In division stages of the cell, the central grain first divides forming an amphi- — I — k "c.Bm -p.f. Fig. 65. — Lepto m onas ctenocephali. Parabasal ap- paratus consisting of peri- flagellar ring and posteriorly directed filament; "kineto- nucleus" and "mastigosome" (basal body). (After A. and M. Lwoff, Bull, biologique de la France et de la Belgique, courtesy of Prof. N. Caullery and Les presses Universitaires de France.) 120 BIOLOGY OF THE PROTOZOA aster consisting of centrosomes, centrodesmose and astral rays made up of the radiating fibrils (Fig. 50, p. 95 — see also Trichonympha cam- panula) . Stern (1914) , however, found that mitotic spindles may arise in Acanthocystis without any connection with the central granule (Fig. 67) . The central grain, however, takes no part in reproduction by bud- ding, whereby ameboid or flagellated buds are formed which contain a nucleus derived from the parent cell nucleus, but no central grain. This nucleus, however, contains an endobasal body which divides and one of the daughter granules emerges from the nucleus as it does in Dimastigamoeba gruberi (p. 34), but retains its eentrodes- mose for some time and ultimately forms the central grain of the Fig. 66. — Relation of axial filaments to nuclei. Section of Actinophrys sol with axial filaments arising from intranuclear granules in recently divided nuclei. (After Schaudinn.) adult organism (Schaudinn, 1896; Zuelzer, 1909; Acanthocystis acu- leata, Wag nerd I a borealis, Fig. 50). Similarity with the centrobleph- aroplast in flagellates is thus shown (1) by its origin from an intranuclear centriole; (2) by its relation to axial filaments which are homologous with rhizoplasts; (3) by its history during mitosis. The analogy is further strengthened by its relation to the flagella and to the axopodia which are simultaneously present in some of the Helio- flagellida {Actinomonas mirabilis, Kent, Ciliophrys marina, Caullery, and Dimorpha m titans, Gruber). In Dimorpha m utans (Fig. 13, p. 34), the central grain lies near one pole of the cell where it forms the basal body of the two flagella as well as the focal point for the axial filaments; here flagella and axial filaments appear to be homologous DERIVED ORGANIZATION 121 Fig. G7. — Acanthocystis aculeata; centroblepharoplasts disconnected from nuclear spindle. (After Stern.) 122 BIOLOGY OF THE PROTOZOA structures. According to Zuelzer the pseudopodia of Wagnerella borealis are withdrawn at times, owing to the contraction of the entire complex of radiating fibrils, and basal bodies lying at the bases of the axopodia become grouped in a zone of granules about the central grain. When the pseudopodia are again formed the granules migrate centrifugally to the periphery and, as basal bodies, give rise to the axial filaments. In Heliozoa without a central granule the axial filaments in some cases center in the nucleus in which there are many distinct and definite granules of uniform size distributed about the outer zone, from each of which an axial filament appears to rise (Fig. 66). In Camptonema nutans the nuclei are multiple and, according to Schaudinn, each one gives rise to a single pseudopodial element, but in Actinosphaerium eichhornii, which is also multinucleate, the axial filaments apparently have no connection with either nuclei or central kinetic elements. Apart from kinetic elements like centroblepharoplasts which, at the same time, are centers of mitotic activity of the nucleus and of kinetic activity of the motile organs, there are comparatively few examples of kinetic elements comparable with centrosomes of Meta- zoa. These are best represented in non-motile organisms such as Sporozoa, whereas in freely-moving types there is always some pecu- liar feature which makes the homology with centrosomes doubtful. A frequently cited example of a centrosome in Protozoa was first described by Hertwig in the case of Actinosphaerium eichhornii (Fig. 6. g., Ophrydium versatile), but gelatinous materials are secreted by all types of Protozoa. Usually, when the secretion is abundant, daughter cells remain embedded in it as a matrix after division, and the so-called spheroidal types of colony result (see p. 38). The ability to secrete gelatinous mantles as a reaction to unusual stimuli appears to be very widely distributed, if not universal amongst Protozoa. Bresslau (1921), using a variety DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 137 of chemical stimuli, was able to demonstrate a voluminous gelatinous envelope secreted by Colpidium campylum. Similar secretions were also demonstrated in other filiates and in certain rhizopods and flagellates as well. The secreted material, which he called "tektin," appears to be a combination of an albumin complex and a carbo- hydrate complex and, according to Bresslau (see also Schneider, 1930) it is instrumental in forming shells and tests of Protozoa, as well as trichocysts of many types. The most characteristic shell-forming material manufactured by Protozoa is chitin and pseudochitin. Chemically chitin is a modified protein (C30H50O10N4 or multiple) and is undoubtedly polymorphic in composition. Its mode of formation is still unproved, but condi- tions in Protozoa support the view of Chatin that it arises by trans- formation or differentiation of the peripheral cellular protoplasm. Not only are cups, tests, "houses" of various kinds formed of these substances, but cyst membranes, spore capsules of the Sporo- zoa and "central capsules" of the Radiolaria as well, while impreg- nated with calcium carbonate, silica, strontium sulphate, etc., or covered by foreign bodies of different kinds, the chitinoid mem- branes furnish the framework for the up-building of the most complex shells and skeletons. In encysting ciliates the animal becomes spherical, much condensed by loss of water and is sur- rounded by an envelope of fluid-like material which condenses more and more with exposure until the definite membrane, impervious to moisture and resistant to all unfavorable conditions of the environment, results. In Radiolaria the central capsule is a spherical wall of chitin, separating the endoplasm from the external proto- plasm and perforated in various ways to permit of communication between the different regions of the cell (see p. 439). In flagellates and ciliates the chitinous houses, tests, cups, etc., are usually colorless and very transparent, but in the rhizopods this is unusual, the chitin shells being colored by oxides of iron usually red or brown (Arcella sp., etc.). In the majority of fresh water rhizopods the outer surface of the chitinoid shell is covered by foreign particles of various kinds, such as sand crystals, diatom shells, or even living algae, which are glued to the membranes by a chitinous cement. Similar shells, which are generally known as arenaceous shells, are found amongst the Foraminifera. In other cases, plates of silica are deposited in the inner protoplasm and passed out during reproduction to be cemented on the chitinous membrane in regular patterns (Euglypha aheolata, Fig. 9, p. 31). Foreign bodies caught up in the wrinkles of withdrawing pseudopodia are similarly stored in the protoplasm to be used for shell-building purposes, Verworn, for example, compelling Bifflugia to build its shell of differently colored powdered glass. 138 BIOLOGY OF THE PROTOZOA The lime shells of Foraminifera are formed in quite a different manner. Here, calcium carbonate is precipitated between two lam- ellae of chitin very much as a cement wall is made between board surfaces. Except for a single mouth opening such limestone shells may form an unbroken wall about the organism (imperforata) or they may be perforated by myriads of minute pores (foramina) through which the pseudopodia pass to the outside, a condition which gave rise to the name Foraminifera. In the more compli- cated types of these lime-stone shells, which may reach a diameter of 2 or 3 inches, the calcium carbonate may be deposited at successive intervals of growth, thus giving rise to chambered structure of the cells. Such polythalamous shells are complicated by the presence of an intricate system of canals which, in life, are filled by moving protoplasm (Fig. 74). Fig. 74. — A complex polythalamous shell of Operculina (schematic). The shell is represented as cut in different planes to show the distribution of the canals and the arrangement of septa and chambers. (After Carpenter.) Skeletons of Ileliozoa and Radiolaria, unlike the more clumsy shells of the Foraminifera, are usually delicate in structure and graceful in design. They are formed for. the most part by a deposit of silica upon a chitinous base. Dreyer has given evidence to indi- cate that such skeletons have their beginnings in spicules which conform in shape and size with the nodal points in the alveolar walls of the cytoplasmic reticulum (Fig. 12, p. 33). Isolated spicules are characteristic of several Heliozoa and Radiolaria where they form a loose or felted covering in the outer protoplasm. Such spicules invariably grow by accretion, that is, by the addition of new sub- stance to the outside of that already formed. If such added material is formed in a limited region of the protoplasm, the result is a con- DERIVED ORGANIZATION— TAXONOMIC STRUCTURES L39 tinued accretion of silica to the end of a spicule which is pushed farther out with each increment, thus giving rise to long bars and spines which are radially arranged in forms like Acanthocystis aculeata, etc. (Fig. 75). The silicious deposit, again, may be made throughout a zone completely surrounding the center, resulting in clathrate or latticed skeletons of varying grades of complexity (Clathrulina elegans, Nassellaria). While cellulose mantles and shells are more usually found in A D Fig. 75. — Types of spicules in Heliozoa. A, Raphidiophrys pallida with curved silicious spicules; B, Pinaciophora rubiconda with tangential plates and forked spines; C, Acanthocystis turfacea, with separated plates and forked spines: D, Pinaciophora fluviatilis. (From Calkins after Penard.) chlorophyll-forming organisms, there are some types in which inter- nal skeleton elements are composed of this or a closely related sub- stance. In the parasitic Ophryoseolecidae skeletal structures are present which are made up of a substance resembling cellulose to which Dogiel gave the name Ophryoscolecin. (b) Motile Organoids.— The organoids by which Protozoa move are to be considered as modifications of the cortex, although some types, as shown in the preceding chapter, are derived in part from internal kinetic elements (flagella and some pseudopodia). Three main types are distinguishable flagella, pseudopodia and cilia, each of which is sufficiently distinct from the others to furnish a natural basis for classification of the Protozoa, a basis of classi- 140 BIOLOGY OF THE PROTOZOA fication which Dujardin first em])loyed to create the three great groups les flagelles, les rhizopodes, and les cilies. Each type is sub- ject to many variations, due to inherent differences in the motile organoids themselves, or to fusion in various ways leading to struc- tures of considerable complexity. It is extremely difficult to decide whether flagella or pseudopodia are the more primitive in type. From most general text-books on Zoology we learn that the matter admits of no question, and are taught that the pseudopodium is the most primitive form of motile organ in the animal kingdom. This certainly has been the most widely accepted view. Many a generalization referring to Protozoa, however, which has found its way into general works on Biology, appears to have been drawn from the conditions in some one organ- ism which is conspicuous by reason of its abundance and ease of study. It would sometimes appear, indeed, that the common species of Paramecium and Amoeba proteus, to many general writers constitute the Protozoa. This seems to be the case with the problem of pseudopodia and flagella, the argument being that a pseudopo- dium of Amoeba proteus is certainly a less complex motile organ than the flagellum of Euglena viridis, and therefore more primitive. Had the comparison been made between the pseudopodia of Actino- phri/s sol or Acanthocystis aculeata and a typical flagellum, the con- clusion would not have been so obvious. There is a good deal of evidence against the generalization as it is usually expressed. In the first place, a pseudopodium of Amoeba proteus cannot be inter- preted as a motile organ. It is not a definite structure in the cell, nor does it cause the body of Amoeba proteus to move. On the con- trary, it exists because of the movement of the body protoplasm and the pseudopodium is merely the visible, physical expression of this movement which, in turn, is due to the transformation of energy in destructive metabolism. This energy finds its vent in that por- tion of the ectoplasm which, for the time being offers the least resist- ance; the ectoplasm gives way at this point, the endoplasm gushes through and a pseudopodium results (see Chapter XII, p. 435). Such pseudopodia are not the source of movements of the cell, they are results, not causes, of movement. The pseudopodia of some Heliozoa, on the other hand, are motile organs, and the axial filaments which they contain are regarded as equivalent in struc- ture and in mode of origin to the kinetic elements of flagella. The pseudopodia of Foraminifera are intermediate between those of Heliozoa and those of testate rhizopods. The problem, then, comes down to a theoretical question of probabilities. Is it more probable that pseudopodia of the type found in Amoeba proteus become pro- gressively differentiated into motile organs through stages like the finger-formed pseudopodia of the testate rhizopods, the reticulate pseudopodia of Foraminifera and axopodia of Heliozoa and Radio- DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 141 laria, to the typical motile organ of the flagellate type? Or is it more probable that a motile organ originating from a definite kinetic center (basal body or blepharoplast) has become progressively indefi- nite with loss of the kinetic elements through the same series of forms, but in the opposite direction, and ending in types like Amoeba proteus? To my mind, the pseudopodia of Amoeba proteus and its immediate relations, have no place at all in such a series; they are merely expressions of the physical conditions of the protoplasm and of the forces operating within, and they may appear in any cell having an appropriate physical make up. Thus we find them in certain types of cell (leukocytes and phagocytes) widely distributed throughout the animal kingdom, and we find them here and there, in every group of the Protozoa. An illuminating illustration in support of this conclusion is afforded by the transitory flagellated stages of one group of ameboid organisms, the Bistadiidae (see p. 108). Here, in Dimastigamoeba gruberi, for example, the organism loses its pseudopodia under cer- tain conditions, and develops flagella, not by metamorphosis of the pseudopodia, but from blepharoplasts which, as centrioles, emerge from the nucleus (Fig. 59, p. 108). Although only a matter of academic interest, I believe that the flagellum type of motile organs is the most primitive type we know while axopodia and myxopodia, the former with kinetic elements of weakened function, the latter with denser axial protoplasm which Doflein also interprets as equivalent to axial filaments, represent stages in the deterioration of the kinetic function coincident with the absence of definite kinetic centers (see also p. 120). For these reasons also, together with others which will be given later, we hold with Doflein (1916), Klebs and many others, that the group of flagellates furnishes more evidence of original ancestry than do the rhizopods (see p. 411). 1. Flagella.— Flagella are widely distributed throughout the animal and plant kingdoms, forming the motile elements of animal spermatozoa and of plant zoospores, or current-producing organs of many types of Metazoa. They are sometimes combined with pseudopodia (Dimorpha mutans, Fig. 13, p. 34, Mastigamoeba inver- tens, Ciliophrys infusionum, etc.), sometimes with cilia (Myriaphrys paradoxa, Fig. 197, p. 478). Flagella are usually excessively fine and delicate fibers extremely difficult to see and to study in the living organism. In the great majority of cases the finer structure has not been made out, but in a few favorable types some progress has been made. In these cases it is known that the flagellum is made up of two definite elements, an axial, highly vibratile filament, which is formed as an outgrowth from the basal body or blepharoplast, and an enveloping elastic sheath which is formed from the periplastic substance of the cor- 142 BIOLOGY OF THE PROTOZOA tex. In some cases the sheath is circular in cross-section (see Plenge), in others ellipsoidal, while the contractile thread which is usually attached firmly to the sheath may run in a straight line the entire length of the sheath, or may follow a spiral course. In the majority of flagellates the sheath undulates and vibrates in unison with the contractile axial thread, but in a few types, such as Per- anema trichophora or certain species of Bodo, the sheath remains passive while the axial thread extends freely beyond the limits of the sheath, where its activity in the surrounding medium results in a steady progressive movement of the cell. Under the influence of somewhat violent stimuli, however, the sheath itself may undergo fibrations in such forms. Owing to the nature of flagella and to their delicacy of structure, there are not many possibilities of variation in type. In addition to those which are circular or ellipsoidal in cross-section, there are some which are band form. Such band-form flagella suggest the possibility that vibratile membranes, which are not uncommon in parasitic types of flagellates, may, morphologically, be regarded as flagellum sheaths which remain attached throughout their length to the cortex while the axial thread forms the contractile margin (Fig. 169, p. 360). Such vibratile membranes are characteristic of the genera Trypanosoma, Cryptobia, Trichomonas, Trichomastix, etc., all of which are parasites in the blood or digestive tract of different animals. There are, however, abundant variations in size, number and position of flagella in the cell. When there is but one it usually emerges from a pit or funnel-shaped opening at the anterior end of the cell (flagellum fissure). When two are present they may be equal in size and length (e. g., Spongomonas splendida, Fig. 49, p. 95), or one may be considerably thicker and longer than the other (heteromastigote types). Both may be directed forward as in Amphimonadidae or one may be directed forward, the other back- ward, as in Bodo, Anisonema, etc. In such cases the posteriorly directed flagellum (trailing flagellum or Schleppgeissel) appears to act as a runner upon which the cell body glides, and has little to do with the actual locomotion of the animal (Fig. 76). Delage and Herouard have attempted to explain the dynamics of flagellum action whereby the comparatively heavy body is moved forward by reason of the vibrations of the exceeding^ delicate thread. In the usual type the extremity of the flagellum describes a rather wide circle so that it is in a certain focus of the microscope for only an instant of time. With this circular movement, which varies in different species, constant undulations pass from the base to the tip. A forward pull results from the combination of such movements and the cell either glides smoothly after its active pro- peller or rotates more or less rapidly on its long axis while freely DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 143 swimming. When two flagella are present a curious shaking move- ment may accompany rotation and translation. With such energetic motile organs exerting a constant strain on the body there would seem to be some danger of their being pulled out, especially in those types with soft fluid bodies without firm Fig 76.— Free-living flagellates with trailing flagella. A, C, D, Bodo caudatus St. B, Bodo globosus St.; E, Ploeotia vitrea Duj. (After Calkins.) 144 BIOLOGY OF THE PROTOZOA periplasts. This phenomenon has indeed been recorded by some observers, the flagellum, freed from the body, moving off like a spirochete (Klebs, Biitschli, Fischer, etc.). Such observations may or may not be well founded, at any rate accidents of this char- acter are guarded against by the manner of flagellum anchorage in the cell. As described in Chapter III a flagellum is derived from a blepharoplast which may be just below the periplast or deeper in the protoplasm, or it may arise from the nucleus (Fig. 59, p. 108). Its anchorage is further assured by rhizoplasts which sometimes run to the posterior end of the cell as in Herpetomonas or species of Rhizomastix (Fig. 62, p. 116), or which form a branching complex deep in the body substance as in Dimastigamoeba (Fig. 59, p. 108). In the various species of Giardia the basal bodies of the eight flagella are connected by a complete system of rhizoplasts (Fig. 17, p. 37). Another type of structure which is regarded by some (e. g., Kofoid) as a modified flagellum is represented by the axostyles or internal motile organoids of the parasitic flagellates. In Trichomonas this appears like a glassy, hyaline curved bar of considerable diameter, extending from the nucleus to the posterior end of the cell where, like a spine, it projects from the periphery (Fig. 77). It is usu- ally interpreted as a supporting axial rod to give rigidity of form to an otherwise soft and variable body (Doflein). Dobell regards it as a remnant of the centrodesmose left in the cell after division of the blepharoplast, a view supported by Hartmann and Chagas (1910) who interpret it as a centrodesmose formed during division of the intranuclear centriole. Martin and Robertson (1909), on the other hand, found that axostyles arise after division quite inde- pendently of the nucleus or of centrodesmose, and regarded them as independent organoids of the cell. Kofoid and his associates discard the assumption that axostyles are supporting or skeletal structures and place them in the category of kinetic elements. They are interpreted as intracellular organoids with a contractile function characteristic of flagella and serve as organs of locomotion in the dense media in which the parasites live and in which the flagella would be ineffective. They are closely connected with the blepharoplasts in all species of Giardia (Fig. 17, p. 37), and are regarded as independent, self-perpetuating organoids which may be the first to divide in the processes of reproduction (Giardia) or the last to divide (Trichomonas). In all cases, according to these ob- servers, but denied by others, the axostyle divides longitudinally throughout its entire length, beginning with divisions of the anterior end in which the blepharoplast may be embedded (Fig. 77). In regard to the two opposing points of view as to the function of axostyles the evidence rather supports the interpretation of Kofoid and Swezy (1915). The necessity of a supporting struc- DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 145 ture, or a form-rectifying organ, in these parasitic types is difficult to conceive. On the other hand, their intimate relation to the blepharoplasts and their activity in reproduction indicate a common function with the kinetic elements. The observations of Kofoid and Swezy on the energetic movements of the axostyle while the organism works its way through the mucus afford a more plausible interpretation of the function of this organoid than the a priori view of those who see in such movements only the efforts of an elastic supporting structure to restore the form of a plastic cell. Fig. ■Trichomonas augusta Alex. Two successive stages in division of the axo- style. (After Kofoid and Swezy.) 2. Pseudopodia. — Pseudopodia are more or less temporary' pro- jections of the cortex which may serve for purposes of locomotion or, more often, as food-trapping or food-catching organoids. Four types are recognized, axopodia, rhizopodia (myxopodia), filopodia and lobopodia, which differ widely in their structural make up. Of these only the first type can be regarded in a strict sense as motile organs (see p. 140), the others functioning as food-catching organoids, or mere protrusions of the semifluid body. Axopodia.— Axopodia are different from other types of pseudo- podia in possessing, like flagella, central axial fibers of specialized protoplasm derived from endoplasmic kinetic elements. They are found only in organisms belonging to the groups Heliozoa and 10 14G BIOLOGY OF THE PROTOZOA Radiolaria, in which they radiate out in all directions from a usually spherical body (Fig. 78). Unlike nagella, the outer coating of an axopodium is not a smooth periplast-like sheath, but consists of fluid protoplasm in which the movements of granules out on one side and back on the other are c '#£<*SW59?*«Wi^. w ^» BO< s3(+*e*»*i,'3K#<"'.- . .. *"■■ t-- 1 *. ;J «-,--*• D Fig. 78. — Types of pseudopodia. ,4, B, Eruptive type of lobopodium; C, myxo- podia type of Foraminifera ; D, axopodia type of Heliozoa. (After Calkins.) clearly discernible. In this manner the outer protoplasm is con- tinually changing about the central axial filament, which alone is constant or fixed. Upon prolonged irritation, or in preparation for division or encystment, the axial filaments themselves, together with the enveloping protoplasm, are withdrawn. Like flagella the axial filaments are formed as outgrowths from DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 147 endoplasmic kinetic elements. Gymnosphaera, Raphidiophrys, Sphaerastrum, Acanthocystis, Dimorpha, etc., possess characteristic "central grannies" which, from their activities in cell division, are unmistakably centroblepharoplasts (see p. 117) from the substance of which the axial filaments are formed (Fig. 50, p. 95). Wagner- ella borealis, in addition to the central granule, possesses a zone of basal bodies which give rise to the axial filaments and which at times of retraction of the pseudopodia are drawn into the central granule. In still other cases, as in Actinosphaerium eichhornii, the axial filaments do not arise, apparently, either from central granules or from nuclei, but appear to start indefinitely in the cytoplasmic reticulum (Fig. 78, D). While the more common forms of Heliozoa are quiescent, floating types, some of the Heliozoa are freely motile. Acanthocystis acu- leata, as well as other species of the same genus, turns slowly over and over in a rolling movement; Camptonema nutans, according to Schaudinn, bends and straightens its axopodia in food-getting and in other activities. Actinosphaerium eichhornii and Actinophrys sol are practically motionless. The active movements are due to the axopodia and the structure of axopodia is strikingly like that of flagella. That the contractile axial filament is the seat of this movement, and not the enveloping protoplasm, is not open to reasonable doubt. Structure, function and mode of origin thus justify the inclusion of axopodia with the kinetic elements of the cell. On the other hand, in type-, with axopodia which are practically motionless, the axial filaments have apparently lost the vibratile function and now serve as supporting elements for the long radiating pseudopodia. There is little reason to doubt that such elements are homologous with the axopodia of motile types and that the latter are homologous with flagella. This is well illustrated by the case of Dimorpha mutatis where two flagella and many axial filaments of axopodia originate from the same blepharoplast (Fig. 79.) Speculations as to phylogeny on purely morphological grounds are not profitable, but in this group of Heliozoa we have pretty good evidence of a close relationship between flagellates and Sarcodina, and equally good evidence of the transition from an active kinetic- element to an inactive, supporting axial rod, as seen in the pseudo- podia of Actinosphaerium eichhornii. This change in type is prob- ably associated with the loss of specific kinetic centers for neither in the cytoplasm nor in the nuclei are such elements to be found. In some forms, finally, notably in Clathrulina elegans, the ends of the axopodia are frequently branched, a condition which points the way to pseudopodia of the rhizopodia type in which the supporting element is not in the form of an axial rod, but in the form of stiff stereoplasm (Fig. 78, C). The pseudopodia of Clathrulina, which 148 BIOLOGY OF THE PROTOZOA have no axial filaments, appear to be transitional to those of the testate rhizopods to which group Valkanov (1928) assigns them. In this stalked form (Fig. 82), however, the stalk takes its origin from the nucleus, as Valkanov clearly shows, and at some stages, at least, has a fibrillar structure. This suggests the possibility that the stalk of Clathrulina (and of Hedriocystis) may represent the con- crescence of ancestral axial filaments. B Fig. 79. — Dimorpha mutans. Vegetative individual with two flagella and axopodia. Axial filaments of axopodia and flagella meet in a common central granule. At division the central granule divides and forms the poles of the mitotic figure, while the axial filaments form astral rays. X 1950. (After Belaf, Allgemeine Biologie, 1927; B. Ergeb. u. Fortschritte d. Zoologie, courtesy of G. Fischer.) Rhizopodia. — This type of pseudopodia differs from others, first, in the tendency to branch and, second, in the tendency to fuse or anastomose when such branches meet. From these characteristics they are sometimes called reticulose pseudopodia and myxopodia. So far as number of species is concerned, this type is the most characteristic form of Sarcodina pseudopodia. They occur in all forms of Foraminifera, Radiolaria and Mycetozoa which include the great majority of Protozoa. As a result of their unlimited power to branch and to anastomose, great meshworks of reticulated proto- plasm are created which make ideal traps for the capture of food. DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 149 In many types, especially in Radiolaria, they may be long and ray- like, with relatively little tendency to fuse; in other cases a main trunk gives rise to so many branches that it is lost in the reticulum, great accumulations of protoplasm collecting at the branching points (Fig. 10, p. 32). Doflein includes axopodia and these branching anastomosing pseudopodia in the one type (rhizopodia), and sees in the axial fila- ment of the former and the inner protoplasm of the latter only Fig. 80. — Clathrulinaelcgans, stalk formation. (After Valkanow, Archiv f. Protisten- kunde, 1928, courtesy of G. Fischer.) different states of the same fundamental stereoplasm. Axial fila- ments, however, derived from the substance of kinetic centers, are quite different from structureless axial stereoplasm which has no relation to kinetic elements. The enveloping protoplasm is appar- ently the same in both types and granule streaming is a common property, but the physical consistency is quite different. In rhizo- podia the outer protoplasm is soft and miscible, leading to fusion on contact with one another, while axopodia never anastomose. The denser core of rhizopodia, while not condensed to a single fiber, serves the same function of support as the axial filament of Actino- sphaerium and gives stiffness and rigidity to long ray-like pseudo- 150 BIOLOGY OF THE PROTOZOA podia of many Foraminifera and Radiolaria which stand out in all directions from the cell. Filopodia. — Structurally filopodia are entirely different from the types described above, being formed of clear hyaline ectoplasm in typical cases, or they contain a few granules indicative of endo- plasm (Fig. 11, p. 33). They are usually long and slender and with rounded ends giving the impression of slender glass rods. In some forms there is a tendency to branch at the ends as in Euglypha alveolata (Fig. 9, p. 31), but there is never anastomosis. Some- times they sway back and forth like a filament of Oscillaria, but usually they creep along the substratum where they serve mainly for food capture. Filopodia are characteristic of the fresh water testate rhizopods, but are sometimes present in naked types like Amoeba radiosa. Lobopodia. — Lobopodia are made up of granular endoplasm and hyaline ectoplasm, and are temporarily projected portions of the body protoplasm not to be compared with definite locomotor organs of other Protozoa. The inner protoplasm of nearly all kinds of Protozoa with granules of various kinds, food substances more or less digested, and waste materials, is in constant movement called cyclosis. In more highly differentiated forms, and in organisms with a firm cell membrane, this movement is confined to the internal protoplasm and the form of the cell is not affected by it. In the shell-less rhizopods, however, there is no such outer covering, and the peripheral protoplasm gives way at the weakest points, and an outward flow of protoplasm with corresponding change in the form of the body results (see Chapter V). If such a weak point is con- stant in position, a constant flow in its direction is the outcome, and the Ameba, consisting of practically one pseudopodium, as in the Umax types, moves in one direction. In Amoeba verrucosa a delicate periplast surrounds a somewhat dense protoplasm which, accumulating on one side (according to Rhumbler, 1898), causes the cell to roll over. Withdrawal of pseudopodia is accomplished by their absorption into the body substance, and is accompanied by a wrinkling of the denser ectoplasm preparatory to its transformation into endoplasm (see Schaeffer). In pseudopodia generally it is evident that we have to do with different types of structure which, in only a few instances, can be regarded as motile organs. Axopodia, with their axial filaments derived from kinetic elements, are closely related to flagella and may be regarded as organs of locomotion, but the other types, which may represent highly modified axopodia, have lost the kinetic elements, if they ever had them, and are useful only as food-catching organs. In most rhizopods the entire organism is the motile element, rhizo- podia, filopodia and lobopodia being expressions of energy trans- DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 151 formations comparable with the rotation of protoplasm in Nitella or circulation in Tradescantia. Axopodia of the motile Heliozoa, axial filaments of the inactive species and stereoplasmic cor.s of the rhizopodia may be regarded as successive phases in the modi- fication of vibratile flagella. These types of pseudopodia have in Fig. 81. — Types of Ciliata. A, Uroleptus pisces (after Stein); B, Cyclotrichium gigas (after Faure-Fremiet) ; C, Stentor polymorpha (after Biitschli) ; D, Nyctotherus ovalis (original). common an enveloping layer of granular protoplasm, but filopodia and lobopodia represent a different type, being made up in large part, or entirely, of ectoplasm and without any evidence whatsoever of kinetic elements. So-called "contractile elements" of this type of pseudopodia are largely figments of the imagination. 152 BIOLOGY OF THE PROTOZOA 3. Cilia.— Cilia are the motile organs of Infusoria and accompany the most highly differentiated types of cortex to be found in the Protozoa. Individually they are shorter, more delicate and less powerful than flagella and owe their importance as motile organs to their large numbers and synchronous beating. Their action may be compared with that of oars in rowing, while flagellum action might be compared with sculling, and the results of cilia and flagella activities bear a relation similar to that between a racing shell and a gondola (Fig. 81). Fig. 82. — Cilia structure. Axial filaments protruding from protoplasmic sheaths in cilia of (1) Coleps hirtus, (2) Paramecium; (3) cilia make up of three lateral cirri of Stylonychia. Silver line technique. (After Klein, Archiv f. Protistenkunde, 1929, courtesy of G. Fischer.) According to the interpretation of several observers, mainly Schuberg, Maier, Schubotz, Schroder, etc., the cortex of ciliates is a composite of zones of differentiated protoplasm. In the majority of cases such zones cannot be made out, for one shades into the other, and the whole into the alveolar endoplasm. In favorable cases, however, we can distinguish: (1) A superficial periplast perforated for the exit of cilia and trichocysts when present; (2) an alveolar DERIVED ORGANIZATION— TAX0N0M1C STRUCTURES 153 layer containing trichocysts if the latter are present; (3) a contrac- tile zone containing the basal bodies of cilia, myonemes and coordin- ating fibers; (4) a denser zone which shades off into the endoplasin and supplies an anchorage for nuclei and contractile vacuoles. A single cilium is constructed on much the same plan as a flagel- lum, consisting of a central axial filament or fiber, and an elastic sheath of protoplasm. Movement is due to the active contraction Fig. 83. — Cyclidium glaucoma. Cilia with axial filaments protruding from plasmic sheaths. Silver line technique. (After Klein, Archiv f. Protistenkunde, 1929, courtesy of G. Fischer.) in one plane of the axial fiber and recovery to the elasticity of the enveloping sheath. The contractile element originates from a basal body in the contractile zone. In many organisms local thickenings occur at intervals along the axial filaments. These are similar to basal bodies and are clearly demonstrated by silver nitrate impreg- nations for bringing out the silver line system (Figs. 82 and 83). The arrangement of cilia on the surface of the body varies in 154 BIOLOGY OF THE PROTOZOA different species; sometimes they form a complete coating for the organism as in the majority of Holotrichida (Fig. 84); sometimes they are limited to certain zones as in Urocentrum turbo, Didinium B Fig. 84.— Types of Ciliata. A, Monodinium balbianii; B, Cyclotrichium sphaericum, C, Dinophrya lieberkuhni; D, Askenasia elegans. (After Faure-Fremiet.) nasutum, etc. (Fig. 205, p. 504) ; or sometimes to the ventral surface, as in generalized Hypotrichida (Fig. 88, p. 159). In all cases they are arranged in longer or shorter rows running straight or spirally, and DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 155 giving the striped appearance characteristic of the ciliates. Waves of contraction pass from the anterior end posteriorly, cilia of the same transverse rows beating synchronously, those of the same longitudinal rows metachronously. The periplast is variously sculptured in different species, giving the appearance superficially of a different mode of origin of the cilia. In some cases they appear to come from the centers of minute cups or dimples as in Paramecium aurelia; in other cases from longitudinal grooves or furrows between ridges of periplast (Fig. 69, p. 124), and in some they appear to come from the ridges themselves. Rhizoplasts or endoplasmic prolongations from the basal bodies are comparatively rare but occur in some cases as in Didinium nasutum (Fig. 98, p. 187). Coordinating fibrils apart from the silver line system have been described in a few types (En plaits, Diplodinium, see p. 129), and center in a specialized neuromotor body, the so-called motorium (Yocom, Taylor, Sharp). In some cases cilia are uniform in length over the entire body (Opalina); in other oases they are longer in the region of the mouth or around the posterior end, but no sharp dividing point separates short from long ones (Fig. 84). In some cases they are uniformly long and vibrate like flagella (Actinobolus radians, Fig. 91, p. 163). 4. Composite Motile Organs.- A well-marked characteristic of cilia is the ability of two or more to fuse into motile organs of vari- able complexity. Such combinations give rise to membranulae, membranelles, undulating membranes and cirri, each of which, although composed of fused cilia, originates or grows as an inde- pendent and complete organoid. In each case also the component cilia may be demonstrated by use of dilute alkalies such as potas- sium or sodium hydrate. It is often difficult to distinguish lines of closely set cilia from fused cilia, and loosely bound cilia are sometimes present, the aggregates being spoken of as "pseudo- membranes." Membranulae.— Membranulae are very long, delicate, finely- pointed aggregates of cilia which differ from the somewhat similar cirri in movement and in composition, while their basal granules, in Didinium nasutum at least, are connected with the vicinity of the nucleus by definite rhizoplasts (Fig. 98, p. 187). Similar mem- branulae form the basal ring in Vorticellidae (Schroder, Schuberg, etc.). Membranelles. — Membranelles are formed by the fusion of cilia in the region of the mouth. In many of the Holotrichida the cilia are longer just posterior to the mouth than in other regions of the body, frequently forming circlets about the mouth as in Lacrymaria olor or L. lagenula (Fig. 85). In the other Orders of Ciliata oral cilia are fused to form membranelles. In the oral regions the body 156 BIOLOGY OF THE PROTOZOA is usually differentiated into a specialized food-collecting, frequently funnel-like structure called the peristome. Cilia on the floor of the peristome are usually longer than in other parts of the body, and in I I mm ' * J : Fig. 85.— Types of Lacrymaria. A, Lacrymaria sp.; 5, and C, retracted and ex- panded phases of Lacrymaria olor; D, Lacrymaria lagenula. (After Calkins.) four of the five orders of ciliates some of these are invariably aggre- gated in triangular, quadrilateral or ribbon-like membranelles and membranes for producing food-bringing currents of water toward DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 157 the mouth. In every order except the Holotrichida a fringe of such specialized motile organs, known as the adoral zone, lies on a margin of the peristome (Fig. 88). Membranelles are usually made up by the fusion of two rows of cilia as shown by the double row of basal bodies (Maier) and their flat or curved faces make powerful sweeps in the water. According to Schuberg, Gruber, Maier and others, the anchorage of these organoids is quite complex. The basal granules form a double row immediately below the periplast; fibrils from these, analogous to rhizoplasts, form a broad triangular basal plate and are then brought together to form an end thread which connects the membranelle with coordinating fibers (Fig. 72, p. 130). While in most cases the membranelles represent the fusion of comparatively few cilia in transverse rows of the peristome, making them relatively narrow at the base, in other cases, notably in the Tintinnidae, such fusion includes practically all of the cilia of the transverse rows, making membranelles as broad as the peristome. In the Vorticellidae there are two rows of membranelles, the double adoral zone winding about the peristome usually in a direction opposite to that of the Heterotrichida and Hypotrichida (Fig. 86.) Undulating Membranes. — Undulating membranes are found in all orders of the ciliates and range in size from delicate aggregates no broader from base to tip than ordinary cilia to relatively enormous balloon-like structures equal in width to more than half the diameter of the body and in some cases, as Lembadion conchoides, almost equal to length of the body (Fig. 87). In the simplest cases these mem- branes are composed of a single row of longitudinally placed cilia, the basal bodies of which form a single basal strand. Since cilia of the longitudinal rows beat metachronously the result of their contrac- tion when fused in these undulating membranes is a series of waves passing from the anterior to the posterior end. In more complex forms undulating membranes may be composed of 3 to 10 rows of cilia, fused in longitudinal rows, the length varying from a few microns to great waving sheets of protoplasm almost as long as the entire cell (Fig. 87). They are usually found in the peristomial area inside the adoral zone and are named preoral, endoral, paroral, etc., according to their positions in relation to the mouth. Pseudomembranes are present in numerous types. Here the component cilia are not firmly united and the membrane is easily disrupted. Such a membrane, which is rather easily disintegrated, is characteristic of Blepharisma undulans. Chambers and Dawson (1925) were able to hold down a portion of the pseudomembrane with a needle whereupon the distal portion broke into fibrils which later reunited after the obstruction was removed. Cirri. — Cirri are the most highly specialized of all the motile organs of ciliates, the most characteristic forms occurring in the 158 BIOLOGY OF THE PROTOZOA Hypotrichida. They are placed more or less definitely on the ventral surface, a group, variable in number, at the anterior end being known as the frontal cirri, a similar group, also variable in number, near the posterior end being known as the anal cirri, while other groups may form caudal cirri, ventral cirri, marginal cirri, etc. (Fig. 88). Vj> P.C. Fig. 86 Fig. 87. Fm. 86. — Structure of typical Vorticella showing the adoral membranes, AM' I 1/ ,• vestibule, 1*.; contractile vacuole, C.V.; food vacuole, FA'., and posterior circlet of cilia. (After Noland and Finley, from Trans. Am. Microscopical Sue, 1931.) Fig. 87. — Lembadion conchoides F.'F. (After Faure-Fremiet.) . ( 'irri are always broader at the base and taper gracefully to a fine point. In cross-section near the base they are either circular, ellipsoidal, quadrilateral or irregular, and always have a basal plate made up of the basal granules of the fused cilia. Under unfavorable conditions of the medium in which the organisms live, and usually after imperfect fixation, the constituent cilia become separated par- DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 159 'A m I I I fllli m liiM W Fig. 88. — Types of Ciliata. A, Amphisia kessleri; B, Uroleptus pisces; C, Histrio pellionella; D, Strongylidium sp. ; E, Oxytricha pellionella; F, Oxytricha fallax. (A, after Calkins; B, C, D, E, after Biitschli ; F, after Stein. j 160 BIOLOGY OF THE PROTOZOA ticularly near the tip, and the cirri then present a most frayed-out or ragged appearance. They vary in size from extremely minute cilia-like marginal and ventral cirri to great ventral brushes in forms like Aspidisca (Fig. 90) or huge hooked structures as in Uronychia, Diophrys and other Euplotidae (Fig. 89) (see also p. 221). Fig. 89.— Types of ciliates. A, Perilromus i mmae; B, Kerona pediculus; C, Diophrys appendiculatus; I), Euplotes charon. (A, C, D, after Calkins; B, after Stein.) ( !irri are preeminently organs of locomotion, but, unlike other motile organs of the ciliates, their stroke is not confined to one plane but may be in any direction. This gives to the Hypotrichida an extreme variety of movements unparalleled by any other group of Protozoa. Many of them walk or run on the tips of their frontal and ventral cirri (Stylonychia) ; others swim with a peculiar jerky DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 161 movement (Aspidisca) ; others combine swimming due to the adoral zone with sudden jumps or springs due to the anal or caudal cirri (Uronychia, Euplotes, etc.). Such saltations are not limited to the Hypotrichida, however, but are characteristic of organisms in all groups where cirri are developed as in Halteria grandinella among Oligotrichida, Mesodinium cinetum among Holotrichida, etc. In some cases cirri are differentiated as tactile organs, especially the more dorsal ones of certain Hypotrichida. It is probable that such cirri are no different from other motile organs of the ciliates in this respect, extreme irritability being a common characteristic. Few observers can have failed to note the instantaneous effect of a slight local irritation on a quietly resting Pleuronema chrysalis, for example, with its long cilia radiating out in all directions, yet there are no cirri here. Fig. 90. — Two species of Aspidisca. (Original.) The synchronous and metachronous vibrations of cilia and cilia aggregates are probably regulated by coordinating fibers with highly developed irritability. This is the interpretation given by Schuberg to the basal fibrils in the contractile zone of Paramecium caudatum; by Neresheimer (1903) to certain fibers distinct from the myonemes in Stentor coerulens, and by Sharp, Yocom, Taylor and others, to conspicuous fibers in Diplodinium ecaudatum and Euplotes patella (see p. 127) ; others, however (e. g., Jollos, and Belaf ), interpret them as supporting structures. In the latter organism Yocom (1918) and Taylor (1920) found fibers running from the posterior anal cirri and from the adoral zone of membranelles to a common anteriorly placed structure termed the motorium, which 11 162 BIOLOGY OF THE PROTOZOA they regard, with Sharp (1914), as a center of the neuromotor sys- tem (see p. 129). The ventral and frontal cirri, however, are not connected by similar fibrils with this motorium, but possess bundles of fibrils, described earlier by Prowazek in Euplotes harya, and by Griffin in E. ivorcesteri, which may run in any direction until lost in the endoplasm. The inference is that these cirri are independent of the coordinated system of fibrils which regulate the adoral zone and the anal cirri, and that their movements, which are always irregular, are not affected by cutting the coordinating fibrils of the motor system (Fig. 72, p. 130, also see p. 131). (c) Other Organoids Adapted for Food-getting.— Mention may be made here of a few special types of cortical differentiation apart from the cell mouths, which Infusoria use for purposes of food- getting. The most striking of these are the tentacles of Actinobolina radians, the "tongue" or "seizing organ" of Didinium nasutum and the tentacles of the Suctoria. Contractility due to myonemes is a widely-distributed phenome- non in ciliated Protozoa and in most cases involves the activity of the entire organism (see p. 124). When it is limited to restricted portions of the body, such as the peristomial complex of Diplodi- nium ecaudatum, or the "vestibule" of Vorticellidae, it acquires a special interest. Even more remarkable than these, however, is the power, possessed by Lacrymaria olor, of projecting its mouth- bearing extremity any distance up to three times the length of the flask-shaped body, or until the rubber-like neck is reduced to a mere fibril. The "head" thus projected dashes here and there with amazing rapidity, the body meantime remaining quiet and unmoved, until finally the head and neck are withdrawn and the cell swims off with no visible trace of contractile structures (Fig. 85, p. 156). No special myonemes have been described in this form and the projection and retraction of the "head" must be due to the elasticity of the cortex of the "neck" region, combined with activity of the oral circlet of cilia while the body cilia are at rest or relatively quiet. Another remarkable and special phenomenon, seen apparently by few observers, is the method of- food-getting by Actinobolina radian:-!. This organism, when at rest, protrudes a forest of radiat- ing tentacles which stand out like axopodia, sometimes stretching a distance equal to two or more times the body diameter. The ends of these tentacles carry trichocysts (Entz, Calkins, Moody) which upon penetrating an individual Halteria grandinella com- pletely paralyze it. The tentacle, then, with prey attached, is withdrawn entirely into the body, the Halteria is worked around to the mouth and swallowed (Fig. 91). Actinobolina vorax (Wen- rich) has a similar food-getting mechanism but is not as fastidious about its food as is .1. radians. DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 163 In Didinium nasutum the proboscis bears a peculiar protrusible plug or tongue of protoplasm termed the "seizing organ" by Thon (1905) and Prandtl (1907) (Fig. 98, p. 187). A zoneof trichocyst-like fibrils lies near the extremity of this plug and when certain types of ciliates, preferably Paramecium, are struck by Didinium the plug, with trichocysts penetrates the cortex of the prey, paralyzing it. While this process takes place too rapidly to be seen, the results show that it must have taken place for, after striking and anchoring in the Paramecium, the seizing organ with prey attached is retracted and the prey, often larger than the captor, is swallowed whole (Fig. 98, p. 187). No satisfactory explanation of this phenom- enon has yet been given. Fig. 91. — Actinobolina radians St. (After Moody.) Still another type of cortical organs is illustrated by the various kinds of tentacles of the Suctoria. Some of these are constructed for piercing, while others are hollow, forming sucking tubes through which food is taken into the body. They are evidently provided with some type of poison, for active ciliates, coming in contact with these tentacles, become suddenly quiet and remain so while the suctorial tentacles penetrate the cortex and suck out the endoplasm of the prey which can be followed through the feeding tubes to the endoplasm of the captor (Maupas, 1883). Like the tentacles of Actinobolina radians, these suctorial tentacles are retractile, but again there is no satisfactory explanation of their activity and no description or mention of specialized motile apparatus. 164 BIOLOGY OF THE PROTOZOA Like the majority of formed organoids of the cell, the more com- plicated of the motile organs described above are formed anew at each division of the cell. This does not apply to the majority of pseudopodia nor has it been observed in the case of cilia, but is well-established for flagella and for the aggregates of cilia, such as membranelles, undulating membranes and cirri. In a few cases the flagella themselves are said to divide, but this is questionable, the flagella probably arising in all cases from the substance of blepharoplasts or basal bodies which have divided. Young (1922) has shown that a cirrus of Uronyckia transfuga if cut does not regenerate, but if the protoplasm is partly included in the opera- tion a new cirrus is regenerated. Demboska (1925) has shown that if a single cirrus of Stylonychia is cut out all of the cirri are renewed. (d) Oral and Anal Cortical Modifications. In all naked forms of Protozoa and in corticate forms which, like Opalina, take in food substances by osmosis through the general body surface, there are no portions of the ectoplasm differentiated as cytostomes or cell mouths. In such forms, furthermore, where there is no undigestible matter, there is no modification as cytopyge (cytoproct, or cell anus). In testate forms, obviously, there is only a limited region of the body substance which is open for the reception of food. In testate rhizopods the shell openings are due to the physical condi- tions under which the lifeless shell materials are deposited and no definite mouth parts as protoplasmic differentiations are present. In all Protozoa, on the other hand, which take solid food and which are covered by more or less highly differentiated cortical plasm, there are permanent openings in the cortex serving for the intake of solid bodies and for defecation of undigested remains. In many cases such openings in the cortex merely expose a limited region of soft receptive protoplasm as in Oikomonas termo (Fig. 97, B, p. 186), but in other cases complicated cortical differentia- tions with supporting and food-procuring adaptations give rise to complex and permanent cytostomes and cytoprocts. In flagellates such an area of softer protoplasm is situated at or near the base of the flagellum, or two such areas may be present, each at the base of a flagellum or group of flagella, as in Trepomonas and Ilexamitus. In one group, the Choanoflagellidae, a collar-like membrane arises as a protoplasmic fold around the base of the flagellum and forms a cuff or funnel surrounding the flagellum for a distance equal to one-third or one-half its length (Fig. 92). These are extremely delicate, the margins alone in many cases indicating their presence and dimensions. According to France, they are somewhat spirally rolled like a cornucopia, the free mar- gin arising from the softer food receptive area and by its move- ments directing food particles toward this area. This, according to de Saedeleer (1929), is an erroneous interpretation, the appar- DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 165 ent spiral roll of the collar being due to the presence of two pre- hensile tentacles. In some cases two such collars, one within the other, are present as in Salpingoeca entzii or S. marinus (Fig. 92). The second, outer, collar in some types is regarded by Doflein as a periplastic rigid structure which forms a part of the cup or Fig. 92. — Types of choanoflagellates. A, Codosigapulcherrimus; B, Diplosigasocialis, C, Salpingoeca marinus; D, collar type according to France. (After Calkins.) house and is not morphologically equivalent to the inner collar, which, like a pseudopodium, may be shortened or lengthened, or drawn in and formed anew by the living cell. According to the older interpretation these protoplasmic collars assist in food-taking by forming a sticky directive course for particles down the inside 166 BIOLOGY OF THE PROTOZOA to the receptive area at the base of the flagellum (Kent), but accord- ing to France granules on the inside of the collar are moving away from the cell as defecatory material while the food particles move down the outside to a receptive area not included by the collar base (Fig. 92, D). In the majority of corticate flagellates the food-taking receptive area is continued as a pit or groove known as the flagellum fissure, or as the cytopharynx. The flagellum arises usually at or near the base of such a pit and in many cases the contractile vacuole empties into it. It is in the ciliate group, however, that we find the most character- istic and most complicated types of cytostome. Here they may be mere pores in the cortex which remain closed except during the process of ingestion and without accessory current-producing motile organs, or they may be permanently open and provided with undu- lating membranes or other vibratile elements. The former type, known as the Gymnostomina, eats only occasionally and then by a definite swallowing process, the soft mouth region widening into a huge opening to receive the prey. Thus Didinium nasutum ordi- narily swims about with little evidence of a mouth at the extremity of the conical proboscis (Fig. 98, p. 187), but when swallowing a Paramecium which may be larger than itself, the entire anterior end appears to be nothing but mouth, the body wall of the Didinium being reduced to a thin enveloping sheath about the Paramecium (Figs. 98, 5). Similar, but not so spectacular cytostomes are present in other types of Gymnostomina. Spathidium spathula may swal- low smaller ciliates like Colpidium (Fig. 99, p. 188); Nassula aurea, Chilodon cucullus, etc., still smaller forms. In all such forms the protoplasmic region around the mouth is strengthened by simple or complex metaplastic structures— the trichites (Fig. 195, p. 475). The Trichostomina are always provided with food-getting motile organs and a constant stream of water with suspended bac- teria and other minute living things passes through the permanently open mouths making these creatures, according to Maupas, gluttons par excellence of the animal kingdom (see, however, p. 190). The complications in regard to structure in these two types of cytostome have to do with the support of the walls of the mouth and of the gullet into which the mouth opens, and for the perfection of the current-producing apparatus. Such support is obviously important in preventing rupture of the soft protoplasmic bodies of forms like Didinium nasutum, Enchelys farcimen, Prorodon tires or Spathidium spathula (Fig. 99, p. 188). In all of these cases there is an armature of elongated rods, trichites, formed of stereoplas- mic substances, embedded in the walls of the mouth and gullet, and these, like spiles in a ferry slip, take up the strain when the mouth is opened. In many cases, however, the perfection and DERIVED ORGANIZATION— TAXONOMIC STRUCTURES U\i strength of these cytostomial supports seem to be entirely out of proportion to such hypothetical needs of the organism. Thus in all of the Chlamydodontidae the trichites form a tubular armature, the ends making a circumoral ring which may project beyond the ventral surface (Chilodon cucullus). Such an aggregate, known as an oral or pharyngeal basket, or pharyngeal armature, forms a more or less definite cytopharynx. In some cases the trichites are re- placed by a compact corneus tube which extends dee]) into the endoplasm as in Nassula aurea, Orthodon hamatus, Trachelitis ovum, etc. (Fig. 93). mm >' A B C Fig. 93.-^4., Orthodon hamatus with oral tube; B, Frontonia leucas. with undulating membrane on left margin of mouth; C, Trachelitis ovum. (A and C, after Biitschli; B, after Calkins.) In the Trichostomina the permanently open mouth always leads into a more or less highly-developed gullet or cytopharynx, while peristomial cortical differentiations of various kinds lead to it. The cytopharynx is usually provided with one or more undulating membranes, while membranelles, undulating membranes and cirri may also be present in the peristome. These are well illustrated bv the complex oral apparatus of Glaucoma {Dallasia) frontata (Fig. 8, p. 29). The mouth region of the ciliates appears to be the focal point of the longitudinal rows of cilia. In the generalized forms, such as Actinobolina radians, Prorodon teres, Holophrya discolor, etc., the mouth is exactly terminal and the rows of cilia run symmetrically 168 BIOLOGY OF THE PROTOZOA to the posterior end (Fig. 84, p. 154). In the majority of cases, however, the mouth is not terminal but may be found at various points on the side or upon the ventral surface. Thus it may be on the side in forms like Nassula aurea, or Glaucoma (Dallasia) frontata (Fig. 8, p. 29), on the ventral anterior surface in Frontonia leucas (Fig. 93, B), or various species of Chilodon, or at the extreme pos- terior end as in Opisthodon mnemiensis (Fig. 191, p. 472). Where- ever the mouth is found the rows of cilia are correspondingly altered from symmetrically placed lines as in the generalized forms, to all kinds of asymmetrical arrangements. This has led to the view, first elaborated by Biitschli, that the ancestral position of the mouth in ciliates was terminal at the anterior end, and that by adaptation to different modes of life, and to various types of food, the mouth has shifted from the anterior end to the various positions as now found in different types. With this shifting the focal points of the ciliary rows have similarly shifted, and the positions of the lines of cilia in some forms are used as evidence to indicate the path of this shifting and the mode of evolution of the present-day cytostomes. A familiar illustration of such shifting is the series of forms repre- sented by the genera Holophrya, with terminal mouth, Spathidium, with oblique mouth, Colpidium, Glaucoma (Dallasia) and many others, with subterminal mouths, Amphileptus and Lionotus with elongated slit-like mouths extending from the anterior end far down the ventral surface, such types leading to the various proboscis- bearing genera like Dileptus in which the mouth is limited to the posterior end of such an ancestral slit-like aperture, now represented for the most part by a row of trichocysts (Figs. 6, 13, 203). In Chilodon there is an oblique line of cilia running from the anterior left-hand margin of the ventral surface to the circular mouth which in some species may be shifted well over on the right side. The lines of ventral cilia begin at this line and not at the mouth, while an oblique row of specialized cilia suggests the begin- nings of adoral zone formations characteristic of the majority of Trichostomina, while the line itself may well represent the positions held by the mouth in ancestral forms. In many types of ciliates, a special region of the body, not found in the more generalized forms, is developed as a feeding surface. Such regions, known as frontal fields, are characteristic of ciliates which live permanently or temporarily as attached forms. There is some evidence to indicate that such frontal fields as occur in Stentor, and the Peritrichida, are derived from the anterior ventral surface of more actively moving forms. In Pcritromus, for example, the line of the peristome cuts out a definitely limited frontal region of the ventral surface, which is provided with special motile organs, the frontal cilia. Biitschli (1888) suggested that such a peristome, if continued around the right side of the organism, would completely DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 169 separate an anterior frontal field from the remainder of the body, as seems to be the case in Climacostommn virens (Fig. 71, p. 128). With the development of an attaching portion of the body as in Stentor, and in the interest of feeding, such a frontal field becomes directed upward, reaching its most perfect development in types like Vorticella and its allies (Fig. 86, p. 158). Fig. 94. — A, Bursaria truncatella, frontal field deeply insunk; B^Folliculina ampulla, with frontal field drawn out into two flexible arms. (.4, original; B, after Doflein.) Such frontal fields are flat in the various species of Stentor, or they may be greatly invaginated as in Bursaria truncatella, or drawn out into two ciliated food-getting arms as in Folliculina ampulla (Fig. 94), or into a tripartite frontal field in Triloba paradoxa, or rolled up in. spiral folds as in Spirochona gemmi para .and Bursalinus synspiralis. The cytoproct is rarely differentiated as a definite opening in the cortex. In many cases, especially in the flagellate group, the cyto- pharynx and anus are the same. In the majority of ciliates, on the other hand, there is a constant opening or pore, usually in the pos- 170 BIOLOGY OF THE PROTOZOA terior region of.the body, which is closed and invisible except during the process of defecation (Fig. 44, C,p. 86). In some forms, notably in Pycnothrix monocystoides and Diplodinium ecaudatum, a definite anal apparatus is developed. In the latter case Sharp describes a " rectum" with distinct walls opening to the outside by a permanent cytopyge, while at the inner end there is a "cecum" which acts as a collecting vacuole for the fecal matter (Fig. 2, p. 20). (e) Contractile Vacuoles.— In the rhizopods and most of the soft- bodied flagellates the contractile vacuole can scarcely be called a cortical differentiation. In these cases they are more or less casual organoids, moving freely with the endoplasmic granules. In the corticate flagellates and ciliates, however, there is a permanent spot in the cortex through which the contents of contractile vacuoles, fixed in position, are emptied to the outside. As a rule, the salt water forms of Protozoa do not have contractile vacuoles (see p. 176) and the number in fresh water forms is variable, sometimes in the same organism (testate rhizopods and Heliozoa). In many types, how- ever, the number as well as the position is fixed; one, as a rule, in Hypotrichida and Peritrichida, and variable numbers in the Holo- trichida and Heterotrichida. In rhizopods the roving vacuole adds to its volume by picking up fluid substances from all parts of the endoplasm until it becomes too heavy to be easily moved with the flowing endoplasm. The vacuole is thus gradually left behind, so to speak, until it finally breaks through the thinning wall of protoplasm and empties its contents to the outside, usually at that part of the body which for the time being is posterior. In the fixed forms of vacuoles the fluids to be excreted are brought to the excretory organoid by more or less definite routes or canals, through the endoplasm. Such canals are highly characteristic of many types of ciliates. A familiar example is afforded by the different species of Paramecium where the five to ten radiating canals form a characteristic rosette about each of the two contractile vacuoles (Fig. 95). In the Hypotrichida there are usually two such canals leading to the dorsally placed vacuole, and two in Stentor, one following the margin of the body to the "foot," the other following the rim of the peristome in a circular course around the body. In Ophryoglena flava there may be as many as thirty fine feeding canals leading from all parts of the body to the centrally placed vacuole, and in Fronton in leucas eight to twelve such canals follow a tortuous course throughout the body substance. In Pycnothrix the canals form a branching network through the endoplasm. Such canals are replaced by a ring of feeding vacuoles in many of the corticate flagellates. In corticate Protozoa the contractile vacuole usually opens to the outside in the vicinity of the anus when such a structure is present. In many cases it opens into the cytopharynx as in the majority of flagellates or in the vestibule of forms like Vorticella. DERIVED ORGANIZATION— TAXONOMIC STRUCTURES 171 In Campanella umbellata such a reservoir is replaced by two definitely walled evacuation canals, while in Pycnothri.v the excretory canal is said to be provided with special cilia. c. v. Fig. 95. — Golgi bodies in Chilomonas Paramecium (B) and Paramecium cau- datum (A and C). c.v., Contractile vacuole; r, radial canals of Paramecium. (After Nassonov.) In some types of parasitic ciliates (Biitschliidae and Paraiso- trichidae) a peculiar type of "concrement vacuole" has been de- scribed by Dogiel (1929) which appears to be a normal part of the derived organization. These are interpreted, not as excretory, but as special structures with a statolith function. CHAPTER V. GENERAL PHYSIOLOGY. There is no doubt that our knowledge of the structures of Protozoa far outstrips our knowledge of their functions. The minute size of the individuals and the inadequacy of micro-chemical tests make it extremely difficult to follow out any physiological process to its end. Furthermore, it must not be overlooked that physiological problems here for the most part begin where similar problems of the Metazoa leave off, namely in the ultimate processes of the single cell. Here the functional activities have to do with the action and interaction of different substances which enter into the make-up of protoplasm and, for the most part, these are beyond our powers of analysis. A few of these activities may be dupli- cated individually and apart from correlated functions, in the laboratory. Or specific reactions between specific chemical sub- stances may be obtained as, for example, the digestion of fibrin by fluids extracted from the protozoon protoplasm; or in a physical sense the reversal of the sol and gel states in colloidal mixtures. Such individualized processes, however, give little idea of the infinite play of forces continually operating in living protoplasm, all of which, harmoniously working together, make up the phe- nomena of vitality and distinguish living from lifeless matter. As Mathews points out, the essential differences in chemical actions in protoplasm and in physical nature are: (1) The order- liness with which they are carried on, and (2) the speed of the reactions. A starving Dileptus gigas will slowly decrease in size although its form remains about the same (Fig. 6, p. 27). This is due to disinte- gration through continued oxidation and other catalytic processes which lead to the exhaustion of protoplasmic constituents unless new food is added. If the process is continued the organism will ulti- mately die in from one to three weeks. If a Dileptus is accidentally crushed its protoplasm will completely disintegrate within a few sec- onds. The process of disintegration in the first case is orderly, in the latter completely disorganized. Other normal vital activities are equally orderly; the orderliness dependent possibly on the regulation of permeability by the colloidal membranes, the alveolar membranes, nuclear membrane and investing membrane of the cell; and regula- tion of permeability in turn is dependent upon the chemical make up GENERAL PHYSIOLOGY 173 of the constituent parts, and salts or electrolytes and the continued activity between them (cf. Clowes, Overton, Mathews). The speed of specific chemical actions is a characteristic vital phenomenon due to the participation of subtle and elusive, but specific, catalytic agents, the enzymes. This aggregate of colloidal substances forming polyphasic physical systems in protoplasm is the seat of the multitude of activities characteristic of life. Huxley's definition of protoplasm as the physical Basis of Life does not carry us very far in the analysis of living matter. In a moving protozoon there is a constant interaction of the various substances making up its protoplasm— oxidation, enzyme formation and action, amidization and deamidization, dis- integration and regeneration, protein break-down and protein recon- struction, all taking place simultaneously or seriatim. Substances in this whirlpool of action may be regarded as living so long as they are, or may be, drawn into the vortex of protoplasmic activi- ties. The results of these multitudinous activities contribute to the well-being of one organism. In another moving protozoon a similar bewildering complex of activities likewise results in the well-being, in this case of a distinctly different type of protozoon. The first protozoon, let it be a Didinium nasutum, captures and swallows the second, say a Paramecium caudatum. It is well known that a frag- ment of a protozoon will regenerate into a perfect organism of its type and we might well be perplexed by the problem why is it that the Paramecium protoplasm in Didinium does not manifest itself as Paramecium and not as Didinium. The answer to this apparently simple problem is a matter of organization or the manner in which the fundamental substances making up the protoplasm in the two organisms are put together and interact. The architectonic of Driesch, or protoplasmic architecture, is specific for each type of organism and the form and structures of the organism are expres- sions of this architecture. When this organization disintegrates, life and the possibility of controlled reactions are lost and the erstwhile living protoplasm becomes dead matter. This happens when Paramecium is paralyzed by the seizing organ of Didinium (see Fig. 98, p. 187). The vital activities of Paramecium are sud- denly stopped, and disintegration of its organization, through hydrolysis, continues with the digestive processes in Didinium. The inert proteins, probably as amino-acids, are re-integrated in the Didinium protoplasm, and what was living substance in Para- mecium, now enters again, through a form of transmigration, into the vortex of vital activities of quite another type of organism. The sum total of the various physiological processes of the in- dividual may be grouped for the Protozoa, as they are for the Metazoa, inter aggregates of special activities which we call the fundamental vital functions, and distinguish as respiration, nutri- 174 BIOLOGY OR THE PROTOZOA tion, excretion, irritability and reproduction. In Metazoa these are performed by specialized cells, grouped into tissues, organs and organ systems, the complexity varying with the specialization of the organism. In Protozoa they are all performed by the single cell and all are more or less dependent on the activities of the diverse substances and structures which compose it. All work together in a harmonious cycle of matter and energy. A. Respiration.— The scientific beginnings of the modern mech- anistic conception of vital activities is traced to Lavoisier and his comparison of animal heat with physical heat due to combustion through oxidation. The utilization of chemical energy, or energy of combination liberated by oxidation, is possibly the keynote to the multiple vital harmonies of animal life (see Yerworn, 1907). Oxygen necessary for such physiological combustion is obtained by all protozoa without the aid of specialized respiratory organs. It is readily absorbed through permeable membranes from the sur- rounding water, or obtained by reduction from oxygen-holding substances, as in anaerobic forms. In one way or another it is ever present to initiate the round of vital functions. Oxygen may be taken into the cell directly from the surrounding medium as in the aerobic forms, or it may be obtained by breaking down Oxygen-holding substances, in protoplasm, so-called reducing processes of all types but especially of anaerobic forms. Through the use of chemical indicators the degree of oxidizing power of a cell, including both direct oxidation and reduction, may be deter- mined and is expressed by the symbol rH in values from one to forty. This factor, known as the "oxidation-reduction potential," varies from time to time and is used in much the same way as the expression pH, indicating the hydrogen-ion concentration from intense acidity (pll 1 or 2) to strong alkalinity (pll 10). It is « highly probable that a definite rH is as important for cell activity as a definite pH, and that this oxidation-reduction potential is maintained by the 11 SI I compounds (cystine, cysteine and gluta- thione) of the protoplasm (Krogh, 1916; Hopkins, 1921; Meverhof, 1924). The intake of oxygen and the voiding of ( !0 2 constitute the essen- tial needs of the cell in respiration. The relationship between the oxygen taken in by an organism and the C0 2 produced by its metabolic activities is indicated by the expression R. Q. (respiration quotient). Daniel, 1931, found that the R. Q. of Balantidium coli under aerobic (sic) conditions is 0.84, which is very nearly the same as the usual R. Q. for man (0.85). For Amoeba proteus and Bleph- arisma undulans Emerson (1929) found the R. Q. to be "about unity." To a certain extent the oxygen intake and ( X) 2 output are measur- able, but always with a large experimental error. Kalmus (1927), GENERAL PHYSIOLOGY 17.") for example, by an ingenious method, made out that a single Para- mecium consumes 0.0052 c.mm. of 2 in one hour at 21° C, a figure which Howland (1931), using the same method, slightly modified, changed to 0.00049. Adolph (1929) made out a typical rate of 0.55 cc. of oxygen intake per million individuals per hour at 19.7° C, In a similar way R. Emerson (1929) obtained results with Amoeba proteus and Bkpharmna undulans; Peters (1921) with Colpidium colpoda; Hulpieu (1930) with Amoeba proteus found that the rate of movement is not noticeably affected by changes in the amount of available oxygen from 0.005 to 100 per cent ; below or above these limits the animals are slowly killed. He found, furthermore, that amebae are able to move for some time in the absence of oxygen #vhich indicates that its energy is not derived by direct oxidation. Verworn (1896), on the other hand, found that Rhizoplasma kaiseri in an oxygen-free medium ceases its centrifugal pseudopodial move- ments while centripetal movements continue for some time but ultimately stop. Addition of oxygen restores both types. It is the function of catalytic enzymes to expedite chemical processes which are under way and catalases of different kinds result from metabolic activities going on in protoplasm. Amongst these are the oxydases which aid in oxidation and reduction in the cell. Indications of such agents as the "reducase" of Becker (1926) and the extraction of glutathion have been obtained, while Joyet-Lavergne (1929) adduces considerable evidence in support of his view that glutathion is intimately associated with the mito- chondria of the cell. Correlated with the intake of oxygen is the output of C0 2 and water. While these are perhaps more properly treated in connection with the functions of excretion there is good evidence of a gaseous exchange, but quantitative results are not altogether satisfactory. The energy of combination, released by oxidation, is paid for by loss in the chemical compound oxidized. Other compounds may be formed with lessened energy of combination, and end-products, notably C0 2 and urea ((NH 2 ) 2 CO), are not only useless to the organ- ism but positively harmful unless voided. Excretion, therefore, must follow oxidation. To make good the loss of substance new food materials must be taken in, digested and assimilated, but this is possible only through movement, and movement in turn is an expression of irritability. Excretion and irritability thus are funda- mental vital functions, while a third, nutrition, is closely correlated. Excess of food intake over waste by oxidation leads to growth of the diverse protoplasmic substances and to their reduplication by division, while the aggregate of such divisions, expressed visibly by division of the cell, constitutes reproduction. The funda- mental vital functions are intimately bound together; external con- 176 BIOLOGY OF THE PROTOZOA ditions, such as decrease in temperature of the medium in which a protozoon lives, means decreased oxidation, retarded movements, less food and a lower division rate. Increase in temperature involves a speeding up of all activities and, if food is abundant, a higher division rate. External conditions involving absence of food lead to starvation and death of the cell through uncompensated loss by oxidation. In short, interference with any one of the fundamental functions leads to disturbance of them all, and the various phases of vitality of the protolasm during a typical life cycle may be due to inadequate functioning of one or another or all of these activities. B. Excretion of Metabolic Waste.— The waste matters of oxida- tion and continued metabolism are frequently voided in the same manner that water and oxygen are taken in, namely, by osmosis. In such cases there is no physiological need of specialized excretory organs. It is possible that all Protozoa excrete in this way, although the majority of fresh water Protozoa possess contractile vacuoles which are generally regarded as excretory organs. In marine forms and in parasites they are generally absent. If the latter forms, and these are in the majority of Protozoa, are able to dispose of the products of destructive metabolism without definite organs for the purpose, why are the latter necessary in fresh water forms? Hartog (1888) has long maintained that contractile vacuoles are not obligatory excretory organs, but are primarily hydrostatic organs for the purpose of maintaining a pressure equilibrium between the fluids within the cell and those in the surrounding water. Degen (1905) interprets the vacuole in a similar way, its variations in size and pulse depending upon permeability of the membrane which varies with the environmental salts. Here difference in density of^ the surrounding medium is largely responsible for loss of the organ characteristic of fresh water forms, but changes in permeability of the cell membrane due to salts in the new medium undoubtedly play an important part. Other experiments by different observers bear out the same principle. Thus dilution of the normal neutral salts in the medium causes enlargement of the contractile vacuoles in ciliates according to Massart (1891), while increased concentra- tion leads to reduction in size, retardation in rate of contraction, or total disappearance of the vacuole. While there is justification for Hartog's view of the purely physical significance of the vacuole, there is every reason for believing that water in protoplasm picks up any soluble waste matter that may be present, and holds it in solution. Early experiments to prove this, by Brandt (1885), Griffiths (1889) and others using chemical indicators, or the murexid test for uric acid, were not convincing, and the function of the contractile vacuole as a primitive type of excretory organ remained an hypothesis. Not only water, C0 2 (see Lund, 1918) and urea, but other prod- GENERAL PHYSIOLOGY 177 nets of metabolism as well, are found in the protoplasm of differ- ent Protozoa. These are usually present in crystalline form or in amorphous heaps, which are rather loosely spoken of as "excretory stuffs" without evidence as to their origin or significance. The crystals often seen in Paramecium were identified by Schewiakoff (1893) as calcium phosphate combined with some organic substance. Similar crystals have been described by Schaudinn, Schubotz and others from the protoplasm of different kinds of Protozoa. Schewia- koff found that the crystals of Paramecium are not defecated as are undigested food substances, but are first dissolved and then disposed of— presumably with the water of the contractile vacuoles. The function of the contractile vacuole in Protozoa thus has long been a disputed problem. The views of the older students of the group, with their conceptions of structural complexity of these uni- cellular organisms, fantastic today, nevertheless have a certain his- torical interest. The idea that a vacuole is a rudimentary beating heart as interpreted by Lieberkuhn (1856), Claparede and Lachmann (1854 and 1859), Siebold (1854) and Pritchard (1861) was no less incongruous than the supposition of Ehrenberg (1838) that the contractile vacuole is an organ connected with the gonadal system. With development of knowledge of structure and function of the Protozoa, and particularly of the mechanism of vitality, more reasonable hypotheses of the function of the contractile vacuole have been developed. There is, first, some ground for the belief of Spallanzani (1770), Bossbach (1874) and Dujardin (1841) that it is an organoid having to do with respiration of the organism, together with other possible functions, a view supported in modern times by Biitschli (1877, L888) and Degen (1905). There is, second, ground for the belief held by Stein (1859), Gruber (1889) and the majority of modern students of Protozoa, that it is an organoid for the excre- tion of katabolic waste, despite the unconvincing experimental evi- dence by Brandt (1885), and by Griffith (1889). Howland (1924), however, by using a much more delicate test (the Benedict blood- filtrate test) obtained unmistakable evidence of the presence of uric- acid in cultures of Protozoa; in P. caudatum analyzed by Benedict, a color reaction was obtained equivalent to 4 to 5 mg. of uric acid per liter. There was no proof here, however, that the uric acid came from Paramecium. Weatherby (1929) showed that the usual ingredients of a culture medium contain measurable quantities of uric acid. He found, however, that the extracted fluids of con- tractile vacuoles of Paramecium and Spirostomum contain urea, whereas the vacuole of Didinium nasutum contains ammonia, but in no case does the nitrogenous waste of the vacuole represent all of the nitrogenous output of the cell, much being voided by exosmosis. There is, third, ground for the belief that the contractile vacuole is an organoid for the regulation of osmotic pressure in the cell, a view 12 178 BIOLOGY OF THE PROTOZOA first advanced by Hartog (1888) and supported by Degen (1905), Stempell (1914), Khainsky (1910) and by Nassonov (1924). These three beliefs are not necessarily exclusive and the possibil- ity of all three functions is still open. The osmotic function is well supported by evidence furnished by Gruber's (1889) experiments in transferring fresh-water, vacuole-holding Actinophrys sol and Amoeba crystalligera to salt water, and vice versa, or by Zuelzer's similar experiment with Amoeba verrucosa, the protoplasm becom- ing more condensed and the vacuole lost in salt water. Hogue (1923) found that Vahlkampfia calkensi when transferred from salt water to fresh water media developed 1, 2, 3, or even 4 contractile vacuoles. More extensive experiments by Degen (1905) with salts of different kinds and w T ith varied conditions of the environment show that the contraction of the vacuole is a function of osmotic pressure, and irrespective of the type of salt or neutral solution introduced. With Hartog, he concludes that protoplasm of fresh water forms, with its salts in solution, has a higher osmotic pressure than the surrounding medium, which leads to continued intake of water. Such intake, if not balanced, would lead to inflation and to diffluence, a conclusion strengthened by Botsford's (1926) mer- otomy experiments with Amoeba proteus in which it was shown that the size of the vacuole depends upon the size of the fragment cut off. According to Degen and Hartog it is the function of the contractile vacuole to establish this balance. This hypothesis, with further evidence supplied by the absence of contractile vacuoles in marine forms where osmotic relations of protoplasm and environment are more evenly balanced, is theoreti- cally correct. There is no reason to doubt, however, the further possibility that the water expelled by the contraction of the vacuole contains water-soluble, katabolic excretory substances such as C0 2 and nitrogenous waste, positive evidence for which is supplied by several observers. This indeed was admitted by Degen although he obtained no evidence of the nature of the substances excreted. He saw in the membrane of the vacuole the possibility of an excretory mechanism. The actual existence of such a membrane, however, is still in dispute, indeed the majority of investigators deny its existence (Biitschli, Khumbler, Schewiakoff, Taylor). Others, however, give evidence to show that a true membrane, although very delicate, is actually present. Howland (1924, 1) for example, by micro-dissection methods has been able to remove the contractile vacuoles of Amoeba verrucosa and of Paramecium caudatum after which they retain their integrity for considerable periods as free vacuoles in the surrounding water. She also has punctured the vacuole with needles while in the endoplasm, causing the expulsion of its contents into the surrounding endoplasm and resulting in the wrinkling of the vacuole membrane. Nassonov (1924) also not GENERAL PHYSIOLOGY 179 only demonstrates the presence of a membrane in various types (Paramecium caudatum, Lionotus folium, Nassula lateritia, Cam- panella umbellaria and other VorticelUdae) but, by use of fixation methods employed for demonstrating the Golgi apparatus in meta- zoan cells, comes to the conclusion that the membrane of the con- tractile vacuole is a part of the Golgi apparatus. This, in Metazoa, he had earlier (Nassonov, 1923) identified as an organoid intimately bound up with secretory activities of the cell (see also Bowen). In different Protozoa the contractile vacuole, which he unhesitatingly calls an excretory apparatus with a definite lipoid membrane, is variously complicated, from a simple vesicle with osmiophilic mem- brane in forms like Chilomonas paramecium (Fig. 95, B, p. 171), to complex aggregations of vesicle and canals as in Paramecium (Fig. 95, A, C). In the latter case the canals appear to contain the ma- terial by activity of which substances are chemically differentiated for secretion and these are passed on to the vesicle 1 >y which they are excreted. According to Nassonov the lipoid-containing membrane (confirmed by Chatton, 1925, and by Gelei, 1928) must be semi- permeable and its contents must have a higher osmotic pressure than the surrounding plasm. Hence fluids would flow into the vacuole completely distending it until the pressure would burst the retaining membrane and the fluid would be ejected. The highly viscous membrane would mend but for a new flow into the vacuole a new supply of osmotically active stuff would be necessary. This, Nas- sonov assumes, is formed by secretion from the osmiophilic mem- brane into the canals and vacuole. This secreting activity is com- pared with the secretory activity of the Golgi apparatus in Metazoa. Gelei holds, however, that the function here is to condense and to conduct concentrates from the plasm into the canals, not a secre- tory function, but excretory. (See also Lynch, 1930.) With this work of recent investigators we have a very definite argument for the excretory functions of the contractile vacuole and for the pres- ence and function of the lipoid membrane. In quite a modern way it brings us dangerously near to an Ehrenbergian conception of a kidney and bladder in Protozoa. C. Irritability. — In the absence of all knowledge as to the manner in which protoplasmic particles respond to stimuli of different kinds, we are constrained in speaking of irritability of Protozoa, to limit descriptions to aggregates of such responses as manifested through movement, as energy transformed by oxidation from the poten- tial or stored chemical energy to the active or kinetic condition, or as manifested by adaptations to changes in environment. But the manner in which such kinetic energy is utilized in pseudopodia formation or by the elements of rlagellum, cilium or myoneme, is a matter of pure speculation. The reactions which characterize the resulting movements, however, can be analyzed and measured 180 BIOLOGY OF THE PROTOZOA and these form the chief basis of our knowledge of protozoan irri- tability. Attempts to explain pseudopodia formation and ameboid move- ment have varied with the changes in our conceptions of the physical make up of protoplasm. The protoplasm of Ameba regarded as a fluid substance was supposed to follow the laws of surface tension characteristic of all fluids. Pseudopodia formation, according to the views of Berthold (1886), is the attempt of one fluid (proto- plasm) to spread out between water and the substratum as Quincke's ' well-known experiments demonstrated for fluids. As physical con- ditions on all sides of the Ameba are not equal, variations in tension result in local diminution, and the tendency to spread is focussed in a local area and the pseudopodium results. Biitschli's (1894) observa- tions and experiments with emulsions of oil, salts and water, and Rhumbler's (1898) analysis of the causes of movement in lobose rhizopods led these observers also to interpret pseudopodia forma- tion as a result of surface tension phenomena. With the more modern conception of protoplasm as a colloidal aggregate in the physical state of an emulsoid in which the external and internal protoplasm of Ameba are in the relation of gel and sol, the difficulty of applying the laws of fluids became apparent and the hypothesis based upon surface tension has been generally abandoned. Rhum- bler himself (1910 and 1914) recognized this difficulty and materi- ally changed his conception of ameboid movement, while Hyman (1917) greatly enlarged and perfected his later point of view. According to Hyman the ectoplasm of Ameba, by virtue of its relatively solid state, becomes tenuous but elastic, as demonstrated by the experiments and observations of Jennings (1904), Kite (1913), Schultz (1915) and Chambers (1915, 1917), and exerts an elastic tension on the inner fluid protoplasm. Bancroft (1913) and Clowes (1916) demonstrated the reversibility of phase in diphasic physical systems through the agency of electrolytes, and the conclusion fol- lowed that \he ectoplasm represents a reversal phase of the more fluid inner protoplasm. Hyman argues that, owing to the tension of the enveloping ectoplasm, if any local region of the more solid ectoplasm becomes liquefied, the resistance gives way at such a point and the fluid endoplasm is pressed out, thus forming a pseudo- podium. The immediate cause of such liquefaction she traces to a local increase of, or change in, metabolic activity resulting in the production of hydrogen-ions which, with the surrounding medium, form an acid appropriate for dissolution of the more solid ectoplasm. By the use of Child's potassium cyanide test for metabolic gradients, she was able to demonstrate that such local regions of greater meta- bolic activity actually occur on the periphery of Amoeba proteus before a pseudopodium breaks out, also that the extreme tip of the advancing pseudopodium is the most actively metabolic part. GENERAL PHYSIOLOGY 181 Whether changes in the nature of protoplasmic response or changes in direction of movement after repeated shocks should be interpreted on the basis of "memory" and "learning" or in some other way is largely a matter of personal idiosyncrasy on the part of the observer. Numerous writers have described processes of food "selection" by Ameba (e.g., Gibbs and Dellinger, 1908; Schaeffer, 1917 and elsewhere; Metalnikoff et al, 1910). Mast and Pusch (1924) interpret an observed change in the protrusion of pseudopodia of Amoeba proteus in respect to a beam of light as evidence of something analogous to "learning" in higher animals, etc. "Learning" involves "memory," and such terms connote processes of an entirely different nature which we associate with the highest types of animals. It is conceivable that fatigue, to use the term in its broad sense implying total or partial exhaustion of pro- toplasmic constituents necessary for a reaction, and therefore a purely physical matter, is adequate for explanation without calling upon any obscure pan-psychic interpretation. Similarly with Kep- ner and Taliaferro's (1913) evidence of "purpose" in methods of food-getting by Amoeba proteus. Many of the reactions of Protozoa are bound up with the coor- dinating mechanism of the cell through which the organism acts as a unit. The specific response of an organism to a stimulus is the result of its particular protoplasmic architecture expressed through its coordinating mechanism and motile organs. This has been elaborately worked out by Jennings (1904 to 1909) in connection with the "motor response" of many different kinds of Protozoa. The discussions and controversies over the matter of directive stimuli or tropisms in Protozoa have evidently been due in large part to a lack of common understanding of the definition. If by "tropism" is meant the orientation of an organism in respect to the path of a stimulus, then tropisms, as Jennings was the first to point out, play little part in the activities of the Protozoa. If, however, by "tropism" is meant "the direct motor response of an animal to an external stimulus" (Washburn, 1908), then tropisms play a most important part in such activities. The two definitions are not compatible; the former conveys the idea of a directive stimula- tion upon local motor organs or controlling elements; the latter implies the complex reaction of a definite mechanism character- istic of any specific protoplasm, and the same reaction follows upon stimulation by any type of stimulus (Putter, 1903, Jennings, 1909). It follows further that the reaction is called forth regardless of the particular elements first to receive the stimulus. We owe Jennings the credit for first clearly distinguishing between these two conceptions, as well as for careful analyses of the move- ments of lower organisms (1904 et seq.), and for demonstrating the particular motor response distinctive of specific types of Protozoa. 182 BIOLOGY OF THE PROTOZOA He also showed that the nature of the motor response in some organisms, e. g., in Stentor, is correlated with the physiological state of the organism, and adduced evidence which indicates that phenomena of fatigue are involved. The classical example of a Fig. 96,-Merotomy in Euplotes patella. (After Taylor.) >./., AnaUirri fibers; m., motorium; m. f., membranelle fiber. (See also Fig. 72.) motor response, formerly interpreted as chemiotaxis, is the case of Paramecium caudatum or aurelia in a drop of dilute acid. Casual swimming brings the individual to the outer limit of the drop; the transition from water to drop does not provide a stimulus strong GENERAL PHYSIOLOGY 183 enough to bring about the motor response and the individual con- tinues through the drop until it strikes the farther limit. Here the stimulus is sufficiently strong to cause the motor response which is manifested as a backward swimming, due to reversal of cilia, turning on the long axis and recovery of normal forward swimming movement. Repetition of this procedure keeps the individual in the acid drop. Others enter in a similar way and are similarly trapped until many are confined in the acid drop where they are ultimately killed. Such motor responses unquestionably play an important role in food-getting and in vital activities generally. The stereotyped nature of the motor response in any specific organism may be due in the main to the characteristic silver line and neuromotor systems which the higher types of flagellates and ciliates possess. The observations of Sharp (1914), Yocom (1916) and McDonald (1922) on ciliates, of Kofoid on flagellates, and the experiments of Taylor (1920) in cutting different regions of the neuromotor complex of Euplotes, indicate that the motor response of Protozoa is bound up with coordinating systems possessing some of the attributes of coordinating systems in Metazoa (Fig. 96). Knowledge of these complex systems and their reactions is quite sufficient to dispel any lingering belief in tropisms as due to stimu- lation of special motile elements acting independently in such a way as to orient the organism in respect to the path of the stimulus. Through coordinating fibrils all parts work together; cutting the system at any point leads to inharmonious or uncoordinated move- ments of the motile organs as Taylor has demonstrated. All reac- tions depend upon the organism as a whole; enucleated fragments are unable to react as do nucleated fragments (Hofer, 1890, Willis, 1916). Jennings' careful observations, which led him to the con- clusion that the protozoon organism always acts as a whole is fully confirmed by these later observations and experiments. 1 D. Nutrition. — Under the heading nutrition are included all physiological processes involved in the replacing of substances exhausted by destructive metabolism. Groups of activities includ- ing: (1) food-getting; (2) secretion and digestion; (3) assimilation; (4) defecation, find their place here. Certain specialized structures adapted for these various activities have been described for the most part in the preceding chapters, and the following is supple- mentary in nature dealing with the functions which these structures perforin. 1. Food-getting.— The varied methods by which Protozoa acquire the needed materials for replenishing protoplasmic substances reduced by oxidation are all correlated with the phenomena of 1 For discussion of different types of stimuli and the resulting reactions by Pro- tozoa see Minchin (1912), Khainsky (1910), Mast (1910-1918), Putter (1900, 1903), Jennings (1904, 1909). 1S1 BIOLOGY OF THE PROTOZOA irritability. The particular method employed by any one type of organism is probably the result of many factors of organization and adaptation combined with mode of life, all of which are traceable to adaptations resulting from the effects of external stimuli and response through irritability. It would indeed be remarkable, considering the endless variety of endoplasmic and cortical differen- tiations, were we to find a common method of food-getting amongst the Protozoa. On the contrary, it is probable that no two types of organism follow an identical method. Nevertheless it is possible, and it is certainly convenient, to group these manifold activities under a comparatively few main types which are designated: (1) Holozoic nutrition; (2) saprozoic nutrition; (3) autotrophic or holophytic nutrition; (4) heterotrophic nutrition. Many authori- ties introduce a fifth type under the caption parasitic nutrition, but as this does not differ in principle from saprozoic nutrition, it is included with the latter type. While these terms apparently indicate different modes of nutri- tion they are more applicable to methods of food-getting, and the differences have to do in the main with the nature of the raw materials taken in and the subsequent processes necessary for their elaboration. Thus holozoic nutrition in Protozoa as in Metazoa involves the ingestion of raw materials in the form of proteins, carbohydrates and fats which are usually combined in the proto- plasm of some other living organism eaten as food. It is an expen- sive method of acquiring raw materials for it necessitates capture and killing of living prey, preparation and secretion of digestive fluids and ferments necessary to make the proteins and carbo- hydrates soluble, and disposal of the undigestible residue. On the other hand, it assures the supply of capital in the form of chemical energy without the labor of storing it up. Saprozoic nutrition is, so to speak, a more economical method, for the organism does away with the elaborate processes of secretion and digestion and relies upon the activities of other organisms for the preparation of its raw materials and the "storage of energy." Dissolved pro- teins and carbohydrates made soluble through the agency of bac- teria agjjj other organisms in infusions, or prepared by the digestive processes of the host in the case of parasites and some commensals, are absorbed directly through the body wall or through special receptive regions, by endosmosis. This type of food-getting may be regarded as a degeneration or adaptation of the holozoic method, the specialized absorptive areas being reminiscent of former mouths, while the pathogenic effects of some types of parasites are inter- preted as due to the secretion by the parasite of digestive fluids which cause cytolysis of the host cells. Holophytic or autotrophic nutrition, characteristic of plants, is quite different in principle from the other two. Digestive processes typical of the majority GENERAL PHYSIOLOGY 185 of animals, as well as the intake of solid or dissolved food, are absent. A highly labile substance, chlorophyll, is manufactured in the presence of light and usually by specialized plastids— chromo- plastids— of the cell. Chlorophyll is very sensitive to light and in some way not yet understood is instrumental in utilizing the radiant energy of the sun to form complex, energy-holding com- pounds. Plants thus become the great banking house for animals and their capital is the apparently inexhaustible energy of the sun. Heterotrophic nutrition, finally, is characteristic of those Protozoa which combine any two of the above methods of acquiring raw materials. The great majority of Protozoa are holozoic in their methods of food-getting, and w T e may distinguish two main groups, the con- tinuous feeders, and the occasional feeders. Continuous feeders are those forms with permanently open mouths through which a constant current of water is maintained by action of the peri- stomial motile apparatus (see p. 164). Minute forms of life, espe- cially Bacteria, are carried by these currents into the endoplasm where they undergo digestion in improvised stomachs or gastric vacuoles (see p. 193). Chejfec (1929) estimates that Paramecium caudatum may thus ingest and digest from two to five million Bacterium coll in twenty-four hours. The majority of ciliates, including many of the holotrichous, hypotrichous, heterotrichous and peritrichous ciliates, belong in this group. The occasional feeders, like carnivorous types of Metazoa, feed whenever chance brings prey within the radius of their activity, and many of them, like cannibals, are guilty of feeding at times upon their close relatives (Maupas, 1883, Joukowsky, 1898, Dawson, 1919, Lapage, 1922). In some cases balloon-like membranes are unfolded and spread out like sails for the direction of food currents to the mouth as in Pleuronema chrysalis (Fig. 199, p. 482). Such forms are intermediate between the constant and occasional feeding types. In other cases great net-like traps are spread for the capture of unwary diatoms, desmids or smaller Protozoa, as in the Foramin- ifera (Fig. 10, p. 32). In other cases the microscopic hunters, like men in shooting boxes, lie in wait for their prey. Here long ten- tacles usually radiate out from the body in the surrounding water as in Actinobolina radians or in Suctoria, until a victim comes in contact with one or more of the outstretched processes (Fig. 91, p. 163) ; in the same way axopodia of the Heliozoa capture chance organisms which serve as food (Fig. 97). The most interesting of these holozoic types are the predatory forms which hunt their prey and capture them, while in full motion. The small but powerful ciliate, Didinium nasutum, belongs in this group. It darts here and there with an erratic movement while rotating at the same time on its long axis. In its sudden darts, lSli BIOLOGY OF THE PROTOZOA it strikes a Paramecium or other ciliate purely at random; the proboscis with seizing organ is buried in the victim which is then swallowed whole (Fig. 98, 1-6). Lionotus fasciola, Spathidium spathula and other gymnostomatous ciliates capture living organ- isms in a similar way (Fig. 99) while less spectacular methods are employed by Frontonia leucas, Ophryoglena flava, Prorodon niveus, etc., in swallowing diatoms, desmids and other relatively stationary organisms. A special type of food-getting, illustrated by the Rhizopods, may be interpreted in some cases as the result of physical properties of semifluid bodies. Rhumbler has made the most exhaustive studies >M Fig. 97.— Types of food getting. A, Acanthocystis (after Penard) ; B, Oicomonas termo (after Biitschli). of food ingestion in these forms and distinguishes four types, viz.: Ingestion by (1) "circumvallation," (2) "circumfluence," (3) "invag- ination" and (4) " importation." Food-taking by " circumvallation" is illustrated by Amoeba yroteus and usually takes place at that por- tion of the body which, for the time being, is posterior. According to Hofer (1889), Schaeft'er (1917) and others, the body becomes anchored to the substratum by the secretion of an ectoplasmic gelatinous substance; then, through the physical stimulus (Schaeffer, 1917) produced by a moving object (even a moving needle point according to Verworn, 1889), walls of protoplasm flow out on either side of the object and meet around it, thus enclosing a rotifer, an GENERAL PHYSIOLOGY 1ST :■,-■ •• h \^ * Fig. 98. — Didinium nasutum O. F. M. capturing and swallowing Paramecium caudatum. 1 to 6, Successive stages in the ingestion of Paramecium; 7, section of conjugating form of Didinium with spindle-form gastric vacuoles (?), and two micro- nuclei in mitosis; 8, section of Didinium just prior to encystment. The seizing organ with zone of trichocysts is protruded from the mouth; and rhizoplasts run from the membranulae (motile organs) deeply into the cell. (After Calkins.) 188 BIOLOGY OF THE PROTOZOA Arcella, a diatom or other food body. Ingestion by "circumflu- ence" appears to be due to a stimulus emanating from a living food body, the effect of which through the motor response (Jennings, 1904) is to cause pseudopodia to flow toward the prey and to entrap it while still at some distance from the body of the captor as in the testate rhizopods, Foraminifera and Choanoflagellates where an endoplasmic projection forms a pseudopodium which engulfs the prey and then withdraws within the endoplasm where the prey is Fig. 99. — Two types of ciliated carnivores. A, Spathidium spathula about to ingest a Colpidium colpoda; B, Lionotus fasciola swallowing a Colpidium colpoda. (Original.) digested (De Saedeleer, 1927 and 1929; Ellis, 1929). "Invagina- tion" occurs in forms having a somewhat resisting periplast-like ectoplasm such as Amoeba terricola according to Grosse-Allermann (1909). When a living organism comes in contact with the surface at any point, the local ectoplasm with prey attached sinks into the endoplasm as though " sucked "in, the ectoplasmic walls being trans- formed into endoplasm, while the ectoplasm about the area of ingestion comes together sphincter-like, and fuses again to a smooth surface. So, too, in A. proteus where, according to Mast (1916 and GENERAL PHYSIOLOGY 189 1923) and Beers (1924), the sphincter-like ingesting area is powerful enough to cut in two organisms like Paramecium and Frontonia. Ingestion by "importation" finally occurs where a food body, with- out apparent movement on the part of the Ameba, merely sinks into the protoplasm of the captor as in Amoeba dofieini according to Neresheimer. In most of these types, which grade more or less into one another, the process of food ingestion may be interpreted as due to local liquefaction in the more solid ectoplasm, and to special conditions of capillarity in the more fluid endoplasm. Rhumbler has shown that a filament of Oscillaria which enters Amoeba verrucosa by " importation " and is too long to be entirely engulfed, becomes coiled up as a result of the physical properties of the protoplasmic mass. In a similar way a filament of shellac may be drawn from water into a chloroform drop in which, by variations in surface tension, it becomes rolled up in a strikingly similar manner. Some of these methods of food-getting in holozoic types are sug- gestive of "conscious" activities to a given end. Thus ingestion by " circumfliience " suggests preliminary activities in anticipation of a "square meal." Or traps formed by pseudopodia or by tentacles, or the balloon sails of Pleuronema chrysalis, etc., might be regarded as "set" by Protozoa for the purpose of catching food. Such inter- pretations, however, are more probably evidences of a tempera- mental imagination on the part of the observer than of purposeful activities on the part of these minute organisms. "Sensing" at a distance has been described for Ameba (Schaeffer, 1912), and for Spathidium spathula (Woodruff and Spencer, 1922), and until these phenomena are explained they will continue to serve as a basis for such speculations. Losina-Losinsky (1931) gives good reasons for interpreting all such phenomena as chemiotactic and dependent upon the organizations of captor and prey. The so-called "selective" activities of some Protozoa in their apparent choice of food or of building materials for their shells are likewise better interpreted as the outcome of physical conditions of the protoplasm than as purposeful actions of the organisms. Schaeffer (1917) attributes the power of discrimination in food- taking to Ameba, as does Metalnikoff (1908) to Paramecium, a conclusion vigorously opposed by Wladimirsky (1916), who inter- prets negative reactions as a result of depression (fatigue?) in their physiological condition. Actinobolina radians apparently chooses, from a great number of miscellaneous forms, one particular species to harpoon, paralyze and swallow. "This remarkable organism possesses a coating of cilia and protractile tentacles which may be elongated to a length equal to three times the diameter of the body, or withdrawn completely into the body. The ends of the tentacles are loaded with trichocysts. When at rest the mouth is 190 BIOLOGY OF THE PROTOZOA directed downward and the tentacles are stretched out in all direc- tions, forming a forest of plasmic processes among which smaller ciliates, such as Urocentrum turbo, Gastrostyla steinii, etc., or flagel- lates of all kinds may become entangled without injury to them- selves and without disturbing the Actinobolina or drawing out its fatal darts. When, however, an Halteria grandinella, with its quick, jerky movements, approaches the spot, the carnivore is not so peaceful. The tentacles are shot out with unerring aim and the Halteria whirls around in a vigorous, but vain, effort to escape, then becomes quiet, with cilia outstretched, perfectly paralyzed. The tentacle with its prey fast attached is then slowly retracted until the victim is brought to the body and swallowed with one gulp. Within the short time of twenty minutes I have seen an Actinobolina thus capture and swallow not less than ten Halterias." (Calkins.) While these observations do not prove that Actinobolina radians eats nothing else, it is certainly true that the usual food is Halteria grandinella, a fact which may account for the rarity of Actinobolina. That it thrives on Halteria is proved by the fact that isolation cul- tures of Actinobolina have been maintained for a period of eight months and through 375+ generations by division during which the only food supplied was a daily ration of 1 to 3 dozen individuals of Halteria, grandinella independent pure "mixed" cultures of which, with bacteria, were maintained at the same time. In these cases it is quite probable that the motor response brought about by some specific chemotactic stimulus is responsible for the apparent "choice" of food by Actinobolina, and chemotactic or thigmotactic stimuli for food capture by "circumfluence," " circumvallation " and "importation." A certain degree of selection is forced upon some Protozoa by the limitations of their mouth parts. Forms like Didinium, Spathidium, Lionotus, etc., with distensible mouths, can handle organisms of various sizes, but forms like Paramecium, Dileptus, Spirostomum, etc., with small inelastic mouths are constrained to "select" small objects for food. Here there is no apparent choice between nutri- tious and innutritious particles, carmine or indigo granules being taken in with the same initial avidity as bacteria or other useful foods. A certain so-called "hunger-satisfaction," however, leads to the cessation of ingestion in many organisms. Thus Actinobolina radians often captures and paralyzes more Halterias than it actually eats; on one occasion, for example, an individual was seen to catch 18 Halterias, 11 of which were swallowed while a small group of 7 were abandoned uneaten, when the Actinobolina swam away. Amoeba proteus, after a period of eating no longer reacts to the stimulus of living food substances, and apparently ignores types which were previously engulfed (Schaeffer). So, too, in Paramecium and Stent or, Metalnikoff and Schaeffer describe an apparent selection GENERAL PHYSIOLOGY 191 of food as illustrated by the rejection of carmine granules after a period during which such granules were actually taken in. It seems probable that such phenomena indicate a type of fatigue involving the temporary loss of irritability through which the organism responds to stimuli produced by the chemical make-up of foreign substances, a period of rest being necessary for the restoration of this form of irritability. Selection in another sense, however, is quite important. All kinds of food substances are not equally suit- able for Protozoa any more than they are for individual men. This may be due to the fact that digestive fluids of a given type of ciliate or rhizopod are not adequate to dissolve all kinds of protein; or it may be due to deleterious substances in the protoplasm of the prey. All observers who have attempted to raise Protozoa in pure cultures are familiar with the difficulty of providing the proper food materials and excluding the harmful. Unsuccessful culture experi- ments indicate that these conditions have not been met. Further- more, a culture medium is suitable only when the organism under cultivation continues to live during all phases of its life cycle. Apparent selection of foreign objects used in shell-building may be due to the physical consistency of the protoplasm and to its ability to pick up foreign bodies like sand crystals, diatom shells, etc., or in part to the size of the shell-opening through which such objects must pass for storage in the protoplasm. Mud and other fine particles of inorganic matter, like carmine granules, are engulfed with bacteria and other microorganisms which produce the stimulus necessary for the operation of food-taking. After the useful sub- stances are digested the residue, like castings of worms, may be voided to the outside or they may serve a useful purpose in the construction of shells. A special kind of holozoic food-getting is illustrated by the Suc- toria which, instead of cilia, are provided with suctorial tentacles (Fig. 100). The prey, usually some form of ciliated Protozoa, comes in contact with one of these tentacles and is paralyzed through the action of some kind of poison contained in it. The cortex of the prey is perforated by the end of the tentacle and the fluid endoplasm is sucked into the body of the captor, a stream of granules being visible within the tentacle. In some cases it is said that the endo- plasm of the captor flows through the tentacle and into the body substance of the prey where the latter is digested (Maupas, 1883). The body of the victim gradually collapses until nothing remains but the denser walls and the insoluble parts. Many of the Protozoa, while parasitic in the cavities and cells of different animals, retain the holozoic method of food-getting, feed- ing upon parts of the protoplasm of the host or upon other living organisms such as bacteria of the digestive tract, or solid detritus of one kind or another. Thus Endamoeba coll lives on intestinal 192 BIOLOGY OF THE PROTOZOA Fig. 100.— Types of Suctoria. A, Trichophrya salparum on a gill filament of Salpa; B, Acineta sp.; C, Podophrya sp. (Original.) GENERAL PHYSIOLOGY 193 bacteria, while Endamoeba dysenteriae, Dientamoeba fragilis, etc., engulf, with other food substances, red blood corpuscles and digest them. According to Haughwout (1919), the flagellate Pentatricho- monas sp. likewise ingests red blood corpuscles. In the majority of protozoan parasites, however, the organisms do not digest the food necessary for the growth of their own protoplasm. They practically live in a huge gastric vacuole and are surrounded by food already digested or partly digested, which is absorbed by osmosis through their body walls. Doflein thinks that such food substances, if not appropriate for the up-building of protoplasm of the parasite, may be made suitable by the secretion from the parasite of special diges- tive substances and is ready for absorption after the action of such secretions. He further suggests that the cytolytic action upon cells and tissues of the host may be due to such secretions (for example Endamoeba dysenteriae) and that other toxins of pathogenic Pro- tozoa, probably enzymatic in their activity, may be similar digestive secretions from the parasites (see p. 362). Secretions and Digestive Fluids.— Products of metabolic activity in the form of secretions and precipitations play most important roles in structure and activities of all kinds of Protozoa. Skeletons, shells and tests, gelatinous mantles, stalks, cyst and spore membranes, and the like are all evidences of the secretory activity of the proto- zoan protoplasm (see Chapter IV). There is evidence that these activities, like secretory activity of the gland cells in Metazoa, are dependent upon the general function of irritability and that specific secretory response follows a specific stimulus. Thus Bresslau (1921) finds that gelatinous mantles or tubes about Colpidium colpoda may be called forth at will by the use of certain chemicals (iodine, fatty acids). If fatty acids are used, the individuals, as in artificial parthenogenesis, must be replaced in a suitable medium before the membranes are formed. Enriques (1919) gives evidence to show that the secretion of stalk material in Anthophysa vegetans depends upon the quantity of food available. Stimulation, through the agency of foreign proteins, is without much doubt responsible for the secretion of digestive fluids and ferments in holozoic nutrition, and considerable advance has been made in our knowledge of intra- cellular digestion. This advance has been due mainly to the appli- cation of the method first devised by Gleichen (1778) of introducing into the body with food substances inorganic, usually colored par- ticles which clearly outline the limits of the digestive cavities. These cavities, early termed gastric vacuoles, were recognized as digesting centers of the organisms, and Gleichen's method, employed by Ehrenberg (1833-1838) led to his elaborate and at first widely accepted, but erroneous, conception of the Polygastrica. Modern applications of this method consist in the introduction with the food of delicate chemical substances, or indicators, which change 13 194 BIOLOGY OF THE PROTOZOA in color according to the acid or alkaline nature of the fluids in which they lie. The observations of le Dantec (1890), Fabre- Domergue (1888), Metschnikoff (1889), Greenwood (1887-1894), Nirenstein (1905), Khainsky (1910) and Metalnikoff (1903, 1912), together with the study of extractives by Mesnil (1903), Mouton (1902), Metschnikoff '(1893), Krukenberg (1886), Hartog and Dixon (1893), etc., have given a fairly comprehensive idea of the processes of intracellular protein digestion in Protozoa. Another group of observers including Meissner, Greenwood and Saunders, Stole (1900), Wortmann (1884), Celakowski (1892), Nirenstein, etc., have shown the digestive possibilities in relation to carbo- hydrates and fats. Fig. 101. — Colpidium colpoda and Paramecium aurelia after feeding with amylo- dextrin and treatment with iodide. (After Cosmovici, courtesy of Annales Scien- tifique de l'Universite de Jassy.) An interesting conception of the gastric vacuoles in ciliates has been given recently by Cosmovici (1932). Using an ingenious method of dissolving rice starch with saliva and immersing ciliates in the dextrin thus formed, he found, upon treating them at differ- ent intervals with iodide, that a canal, colored blue, often con- voluted or swollen into "gastric vacuoles," runs from mouth to anus (Fig. 101). Further investigation of this remarkable canalic- ular system is needed. The majority of Protozoa which ingest "solid" food take in at the same time more or less water, which forms the gastric vacuole. Thus in trichostomatous ciliates a vacuole is formed at the base of GENERAL PHYSIOLOGY 195 the cytopharynx which varies in size according to the abundance of food particles present. In Paramecium caudatum the vacuole, when formed, becomes spindle-shape as though pulled away from the gullet by endoplasmic force, but it soon becomes spherical as it moves about in the fluid endoplasm (Nirenstein, 1905). With the ingestion of larger food bodies such as infusoria, flagellates of larger size, diatoms, rotifers, etc., comparatively little water accompanies the prey. Paramecium caudatum when eaten by Didinium na.su- tum, for example, lies in close contact with the protoplasm of its captor and no water at all can be made out (Fig. 98). In such cases the ingested organism is paralyzed and therefore motionless when swallowed, but it very often happens that resistant food bodies continue to struggle after they have been taken into the protoplasm; rotifers, for example, are usually not motionless when engulfed by Amoeba proteus. In such cases a considerable volume of water gives the prey ample room to move without danger to the make up of the captor. In other cases in which water does not appear to be taken in with the food, the latter becomes surrounded by fluids secreted by the protoplasm. With many types of Protozoa the process of digestion begins before the living prey is taken into the protoplasm of the captor. This is manifested in most cases by the paralysis of the victim when it comes in contact with pseudopodia of many rhizopods and Heliozoa, Ehrenberg (1833) for Actinophrys sol; F. E. Schultze (1875-1876) for Allogromia and Polystomellina; Winter (1907) for Peneroplis, etc. In some cases, at least, it is not improbable that this paralyzing killing substance is analogous to, if not the same as, the digestive fluids which kill bacteria and other prey after they are taken into the body protoplasm. Thus bacteria become motion- less in about thirty seconds after the gastric vacuole is detached from the cytopharynx of Paramecium caudatum (Metalnikoff, 1903 and 1912). The color changes of chemical indicators, for example alizarin sulphate, show that the killing agent is acid in nature; this was early detected by Greenwood and Saunders (1894), who interpreted it as a mineral acid without further specification. Later observers have confirmed this suggestion, Nirenstein, Metalnikoff and others showing that digestion in the vacuole is a process which is divisible into two periods, in one of which the reaction of the vacuole contents is acid, while in the other it is alkaline. The acid reaction lasts for about fifteen minutes, according to Nirenstein and Metalnikoff, in the gastric vacuoles of Paramecium, but Khain- sky concluded that the acid reaction is maintained during the entire period of digestion, becoming alkaline only after the dissolu- tion of the protein substances is at an end. In other cases, however, no acid reaction at all can be demonstrated. Thus, Metalnikoff, also in the case of Paramecium, found that some vacuoles never give an acid reaction; others much more rarely show an acid reaction 196 BIOLOGY OF THE PROTOZOA throughout, while still others in the same organism are first acid and then alkaline. Minchin (1912) suggests, in connection with this diverse history of vacuoles in the same species, that different food substances incite different responses on the part of the proto- plasm much as different antibodies are formed from cells of the Metazoa in response to toxins from different types of pathogenic parasites. Shapiro (1927) followed the change in pH of the gastric vacuole in Paramecium from an initial alkaline stage (7.6) which quickly changed to a maximum acid stage (pH 4) from which it slowly returned to the alkalinity of the surrounding water (pH 7). In Heliozoa, Howland (1928) shows that the initial pH of a gastric vacuole of Aciinosphacrium eichhornii is about neutral or slightly acid (pH 7 to 6.6). This lasts for a period of five or ten min- utes but changes to pH 4.3 ± 0.1 in all vacuoles in which active digestion is going on, while old vacuoles containing indigestible remains have a pH range from 5.4 to 5.6. In view of the number of different ferments which have been isolated from different types of Protozoa, it is quite probable that digestion does not take the same course in all types. Pepsin-like ferments, which dissolve albumins in an acid medium, were isolated by Krukenberg (1886) from the Mycetozoon Aethalium septi- cum, and by Hartog and Dixon (1893) from the ameba Pelo- myxa pahisiris, while Metsch- nikoff (1889) showed that the food vacuoles in the Plas- modia of Aethalium have an acid reaction favorable to the activity of such ferments. Trypsin- like ferments have likewise been isolated by Mouton (1902), from soil amebae cultivated in large numbers on agar; also diastatic fer- ments were easily obtained from Balautidium coli by Glaessner (1908), and from Pelomyxa palustris by Hartog and Dixon (1893). The typical course oi' a gastric vacuole through the endoplasm of ciliates has been carefully worked out by Greenwood and by Nirenstein for Carchesium and Paramecium caudatum (Fig. 102). Prowazek (1897) staining with neutral red found a collection of red granules about the gastric vacuole; similar granules were observed Fig. 102. — Carchesium polypinum ? History of food vacuole; (c) stage of stor- age and little change; (b) stage of acid reaction; (c) neutral reaction. (After Greenwood.) GENERAL PHYSIOLOGY 197 by him and by Nirenstein (1905) to pass into the gastric vacuole and to mix with the food substances from which circumstance they were regarded by both observers as the bearers of ferments (trypsin- like according to Nirenstein). The so-called Excretperlen (excre- tory granules) first described by Prowazek (1897) and interpreted by him, by Nirenstein and by Doflein (1916) as furnishing evidence of excretion through the general cell membrane, with equal justifi- cation may be interpreted as secretory granules. If the neutral red staining granules about the gastric vacuoles are bearers of ferments as maintained by Prowazek, they certainly are secretory in nature. There is some uncertainty, however, as to the identity of these with the so-called excretory granules. The experiments of Slonimski and Zweibaum (1922) show that there are two types of these granules which they call A and B, and that the peripheral granules (B) which exude from the membrane vary in number and size according to external conditions of temperature and internal conditions of vitality, being rare or absent prior to conjugation. The nature of these varying granules and their function in metab- olism are still unsolved problems. In connection with secretions we may take into consideration the various poisons produced by Protozoa either in the form of toxins exuded by the individuals and soluble in the surrounding medium, or in the form of endotoxins which are liberated only when the individual is disintegrated. What little is known about these secretions is mainly in connection with parasitic forms and here knowledge is limited to the effects produced upon the host (see Chapter X). In general it may be stated that, if we except the toxins produced by the so-called Chlamydozoa (particularly small- pox and rabies organisms), the poisons of protozoan origin are much slower and indefinite in their action on the host than are bacterial toxins, and the course of the specific diseases caused by pathogenic protozoa is relatively much slower than diseases caused by bacteria. Relatively few toxins of protozoan origin have been extracted and used in experimentation. One such, called sarcocystin, was obtained from sarcosporidia by Pfeiffer and Gasparck and by Laveran and Mesnil (1899). The latter found that rabbits are soon killed by the blood injection of sarcocystin in glycerin solution, also that crushed cysts give rise to characteristic pathological effects in the muscles, whereas no such reaction accompanies the presence of uninjured cysts. Filtered blood of malaria victims, if taken at the height of parox- ysm and injected into a malaria-free individual, produces in the individual a characteristic malarial paroxysm according to Rosenau and his co-workers, and analogous "paroxysm toxins" have been detected in connection with other blood parasites. Toxins from organisms of amebic dysentery are more regional in their action, causing local ulceration and abscess formation indi- 198 BIOLOGY OF THE PROTOZOA eating a cytolytic process possibly due to secretions of digestive fluids. There is still some uncertainty, however, in regard to this matter, and the possibility of participation by bacteria in the reactions is not excluded. Notwithstanding the serious diseases in man and mammals generally due to trypanosomes, there is very little positive evidence that secretions are responsible for the effects produced. Experi- ments with extractives from Trypanosoma brucei by Kanthak, Durham and Blanford, and by Laveran and Mesnil, gave no indi- cation of toxic effects. On the other hand, Novy and MacNeal, injecting dead Trypanosoma brucei in guinea-pigs obtained definite fever symptoms, loss of weight and local ulcerations which, however, they did not trace to the effects of a specific toxin. Somewhat more positive evidence is accumulating in regard to the possibility of endoenzymes locked up in the trypanosome proto- plasm and liberated on disintegration. Thus a number of observers, among whom may be enumerated MacNeal, Plimmer, Leber, Martin and others, have interpreted the rise in temperature of organisms with trypanosomiasis as due to the presence of endotoxins, freed in the blood upon death and disintegration of trypanosomes resulting from treatment with medicaments. Also Uhlenhuth, Woithe, Hiibener and others have concluded that endotoxins fatal to rats are liberated if blood containing Trypanosoma equiperdum is first dried, then dissolved again and injected into rats. Schilling, Braun, Teichmann, on the other hand, got no reaction upon injecting dead pathogenic trypanosomes into the peritoneum or subcutane- ously (see pp. 363 and 384). In all of these cases, with the exception of sarcocystin, the evi- dence in favor of the secretion of exotoxins or the presence of endotoxins is purely circumstantial and verification by chemical and biological methods with exclusion of other possible contributing factors has not yet appeared. Digestion of Carbohydrates and Fats. — Specific ferments for the transformation of starch into soluble sugar have not been isolated; nevertheless, the evidence that such action takes place is convinc- ing. Curiously enough, this evidence does not apply to the Infusoria where very little digestion, beyond a slight corroding of starch grains, occurs. In rhizopods, however, especially in the ameboid Pelomyxa and in species of Ameba, starch grains are entirely dis- solved, according to the observations of Stole (1900) who found that the characteristic refringent granules of Pelomyxa palustris have a very definite relation to carbohydrate nutrition. These granules (Glanzkorper) are filled with glycogen, the volume of which increases up to fourfold when the animals are fed with starch, and decreases to entire disappearance when they are starved. GENERAL PHYSIOLOGY 199 Even cellulose is said by Stole to be digested by this organism and Schaudinn made the same observation on the Foraminiferon Cal- cituba polymorpha. In Foraminifera generally, according to Jensen, and in myxomycetes, according to Wortmann, Lister and Cela- kowsky, starch may be similarly digested. The flagellates appar- ently have in some cases, at least, the same power of dissolving starch. Thus, Protomonas amyli and Phyllomitus augustatus eat practically nothing but starch, a fact indicating the action of appropriate digestive ferments. The Hypermastigidae which are abundant in white ants (termites) are unusual in their ability to digest cellulose. It has been shown that these flagellates live as symbionts with their termite hosts digesting the wood eaten by them. The termites die if deprived of their protozoan symbionts by heating or by oxygenation; the protozoa die if the wood diet of the termites is stopped (Cleveland, 1923). In few Protozoa has the actual digestion of fat been observed. Under experimental conditions, ingested fats are usually carried along unchanged in the protoplasm. We cannot state arbitrarily, however, that fats are not emulsified and used as food. On the contrary, it is difficult to account for the presence of oils and fat bodies in varying quantities in all groups of Protozoa under any other assumption, despite the negative results of Stamiewicz (1910) and of Nirenstein (1909). Positive results indeed have been ob- tained by Dawson and Belkin (1928), who injected oils of different kinds into Amoeba proteus; of these 8.3 per cent of cod-liver oil was digested, 8.2 per cent of olive oil, 4 per cent of cotton-seed oil, 3.5 per cent of sperm oil and 1.4 per cent of peanut oil. Saprozoic Nutrition. — In holozoic nutrition the food substances are in the form of complex proteins, carbohydrates and fats, making up the bodies of the various organisms ingested. In saprozoic and saprophytic nutrition the food substances are less complex chemically, consisting of materials dissolved out of the disintegra- ting bodies of animals and plants. These are taken in, not through the agency of specialized oral motile organs, nor through a definite mouth, but are absorbed through the body wall. Many of the smaller types of flagellates obtain their nutriment in this way, extracts or infusions of animal or plant tissues containing various salts and organic compounds forming excellent culture media for such Protozoa. Little is known, however, of the chemical make- up of such fluid substances, nor is it known whether they are prepared for absorption by chemical processes due to the activity of the receptive organism; nor is there any evidence to indicate processes of digestion subsequent to their absorption. The general assumption, based upon the thriving cultures in infusions of dis- integrating animal and plant matter, has been that dissolved 200 BIOLOGY OF THE PROTOZOA proteins are taken into the protoplasmic bodies of many kinds of Protozoa by absorption through the general cortex or through some specialized region for the purpose. From experiments with the green alga, Euglena gracilis, by Zum- stein (1900), Ternetz (1912), et al., it appears probable that some saprozoic forms of Protozoa get their main nourishment from amino-acids derived from disintegration of animal and plant matter through the agency of bacteria, and from carbohydrates in solution. The necessary mineral matters are obtained from the surrounding alkaline medium. Emery (1928), experimenting with Paramecium caudatum, found that a measurable quantity of amino-acids is utilized in place of the normal bacterial food. With a mixture of equal parts of ten amino-acids he figured out that 100,000 Paramecium caudatum in twelve hours would use 48.3 per cent of a 0.1 per cent solution, while different amino-acids used singly gave differing results. 1 In this connection, it is important to consider the possible inter- action of excretion products of different Protozoa upon themselves and upon each other, as well as the effects of products of bacterial action. It has long been known that isolation cultures are fre- quently threatened by the growth of detrimental bacteria. On a 'priori grounds it is not improbable that excretion products of Protozoa themselves may have such an effect. Woodruff (1912, 1913) has studied this problem in connection with Paramecium aurelia and the hypotrichous ciliates, Stylonychia pustulata and Pleurotricha lanceolata, and found that Paramecium, when placed in filtered medium which had contained enormous numbers of Paramecium in pure culture, were manifestly weakened in vitality. Similarly the hypotrichs, when placed in filtered medium which had swarmed with hypotrichs, showed a weakened vitality. When, however, Paramecium was placed in filtered hypotrich culture medium, the result was an increased vitality. Woodruff concluded that excretion products from Paramecium are detrimental to Paramecium, and hypotrich products to hypotrichs, while the latter products have a somewhat stimulating effect on Paramecium. This may be, as Woodruff suggests, of some importance in deter- mining the sequence of protozoon forms in a limited environment such as hay infusion. 1 The degree of absorption of specific amino-acids is as follows: Per Per cent. cent. Mixture of different amino-acids Alanine 15.5 (except arginine) . 48 . 3 Glutamic acid 13.2 Glutamic acid hydrochloride . 45 . 6 Leucine 12.0 Cysteine hydrochloride 26.3 Glycocoll 9.6 Aspartic acid 25 . 1 Tryptophane 9.6 Tyrosin .17.7 Phenylalanine 7.7 Arginine 15.9 GENERAL PHYSIOLOGY 201 Specific structural adaptations, useful in methods of food-getting, are characteristic. Haustoria-like processes, derived from the epimerites of gregarines, in some cases extend deeply in the tissue cell {Stylorhynchus longicollis, Echinomera hispida, Pyxinia moebiuszi, etc., Fig. 103). The coccidian Caryotropha mesnili, according to Siedlecki, shows a significant relation between the nucleus of the host cell and that of the parasite. This organism is a parasite in the spermatozoa of the annelid Polymnia nebvlosa where the sperm cells are aggregated in bundles in the characteristic annelid fashion, usually about a feeding mass or blastophore. The parasite gets into such a cell as an agamete or sporozoite, one only of the bundle, Fig. 103. — Food-getting adaptations of Sporozoa. 1, Pyxinia moebiuszi with epi- merite deeply insunk in the epithelial host cell (after Leger and Dubosq) ; 2, Caryo- tropha mesnili with an intracellular canal from the nucleus of the host cell (ti). (After Siedlecki.) as a rule, being infected, and as it grows the nucleus of the cell is displaced to one side and the cell loses its characteristic structure, becoming hypertrophied and distorted (Fig. 103, 2). Not only the infected cell but all the other cells of the spermatogonia bundle are affected, and none of them continues the normal development, but they become arranged like epithelial cells about the hypertrophied infected cell. The specific effect of the young Caryotropha on the infected cell consists not only of the enlargement of that cell, but of a definite feeding mechanism by which the parasite is supplied with food. That the nucleus is a center of constructive metabolic changes is well assured at the present day, and the conditions in these para- 202 BIOLOGY OF THE PROTOZOA sites suggest the peculiar relation which Shibata (1902) has described in the intracellular mycorhiza, where a mycelium thread is grown straight toward the nourishing cell nucleus of the host, causing marked hypertrophy on the part of the cell. In Caryotropha, the nucleus of the host cell is pushed to one side and the parasite assumes such a form that the nucleus lies in a small bay (Fig. 103, 2n). In the cytoplasm of the cell an intranuclear canal is then formed which runs from the host nucleus to the nucleus of the para- site, and Siedlecki holds that the food of the parasite is all elab- orated by the nucleus of the host cell, while the other spermatogonia form a protective epithelial sheath around it. When the parasite is full grown the cell is destroyed and the bundle degenerates. _ O. Fig. 104. — Ellobiophrya donacis, a peritrich with ring-form attaching organ which passes around the gill bars of the lamellibranch. X 800 and 1350. (After Chatton and Lwoff, Bull. biol. de la France et de la Belgique, 1929; courtesy of Prof. N. Caullery and Les presses TJniversitaires de France.) Other special adaptations in the interest of food-getting are fre- quently spectacular. Thus Ellobiophrya branchiorum (Chatton and Lwoff, 1928), a commensal ciliate on the gills of the lamellibranch Donax sp., has developed a curious, posterior, ring-form process whereby it is firmly anchored to the gill bars (Fig. 104). It is difficult to draw the line between symbionts, commensals and parasites. Symbionts are organisms living with a host in such a relation that both are benefited ; commensals are organisms which live with a host without benefit or injury to the latter but to their own advantage, and parasites are organisms which, to their own benefit, cause injury in one form or other to the host. Symbiosis GENERAL PHYSIOLOGY 203 is well illustrated by the harmonious life of some chlorophyll-bearing forms, Zoochlorella, Zooxanthella, etc., and Protozoa in which the former live (Paramecium bursaria, "yellow cells of Radiolaria and Foraminifera," Stentor viridis, Amoeba viridis, Vorticella viridis, etc.), and it is conceivable that some gut-dwelling forms may perform a useful activity for a host by disposing of pernicious bacteria, or by preparing food substances for use by the host as do Hypermastigidae in termites (Cleveland). Commensals, such as Endamoeba coll, Endamoeba nana, Trichomonas species and other intestinal forms may, on occasions, turn into parasites, as is the case with Tricho- monas (Tritrichomonas, Kofoid), Giardia (Lamblia), etc. 2. Products of Assimilation. — With the majority of forms the products of assimilation vary with the type of food used and are frequently so abundant in the cell as to give a characteristic appear- ance or color to the animal. Thus the refringent granules of Pehmyxa palustris (Stole.) produce a peculiar refringent effect. The brown granules of Plasmodium, species, characteristic of malaria, are products of hemoglobin assimilation. Similarly the coccidin of Coccidia; stentorin of Stentor coeruleus and Folliculina ampulla; the pink of Holosticha; the lavender of Blepharisma undulans or the red of Mesodinium rubrum, are examples of the great variety of colored cellular substances dependent upon the food that is eaten. In the absence of the specific kinds of food which yield these chromic products the organisms are colorless, and colored or colorless indi- viduals of the same species may appear in the same culture (see p. 134). CHAPTER VI. REPRODUCTION. Of all the marvels associated with the Protozoa there is nothing more staggering to the imagination than the fixity of type which their protoplasm manifests. The genotype, represented by the derived organization, subject to minor variations of a fluctuating character in the course of a normal life history, or subjected experi- mentally to all kinds of unusual environmental conditions, remains fundamentally unchanged. Types modified through amphimixis or through permanent modifications of the environment may lead to divergent types. This conservatism or fixity of type is a function of the organization which has been continuous in the past and will be continuous in the future. The activities which take place in the organization, the sum total of which constitute vitality, are discontinuous, they have been and will continue to be dependent upon the interactions between organization and environment. The single individual which we study under the microscope has had no such history in the past and no promise for the future; its span of life as an individual is measured by hours or days only. It is the temporary trustee of a small portion of an organization which has been parceled out among unknown myriads of similar trustees. Its metabolic activities are the interactions within the organization and as a result of these activities the fluctuating variations charac- teristic of the genotype follow one after another in the form of inevitable differentiations which may or may not be visibly indi- cated by structural changes (see Chapter VII). Ultimately its possi- bilities of further vitality as a single individual are exhausted and it undergoes its final manifestation of vitality. The significance of this final act is a function of all genotypes and of all organizations whereby the organization is further parceled out to two or more trustees. It is reproduction by division, which by reason of its universal occurrence is one of the most characteristic properties of protoplasm. There is no doubt that division of the cell is a phenomenon of deep-reaching significance; we shall endeavor to show that the organization as parceled out to the descendants by division is not a mere equal division of the protoplasm of the individual with its load of metaplastids and other modifications of the organization, but a renewed or purified organization such as the individual received when it was formed. Unlike Metazoa, with the processes of division, the old derived organizations of Protozoa are lost by absorption, REPRODUCTION 205 the organization being de-differentiated, and the protoplasm has a renewed potential of vitality. In order to understand the relations of division to the chain of metabolic activities we should know more about the conditions under which division occurs, and the "causes" of division. There is very little real evidence for conclusions in this matter but there have been many theories. The latter for the most part are based either upon analogies with physical phenomena or upon hypothetical "spheres of influence" of morphological elements of the cell. They have been developed in the main to interpret phenomena of division in metazoan cells, particularly in egg cells, and fall completely to the ground when applied to division of Protozoa. So it is with the contractility hypothesis of Heidenhain, Driiner and others, who see in the spindle fibers and astral rays a contractile system whereby the nucleus and cell are divided in a strictly mechanical manner. The intranuclear spindle and the absence of cytoplasmic rays in the great majority of Protozoa are enough to show that such physical interpretations do not reach to the root of the matter. The " spheres of influence" hypotheses, based upon the kinetic center of the cell and its influence on the cytoplasm, was developed by Boveri in the attempt to associate cell growth and the causes of division. The "energid" theory of Sachs and Strasburger was an analogous effort to trace the causes of cell division to increasing volume of the cell through growth, each nucleus having its sphere of influence in the cytoplasm and dividing when the volume of the cell outgrows the sphere of activity of the nucleus. The Kernplasmverhaltnis theory of Hertwig was based upon somewhat similar grounds. Accord- ing to this the volume of the nucleus bears a certain normal relation or ratio to the volume of the cytoplasm in young actively func- tioning cells, evidence of which in Fronionia was given by Popoff (1909) and by Hegner (1920) in the equidistant distribution of nuclei in various species of Arcella. With increasing age this ratio is altered to the advantage of the cytoplasm until division of the cell restores the normal ratio. With uninucleate forms such as Para- mecium or Fronionia there is some evidence of change in relative volumes, and careful measurements by Popoff (1909) and other followers of Hertwig are adduced to support the hypothesis. In these forms the volume of the nucleus is proportionally reduced until just prior to division when the nucleus rapidly increases in volume and divides. Looper (1928) more recently, by mech- anical stimulation, caused Aciinophrys sol to fuse with enucleated fragments from other individuals. This led to change in the nucleus- cytoplasm ratio to the advantage of the cytoplasm. Such forms divided from one-half to two times faster than the controls. If, on the other hand, some cytoplasm is cut away, the reduced cells (100 cases) divided on the average in eighty-eight hours, while 206 BIOLOGY OF THE PROTOZOA the controls divided in twenty-four hours (see Hartmann's simi- lar experiments with Ameba, p. 239). In Uroleptus, Uronychia and similar forms, however, the many nuclei fuse to form one com- pact and relatively small nucleus prior to division. It would seem that such changes in relative volume of nucleus and cytoplasm are better interpreted as the effects of underlying conditions which lead to division rather than as the direct cause of division. None of these theories is of much value in analyzing the antecedent phenomena of division. These must be sought in the reactions of different substances constituting protoplasm. Division of the cell itself is a last step in a progressive series of reproductive changes affecting the entire protoplasm, the constituents of which— micro- somes, mitochondria, plastids, chromomeres, kinetic elements, etc.— have already divided. It is in the division of these fundamental granules in the make up of protoplasm that we must look for the underlying causes of cell division. The dependence upon growth and metabolism of the succession of division processes which char- acterize reproduction is clearly evidenced by simple starvation exper- iments, division ceasing with cessation of metabolic activities. There is a possibility that environmental conditions play a more direct part in reproduction than is indicated by their relations to metab- olism. Thus Robertson (1921) concludes that a catalase (X sub- stance) is secreted by the living cell which directly enhances division. He found that two individuals, or more, of Enchelys farcimen in a drop of culture medium would divide from four to sixteen times more rapidly than a single individual in a similar drop, the result being interpreted as due to contiguity of individuals. This, however, is a direct contradiction of Woodruff's (1911) results with Paramecium and Stylonychia, according to which the division rate is reduced by accumulation of products of metabolism in the medium. Nor is Robertson supported by other observers. Cutler (1924) for example, found for Colpidium colpoda that the division rate depends upon the number of bacteria present as food, and that increase in number of individuals in a drop means a decrease in the individual division rate. Greenleaf (1924) similarly found that solitary indi- viduals of Paramecium caudatum, P. aurelia and Pleurotricha lanceolata isolated in 2, 5, 20 and 40 drops of medium, gave a highest division rate in five days in the 40-drop test, the lowest in a 2-drop test. Also in Uroleptus mobilis, in a sixty-day test in which 1 individual, 2, 3 and 4 individuals were isolated daily in a single drop of medium the highest division rate was shown by the solitary individual in a drop as shown in the following table (see also table on next page) : 10 individuals, 1 to a drop, each divided in the sixty days . . 74. 1 times 20 individuals, 2 to a drop, each divided in the sixty days . 59 . 5 30 individuals, 3 to a drop, each divided in the sixty days . . 54 . 7 40 individuals, 4 to a drop, each divided in the sixty days . . 54 . 2 REPRODUCTION 207 Environmental conditions which alter the permeability of the cell, thereby enhancing or retarding metabolic activities do, how- ever, have a corresponding effect upon the division rate. Age of individuals, or the protoplasmic organization at different periods of the life cycle likewise has a determining effect on the rate of division, the differences, as shown in the following table, being due to the differences in the reactions of the protoplasm to the same medium under different conditions of organization. Series 111 and 112, for Uroleptus Mobilis Division Rate. Experiment from September 2 1 to November 10, 1924. t Age. Genera- tion. Divisions per individual. Series. No. in drop. First ten days. Second ten days. Third ten days. Fourth ten days. Fifth ten days. Sixth ten days. Total, sixty days. f 1 12 7 10 13 9. 9 60 Ill 270 J 2 ) 3 11 9 7 5 6 6 10 7 5 5 5 4 44 36 I 4 10 4 3 6 3 5 31 1 14 14 9 10 7 6 60 112 263 2 1 ;5 11 13 13 8 5 4 8 7 4 2 6 4 47 38 4 8 11 10 7 2 3 41 | 1 11 S 5 9 4 6 43 114 160 1 2 1 3 6 5 8 4 3 3 6 4 1 2 6 30 18 4 8 4 3 2 3 1 21 | 1 14 17 9 10 13 10 73 115 247 i 2 i 3 10 14 13 16 6 7 9 8 10 8 9 4 57 57 I 4 15 13 7 9 10 7 61 1 13 14 10 9 7 7 60 116 189 J 2 3 9 9 10 11 8 5 10 7 9 8 8 7 54 47 \ 4 7 7 3 7 6 4 34 1 16 18 11 10 14 12 81 117 133 l 3 14 14 17 17 7 8 10 9 8 10 9 9 65 67 4 13 17 6 8 10 8 62 f 1 IS 22 12 16 17 14 99 lis 140 J 2 IS 14 8 11 13 13 82 ) 3 15 20 9 12 11 9 76 1 4 14 20 7 12 12 12 77 » [ 1 15 19 10 10 10 8 72 11!) 110 J 2 ] 3 15 11 14 14 7 7 7 8 7 6 9 6 59 52 4 10 14 6 8 7 5 50 1 18 19 11 13 13 12 S6 12 I 12 1 2 1 3 16 17 16 15 6 5 12 8 9 13 10 9 69 67 \ 1 16 15 9 9 13 11 73 18 23 13 16 18 19 107 121 10 J 2 14 24 9 8 15 18 88 3 15 23 10 11 14 16 89 I 4 19 21 10 11 14 17 92 208 BIOLOGY OF THE PROTOZOA example, were 279 and 263 generations old at the beginning of the experiment, the single individual isolated daily in a drop of medium divided 60 times in sixty days; with 4 individuals in a drop, each divided only 31 times. Series 120 and 121 were 12 and 10 genera- tions old, and each solitary individual divided 86 and 107 times in the same sixty days, and with the same medium freshly made each day. From this table it is apparent that the division rate is reduced by the presence of more than one individual to a drop. Furthermore, the reduction of the division rate under such conditions is much less for "y° un g" individuals than for old. Substances making up the composition of living protoplasm are constantly manufactured. Such substances, usually in the form of granules, grow to a certain limit of size and each then divides. Evi- dence for this is apparent only in the more obvious of the proto- plasmic elements such as plastids, kinetic elements, chromomeres, etc., the division of which has been mentioned in the preceding pages. Finally the grand aggregate, the cell itself, divides as a last expres- sion of the series of events that have taken place. It is evident that such division of the cell as a whole constitutes only a small part of the phenomena of reproduction and perhaps not the most important part. While most of the elementary granules, apart from those enumerated above, which make up the bulk of proto- plasm, cannot be followed from their smallest stages to the stage when they become visible, it is not inconsistent with the idea of continuity from generation to generation to regard even the smallest as retaining its integrity and reproducing itself by division. "For my part I am disposed to accept the probability that many of these particles, as if they were submicroscopical plastids, may have a persistent identity, perpetuating themselves by growth and mul- tiplication without loss of their specific individual type" (E. B. Wilson, 1923). While the division of a single granule results in the formation of two probably identical granules of the same substance, the division of aggregates of granules of different substance may or may not result in identical daughter aggregates. The nucleus is such an aggregate which, by ordinary equation division, is probably divided into two identical halves, but in meiotic divisions the products of the nucleus are different, visible evidence of which is shown by the history of the sex chromosomes and by the results in modern genetics. It is entirely possible that differentiations may arise from such inequalities in nuclear division (see Chapter IX). The cytoplasm of the cell, likewise, is such an aggregate, made up of all the different substances variously distributed, which compose living protoplasm. If all the granules were equally distributed at division to the daughter cells, as are nuclei and many kinetic ele- ments, then the products of cell division might be identical. Mor- REPRODUCTION 209 phological evidence that all granules are not thus equally distributed is furnished by all budding and spore-forming types, and by forms like Dileptus gigas or Holosticha multinucleata, where the large chromatin granules, while still in the process of division, are carried bodily to one or the other daughter cell (Fig. 46, p. 92). Reproduction whereby a type of organism is perpetuated and distributed, is thus preeminently a process of division. In the last analysis cell division is the only kind of reproduction known. Potential individuals are contained in every germ cell, but germ cells, like other cells, are formed by division and it follows that every female reproduces as many potential offspring as eggs. Develop- ment of such eggs, however, is usually dependent upon fertilization, which is quite a distinct phenomenon, accessory to reproduction and necessary in most animals, but not itself reproduction. In the present chapter only a summary of the more obvious phenomena of reproduction will be described, leaving the problems associated with fertilization for treatment in a later section (see Chapter VIII). It is division of the grand aggregate of protoplasmic substances, /. e., division of the cell itself, that is usually described as reproduc- tion of the Protozoa. Such reproductions are usually classified as division, budding or gemmation, and sporulation, the inference being that these are different modes of reproduction. In reality, however, they arc different types of reproduction by division, and such modifications would be expressed better by the terms equal division, unequal division, and multiple division. I. EQUAL DIVISION AND EVIDENCE OF REORGANIZATION. In the ordinary metabolic processes of an active protozoon there is evidence of a cumulative differentiation which indicates a differ- ence in organization between a young cell immediately after division by which it is formed and the same cell when it is mature and ready itself to divide. Child (1916) mainly from experiments with cells of the Metazoa, came to the conclusion that "senescence con- sists in a decrease in metabolic-rate determined by the change in, and the progressive accumulation of, the relatively stabile components of the protoplasmic substratum during growth, develop- ment and differentiation" (loc.cit. p. 333) . He further suggested that in every cell division in unicellular animals, with the accompany- ing processes of reorganization, there is some degree of rejuven- escence and, if such rejuvenescence balances the cumulative differ- entiation, continued life of the organisms by division alone may go on indefinitely. By proper conditions of the environment it is ■ conceivable that such a balance may be established. On such an hypothesis it is possible to account for the continued vitality of animal flagellates in which fertilization processes are unknown, for 14 210 BIOLOGY OF THE PROTOZOA the continued life of many of the higher plants, and for the con- tinued life of the tissue cell cultures in the hands of Carrel and others (see Chapter VII). In many Protozoa there is unmistakable evidence of such reorgan- ization processes which will be described in the following pages; in many there is no visible evidence, but in such cases and in the absence of other possibilities of reorganization, it is permissible to assume that reorganization processes which escape the most vigilant watchfulness of the observer, do actually occur. A. Division in Mastigophora. — With very few exceptions cell division in flagellates is longitudinal, beginning as a rule at the anterior or flagellar end, the cleavage plane passing down through the middle of the body. As the halves separate the two daughter cells usually come to lie in one plane, so that final division appears to be transverse. In the majority of forms the individuals divide while freely motile, but this is by no means universal, variations in this respect occurring in the same family and even in the same genus. As there are few details in the structure of a simple flagellate on which to focus attention, descriptions of division processes are practically limited to the history of the nucleus, kinetic elements and the more conspicuous plastids. Here, in the main, are fairly prominent granules of different kinds which divide as granules, and, save for the chromatin elements of the nucleus, without obvious mechanisms. In the simpler cases there is little evidence that can be interpreted as reorganization at the time of division, and the little we find is limited to the motile organs. In the more complex forms, however, there is marked evidence of deep-seated changes going on m the cell. The earlier accounts of cell division in the simpler flagellates described an equal division of all parts of the body including longi- tudinal division of the flagellum, if there were but one, or equal dis- tribution if there were two. One by one such accounts have been checked up by use of modern technical methods until today there is very little substantial evidence of the actual division of a flagel- lum. The basal body and the blepharoplast usually divide, but the flagellum either passes unchanged to one of the daughter cells as in Crithidia, Trypanosoma, etc., or is absorbed in the cell. In some doubtful cases it may be thrown off. If the old flagellum is retained in uniflagellate forms the second flagellum develops by outgrowth from the basal body or the blepharoplast. If the old flagellum is absorbed, both halves of the divided kinetic element give rise to flagella by outgrowths (Fig. 49, p. 95). Similarly, if there are two or more flagella, one or more may be retained by each daughter cell while the other, or full number, is regenerated. In some cases, as in Herpetomonas musca-domesticae, the regenera- REPRODUCTION 211 tion of a second flagellum occurs before division of the cell is evident, a circumstance which evidently led Prowazek (1905) to conclude that this organism is normally bi-flagellated (Fig. 170, p. 368). E V\ F Fig. 105.— Lophomonas blattarum. A, flagellar tuft and nucleus in calyx in pro- phase of division; B, nucleus with chromosomes leaving calyx; paradesmose on side; C-F, stages in nuclear division in the posterior part of the organism and formation of new calyces and flagellar tufts. X 1850. (After Belaf, Erg. u. Fortschr. der Zool., courtesy of G. Fischer.) 212 BIOLOGY OF THE PROTOZOA Reorganization is indicated to some extent by these cases in which the old flagellum is absorbed. It is still better indicated by a number of flagellates in which the cytoplasmic kinetic elements, as well as the flagella, are all absorbed and replaced by new combinations in each of the daughter cells. Thus in Spongomonas splendida, accord- ing to Hartmann and Chagas (1910) the old blepharoplasts and the two flagella are absorbed and new ones are derived from centrioles of the nuclear division figure (Fig. 49, p. 95). The phenomenon cannot be regarded as typical of the simple flagellates, for in the great majority the kinetic elements are self-perpetuating, even the axostyles according to Kofoid and Swezy (1915) dividing in Tricho- monas (Fig. 77, p. 145). This, however, has not been supported by later workers. Fig. 106. -Vahlkampfia Umax. Nucleus in upper cell in full mitosis (promitosis). (From Calkins.) An extreme case of reorganization is apparent in the two species of Lophomonas (L. blattae and L. striata) first described by Janicki (1915). Here the parental calyx, basal bodies, blepharoplasts and rhizoplasts all degenerate during division (Fig. 105). At division a cytoplasmic centriole first divides with a connecting fibril which is retained throughout as a parademose. The nucleus emerges from the calyx in which it normally lies, and moves with the spindle to the posterior end of the cell. The spindle takes a position at right angles to the long axis of the cell; chromosomes, probably eight in number, are formed and divided, and two daughter nuclei result, each of which is enclosed by a new calyx while new basal bodies and blepharoplasts apparently arise from the polar centrioles (Fig. 105). Thus the old kinetic complex, with the exception of the cytoplasmic centriole, is discarded and entirely new_aggregates are formed. REPRODUCTION 213 B. Division in the Sarcodina. — It is questionable whether any rhizopod divides in the very simple manner described by F. E. Schultze for Amoeba polypodia. The "limax" types indeed approach this simplicity (Fig. 106) but new discoveries are constantly at hand to indicate that these are not as simple as they have been described. Thus Arndt (1924) quite recently has given creditable evidence of the existence in a simple ameba, Hartmannella klitzkei, of a definite centrosome with centriole which is permanently extranuclear (Fig. 58, p. 106). At division of the cell the centrosome divides and the daughter centers with their centrioles, take positions at the poles of the nuclear spindle which originates within the nucleus. The mitotic figure is thus made up of cytoplasmic elements, kinetic elements derived from the nucleus, and chromatin. A similar combination occurs in dividing Heliozoa. The original description of division of Acanthocystis aculeata by Schaudinn, a form possessing the characteristic central granule of the Heliozoa, has been consider- ably modified by later observations. According to Schaudinn the central granule or centroblepharoplast, which is the focal point in the cell of the radiating axial filaments, divides to form an amphi- aster (Fig. 50, p. 95) which becomes the central spindle of a typical mitotic figure. The more recent observations of Stern (1924) indicate that, as in the simpler ameba described above, the central granule of Acanthocystis behaves as a cytoplasmic centrosome, forming poles of a mitotic figure which is derived otherwise entirely from the nucleus. Individuals which have been deprived of their skeletons and membranes, which afford resistance to the activities of the enclosed protoplasm, become "sprung," so to speak, and the unusual freedom from restraint results in a separation of the eentro- somes from the remainder of the spindle which completes its division without further participation of the centrosomes (Fig. 0", p. 121). Schaudinn's description of division in Heliozoa was confirmed in the main by Zuelzer (1908) in connection with the aberrant form Wagnerella boreal is. Here the axopodia-bearing portion of the cell is free from the silicious mantle which covers the remainder of the animal, the nucleus being in an enlarged pedal portion attached to the substratum. The central granule is in the geometrical center of the "head" and is the focal point of the axopodial filaments. Each of the latter bears a granular enlargement similar to a basal body. In preparation for division these move centripetally toward the central granule forming a zone about it which divides with the division of the central granule. In the meantime the nucleus migrates from the other end of the body and with the spindle formed by the divided central granule forms the mitotic figure. Complications in the division process accompany the presence of shells and tests. Where these are chitinous or pseudochitinous, they may also divide with the cell body (Pseudodifflugia, Cochlio- 214 BIOLOGY OF THE PROTOZOA podium). In other cases the individual divides within the shell, after which one of the daughter individuals moves out and forms a new shell, while the other one remains in the original test {Micro- gromia socialis, Clathrulina elegans, etc, Fig. 107). In most cases, however, a novel method of shell duplication found in no other divi- sion of the Protozoa, has been developed. This process, known as "budding division," occurs throughout the group of the testate Fig. 107 '. — Microgromia socialis after Hertwig (A), and Microgromia sp. (B), original. rhizopods and is well illustrated by the classical example of Euglypha aheolata first described by Schewiakoff (188S). Here after full growth following vegetative activity of the individual, the pseudo- podia are drawn in; water is then absorbed whereby the protoplasmic density is greatly reduced and the volume increased. This is fol- lowed by a process resembling pseudopodia formation, the proto- plasm emerging from the parent shell opening as a ball or dome which REPRODUCTION 215 assumes the general form of the parent organism. A new membrane of pseudochitin is formed about the extruded mass and on it the silicious shell plates, preformed in the parent protoplasm, are now cemented. In some forms, e. g., Arcella species, the chitinoid mem- brane becomes the permanent shell of the organism, older shells becoming brown or reddish by coloring due to oxides of iron ; in other forms as in the Difflugiinae the chitinoid membrane is covered by foreign objects picked up and stored by the parent organism. In all cases of budding division after the budded individual is fully molded, the nucleus divides and one-half passes into the protoplasm of the new shell. The connecting zone of protoplasm between the old and the new shell breaks out into pseudopodia and the two indi- viduals separate (Fig. 11, p. 33). The various types of foraminiferal shells, nodosarine, frondicular- ine and rotaline— may be interpreted as due to a similar budding division, but without actual separation of the parent and bud proto- plasm, the type being dependent upon the density of the protoplasm at the time of protrusion from the shell mouth (Fig. 19, p. 38). There is very little evidence of reorganization of the protoplasm at division in these rhizopods. The frequent withdrawal of pseudo- podia and rounding of the body may be an indication of changes going on within, as in Chlamydomyxa, Nuclearia, etc., but even such questionable indications are absent in many cases of recent inves- tigation (Belaf, Stern, et al.), where reorganization, if it occurs at all, must be in the make-up of the protoplasmic and undifferentiated elements. C. Division in Infusoria.— Here in the most highly differentiated forms of the Protozoa the processes of equal division are complex and the protoplasmic changes far-reaching. With but few excep- tions the division plane is through the center of the body and in a plane at right angles to the long axis of the cell. The externals of division are similar to division in other groups, with preliminary division of the plastids and nuclei and final division of the cell body. As in flagellates and some rhizopods the cup- or test-dwelling forms divide within the parent cup, one of the daughter individuals migrat- ing and forming a cup for itself. In some forms the daughter indi- viduals may remain and share the old house (Cothurnia ingenita). Where a tightly-fitting cell-covering is present as in Coleps hirtus, it is divided transversely and the missing parts are regenerated by the daughter organisms (Fig. 73, A, B, C, p. 136). In some Infusoria as in the other groups, division in many cases is incomplete, the daughter individuals remaining attached end to end as in Polyspira delagei or Haptophrya gigantea. Or daughter individuals may remain attached by incomplete division of their stalks, thus giving rise to arboroid colonies of different types (Vorticellidae mainly). In some forms, probably in the majority of ciliates, there appears 216 BIOLOGY OF THE PROTOZOA ^, m Fig. 108. — Paramecium caudatum, merotomy. 1, 2, and 3, different experiments, the straight line indicating the plane of cutting; 3, the history of a monster; an original cell, 3a, was cut as indicated; the posterior fragment (b) divided (c) into (d) and (e), the latter formed a monster (3, f-o); enucleated individuals (h, k, and n) occasion- ally separated from the parent mass. (After Calkins.) REPRODUCTION 217 to be a definite and permanent division zone which indicates the future plane of division and which is not displaced even after diverse mutilations of the body. Thus if Paramecium caudatum is cut across either the anterior or the posterior end, the cell ordinarily does not regenerate more than a ciliated surface on the truncated end. It divides like a normal form, but the division plane is not in the geometrical center of the mutilated cell, but in the geomet- rical center of the cell as it was before the cutting (Fig. 108). The same is true of Uronychia transfuga or U. setigera (Fig. 113). In daughter cells of dividing Paramecium the future division zones appear to be formed at an early period, and if a daughter cell is cut in such a manner that the geometrical center is destroyed without, however, destroying the nuclei, monsters of various types are produced indicating a complete upset of the organization (Fig. ION, f-o). In some cases, e. g., Frontonia leucas, the geometrical center, or division zone, has a different physical appearance from the remainder of the cell (Popoff, 1908, also mentioned by Hance, 1917, as occurring in Paramecium), but in the majority of cases there is no morphological evidence of the plane of division during inter-divisional stages. (a) Evidence of Nuclear Reorganization.— The two types of nuclei, macronucleus and micronueleus, complicate the nuclear phenomena at division. The macronucleus is more like a huge plastid of the cell with active functions in metabolism, while the micronueleus is generally interpreted as a germinal or racial nucleus, functioning at division and particularly at conjugation. Reproduction of the macronucleus in the majority of ciliates is analogous to that of a plastid. Division is direct with only a few isolated cases showing evidences of spindle formation or of indefinite chromosomes. In preparation for division, however, there is evi- dence in many forms of profound changes in the make-up of the nucleus destined to divide and some of these afford evidence of a clear-cut reorganization of this important element of the ciliate (see p. 93). In the less complicated types division of the macronucleus is relatively simple. In Dileptus gigas, for example, the nuclear material is in the form of many scattered chromatin and plastin spheres, each of which divides prior to cell division (Fig. 46, p. 92). There is no equal distribution of this chromatin to the daughter cells but the daughter halves may go together to the daughter cell in whose protoplasm they happen to lie. Some of the granules, how- ever, those in the region of the division zone, may be represented in each of the progeny. In forms with a single ellipsoidal macronucleus as in many of the commoner types (e. g., Paramecium, Colpoda, Frontonia, Glaucoma, etc.), the macronucleus simply elongates and constricts to form 218 BIOLOGY OF THE PROTOZOA two equal portions, one passing to each daughter cell (Fig. 35, p. 67). Band-form nuclei characteristic of Blepharisma, Spat Ind- ium, Didinium, Vorticella, Euplotes, etc., condense into spheroidal or ellipsoidal bodies before dividing. Where two macronuclei are present in the usual vegetative cell, as in Oxytricha, Stylonychia, Gastrostyla, etc., each divides independently of the other but syn- chronously. As with band-form nuclei the beaded macronuclei likewise form short rods as in Stentor, Spirostomum ambiguum, etc., the beaded character in all cases being lost. Here the separate beads are usually enclosed in a common nuclear membrane which is constricted at intervals, the contained chromatin massing together at the period of division. This is the condition in Uronychia trans- fuga, also, the twelve to fourteen apparently separate macronuclei are all connected, and the chromatin fuses prior to division to form a relatively short ellipsoidal nucleus (Fig. 113). In other types, however, the multiple macronuclei are independent and entirely disconnected. They arise by division and retain their independence during vegetative life. Thus in Urolepius mobihs and U. halseyi the eight or more macronuclei are formed as a result of a fourth division of the single parental nucleus from which they came (cf. p. 93 and Fig. 110). In preparing for division of the cell each of these eight nuclei of Uroleptus undergoes a remarkable transformation. A nuclear cleft (Kernspalt) appears in each, and in the cleft is a single large granule. The major part of the nucleus lies below the cleft and is filled with densely-staining chromatin; the other part lying above the cleft contains much less chromatin in the form of fine granules (Fig. 47). This latter part, together with the granules in the cleft, is thrown off and the chromatin contents are distributed in the cytoplasm. When each of the nuclei is thus freed from its distal portion the eight remaining parts fuse, forming first a long banded nucleus, and later, by con- densation, a relatively small ellipsoidal and single nucleus. This divides twice or three times before the division of the cell is com- pleted, the fourth division always occurring after the daughter cells have separated (Fig. 110). The micronuclei show no such complicated histories. If they are multiple in the cell there is no fusion, nor is there any elimination of micronuclear material. Each divides with the formation of an unmistakable, but very minute, mitotic figure (Fig. 23, p. 50). They are all represented furthermore by daughter halves in each of the daughter cells. (b) Evidence of Cytoplasmic Reorganization. — Not only is there evidence of change in the cytoplasmic makeup at division through the distribution and absorption of nuclear material as in Urolepius mobilis, but the entire cytoplasm shows other evidence at this period. In all eiliates there is a more or less clearly marked antero- REPRODUCTION 219 posterior differentiation, the anterior part usually bearing the mouth and the more or less specialized motile organs for the capture of food Fig. 109.— Uroleptus mobilis. Stages in the fusion of the macronuclei prior to cell division; rnicronuclei in mitosis. (After Calkins.) or the directing of food currents, while the posterior part is usually much less specialized. Should such a specialized ciliate be cut through the center as Balbiani (1888) did for the first time, the two 220 BIOLOGY OF THE PROTOZOA fragments would be different. The anterior fragment of a Stylo- nychia or Uronychia, for example, would retain the highly differen- tiated parts about the mouth while the posterior part would be ® Fig. 110. — Uroleptus mobilis. Division stages after fusion of the macronuclei. (After Calkins.) relatively undifferentiated. The finer organization or genotype, however, is represented by all of the protoplasm of the cell, and that organization has the ability under proper stimulation, of form- REPRODUCTION 221 ing all of the differentiated parts of the entire adult organism. By regeneration, therefore, such a cut individual replaces the charac- teristic structures of the posterior end by the anterior fragment and the characteristic structures of the anterior end by the posterior fragment (Fig. 113). By their usual method of transverse division the ciliates have quite a different inheritance than do flagellates which divide longitudinally. In the latter the highly differen- tiated anterior ends and the less differentiated posterior ends are equally divided so that the daughter cells have a like inheritance (p. 95). Fig. 111. — Uronychia Iransfuga with giant cirri, membranelles used in swimming, ten macronuelear segments, and single micronucleus. (After Calkins.) The processes through which the filiate cell passes during division indicate that the organism is restored to a generalized condition practically equivalent to an encysted cell. Except for the cyto- stome the entire array of complex cortical organs is withdrawn and a new set is formed from the cortical protoplasm. This significant process first described by Wallengren (1900), later by Griffin (1910) in hypotrichous ciliates, has been observed in many forms and is probably characteristic of the entire group. It is most clearly established in the Hypotrichida w T here the highly specialized and conspicuous motile organs furnish suitable material for study. According to Wallengren's description the membranelles of the adoral zone slowly decrease in length as the process of absorption 222 BIOLOGY OF THE PROTOZOA continues and at the same time minute buds of protoplasm appear at the bases of these disappearing membranelles. These buds grow pari passu with the dwindling motile organs until finally the latter are entirely absorbed and the buds have developed into functional membranelles. In the same way each cirrus is replaced by a new growing bud quite regardless of the position in anterior or posterior half. Undulating membranes are similarly withdrawn and replaced by new ones so that the young cells formed by division of the meta- morphosing parent cell receive a full set of new motile organs com- mensurate with the size of the young organisms. The phenomenon Fig. 112. — Chilodon uncinatus. New mouth and basket replacing the old ones prior to cell division. (N.B.) New mouth and basket; (O.B.) old mouth and basket before degeneration and disappearance; (P.B.) new mouth and basket for the pos- terior individual after division. (After MacDougall.) is very striking in forms with giant cirri such as the jumping types of Euplotidae— Diophrys or Uronychia. In the latter genus the great posterior cirri are the most conspicuous organs of the cell (Fig. 111). The buds which are to grow and replace them are appar- ent before there is other external evidence of the approaching division and even before the nucleus has concentrated into its divi- sion form. At the same time similar buds appear in the division zone, that which is destined to form the giant-hooked cirrus appears first and is always larger than the others which appear one after the other according to ultimate size. Owing to their minute size it has not been determined whether or not the individual cilium is REPRODUCTION 223 withdrawn in like manner and replaced by new ones. In some, at least, according to the observation of MacDougall on Chi lotion uncinatus (1925) such substitution does take place and it is quite probable that it is universal. The interesting experiments of Dembowska (1925) show that removal of a single cirrus of Stylo- Fig. 113.— Uronychia Iransfuga, merotomy and regeneration. 1, cell immediately after division, cut as indicated; 2, fragment A of 1, three days after the operation; no regeneration; 3, cell cut five hours after division; 4, fragment A of 3, three days after operation, no regeneration; 5. cell cut at beginning of division as indicated into fragments A, B, and C; A', B' , C", fragments A, B and C, twenty-four hours after the operation; fragment A regenerated into a normal but amicronucleate individual (A'); B, C divided in the original division plane forming a normal individual (<'') and a minute but normal individual (B'). (After Calkins.) nychia mytilus causes regeneration of the entire motile apparatus, but no such result follows extirpation of any body region that is free from cirri or cilia. The phenomenon is obviously analogous to the absorption and renewal of flagella in the flagellates. Whether or not there is a 224 BIOLOGY OF THE PROTOZOA similar division of the basal bodies of the cilia and grannies of the silver line system has not been fully established. Other evidence of protoplasmic reorganization at division is furnished by the history of some of the functional metaplastids of the cell. Trichocysts are apparently handed down without change Fig. 114. — Glaucoma scintillans. A, individual at beginning of division with silver line system. The beginnings of the month of the posterior daughter cell are seen on striation No. 1. B—F, successive stages in formation of the posterior mouth. (After Chatton, A. and M. Lwoff and Monod, Compt. rend. Soc. biol., 1931, courtesy of Masson et Cie.) (Fig. 35, p. 67), but there is good evidence that the more compli- cated aggregates of trichites are absorbed and replaced by new ones. This is the case for example in the Chlamydodontidae, where the complex oral baskets are replaced by new ones at each division (Enriques, Nagler, MacDougall, et ah, Fig. 112). REPRODUCTION 225 From this brief survey it is quite evident that far-reaching changes of the protoplasmic organization take place at periods of division. Both nuclei and cytoplasm are necessary but the micronucleus apparently may be lost without destroying the power of the cell to divide. Amicronucleate races of ciliates, arising possibly through defective reorganization and division after conjugation (see Moore, 1924), have been maintained in culture for many generations by division, although they are ultimately lost (see (Chapter VII). On the other hand, the power to regenerate is connected in some manner with the micronucleus. Thus young cells of Uronychia transfuga, when transected with a scalpel, will regenerate only that fragment which contains the micronucleus (Calkins, 1911, Fig. 113; Young, 1923). In old cells, however, both fragments regenerate regardless of the presence or absence of a micronucleus, a fact indicating a change in organization with advancing age (Fig. 113, 5). The fate of the motorium and of the coordinating fibrils both endoplasmic and those of the silver line system, at division is still unknown. It is a significant fact that the peristome and the peri- stomial organs appear first in the more specialized anterior half of the ciliate cell, and from this position gradually shift to the region immediately posterior to the division zone (Figs. 109, 110). The relation of the posterior mouth to the silver line system in a dividing form of Glaucoma scintillans is clearly shown by Chatton, Lwoff (A. and M.) and Monod (1931). The complicated oral membranes of this organism are formed as a result of division of the blepharo- plasts at a localized region of certain lines of the silver line system (Fig. 114). In Vorticella according to Biitschli (1888) after Fabre, the peristome and adoral zones are reversed in the daughter cells. II. UNEQUAL DIVISION (BUDDING OR GEMMATION). In reproduction by budding or gemmation, one or more minute fragments of the cell are produced by unequal division of the organism. Parent and offspring are thus distinguished, their rela- tive sizes varying in different cases. In many instances both parent and offspring continue to live after such reproduction. In many other instances the residual parental protoplasm is no longer able to carry on metabolic activities and dies. Illustrations of both types abound in all groups of the Protozoa, the buds being formed either on the periphery of the parent in so-called exogenous budding, or within the protoplasm of the parent in so-called endogenous budding. The minute cells that are formed by budding always contain a por- tion, sometimes one-half, of the nuclear structures of the parent and may develop asexually into organisms similar to the parent, or they may be differentiated as gametes requiring fertilization before development. 15 226 BIOLOGY OF THE PROTOZOA A. Exogenous Budding.— In Acanthocystis aculeata according to Schaudinn (1896) and in Wagnerella borealis according to Zuelzer (1909) the nucleus of the cell divides one or more times by simple constriction and without the formality of mitosis or participation of central granule. The minute nuclei thus formed wander to the periphery of the cell where they are pinched off in minute cells. In Acanthocystis these buds form minute amebae which after four Fig. 115. -Ephelota biitschliana, a suctorian. Budding individual with five exogen- ous buds. N, branching macronucleus. (After Calkins.) or five days of activity settle down and metamorphose into young Heliozoa (Schaudinn). The buds have no central granule, but during metamorphosis a kinetic element emerges from the nucleus and this becomes the central granule of the adult Acanthocystis (Fig. 50, p. 95). In Wagnerella borealis, according to Zuelzer, the buds which are formed in a similar manner are flagellated, but her description in other respects follows that of Schaudinn. In Infusoria, particularly in Suctoria, exogenous budding is not REPRODUCTlo.X 227 uncommon. In Ciliata it is comparatively rare and limited appar- ently to the Conotrichida and some parasitic forms. In Spirochona (ic mini para according to Hertwig a swelling appears at one side of the base of the peculiar funnel-like peristome. The nucleus divides equally, one-half passing into the swelling which, with only partial peristomial development, breaks away from the parent and then completes its peri- stomial differentiations. In Suctoria similar exogenous buds, either single or multiple, are formed from the oral extremity of the cell (Fig. 115). Such buds are dissimilar to the parent which they come to resemble only after a period of metamorphosis and development. In Sporozoa, with the exception of some Cnidosporidia, exogenous budding is lim- ited to unequal division in gamete-forming processes. Thus, in Gregarinida and in microgametocytes of Coccidiomorpha the nucleus of the cell undergoes several divis- ions, the final products arranging themselves about the periphery from which they be- come nuclei of variously formed gametes budded out from the surface (Fig. 173, p. 403). In all such cases the parent protoplasm dies after giving rise to the buds. In some ( 'nidosporidia, on the other hand, budding processes appear to be normal activities carried on during the vegetative life of the organisms. Accord- ing to Cohn (1895) large numbers of buds, each containing several nuclei, may be formed from the periphery of Myxidium lieberkilhni. The phenomenon appears to be an exaggeration of the peculiar process of division termed plasmotomy by Doflein, whereby a multinucleated cell divides spontaneously into two more or less equal parts as in Chloromyxum leydigi accord- ing to Liihe and Doflein, or into several parts, as in the Coccidian Caryotropha mesnili and Klossiella maris and termed "schizonto- cytes," or "cytomeres" by Siedlecki (1902). Terminal exogenous budding is characteristic of some parasitic ciliates and a chain of posterior reproductive bodies is formed as in Radiophrya limnodrili (Fig. 116). Fig. Radiop limnodrili, astomatous fili- ate with terminal budding. (After Cheissin, Archiv f. Protistenkunile, courtesy of G. Fischer.) 228 BIOLOCY OF THE PROTOZOA B. Endogenous Budding. — This type of unequal division is not so widely distributed amongst Protozoa as is exogenous budding and is apparently not represented at all in flagellated forms. It does occur, however, in all of the other groups. In Sarcodina endogenous budding has been described mainly in connection with the testate rhizopods. In Centropyxis aeuhata according to Schaudinn (1903) it leads to gamete formation, but in Arcella vulgaris', according to Swarczewski (1908) and Elpatiewsky (1909) it is a form of asexual reproduction. In Infusoria internal budding is characteristic of many types of Suctoria, but is apparently not represented in the Ciliata. In the simplest cases the budding area at the anterior end becomes internal by insinking of the anterior surface and constriction of the body walls on all sides, so that the reproducing area is enclosed by living ■agjnpjp Fig. 117. — Endogenous budding in Suctoria. A, B, two stages in the formation of a bud (b) and (c), of Tokophrya quadri partita; C, Acincta tuberosa with endogenous buds (e) and (d). (From Calkins after Butschli.) protoplasm which thus becomes a potential brood chamber within which the buds develop. Such buds may be single, as in Toko'phrya quadripartita (Fig. 117, A, B), or multiple as in Metacineta (Fig. 117, C), and are always provided with cilia either as girdles or otherwise. Through the activity of these cilia the buds swim freely about in the brood chamber until they finally emerge through a "birth-pore" and after a variable period as free swarmers or as parasites in other Infusoria, they develop into adult forms of Suctoria. Cilia in Suctoria are thus confined to the embryonic stages and their various arrangements on the buds of different species recall the types of ciliation in the other branch of the Infusoria. A biologically interesting phenomenon of internal budding is described by Collin (1911) in the case of Tokophrya qjchpum. Here a brood pouch is formed by the cortical protoplasm within which REPRODUCTION 229 the rest of the protoplasm becomes metamorphosed into a single bud with cilia. When mature this bud leaves the parent membrane on its old stalk and swims oft" as an embryo (Fig. 118). In Sporozoa endogenous budding is manifested in a number of different ways. In some it is apparently a method of multiplicative reproduction, in others it is associated with gamete formation or with sporulation. Asexual reproduction by internal budding is illustrated by some of the Schizogregarinida where a typical brood pouch is formed through which the internal buds escape through a birth opening as in Suctoria. The Eleutheroschizon dubosqui, accord- ing to Brasil (1906), the nucleus divides repeatedly until many are formed (Fig. 119, A-D). Each is then surrounded by a small portion of the parent protoplasm cut off from the rest of the cell. A B C Fig. 118. — Tokophrya cyclopum, the entire cell, except the membrane, is used in the formation of a single bud which develops cilia (B) and swims off, leaving the old membrane to shrivel up on its stalk (C). (After Collin.) The central portion becomes vacuolated and opens to the outside, the agamonts making their way through the opening, leaving the remnants of the parental protoplasm to degenerate. Similarly in Schizocy.stis sipunculi, Dogiel (1907) described the formation of a brood pouch becoming filled with agamonts derived by internal budding from the parent protoplasm (Fig. 119, E-G). Gametes formed by internal budding are described by Leger (1907) in con- nection with the life history of Ophryocystis mesnili. Here after two ' 'maturation" divisions of the nucleus in each of the gamonts united in pseudoconjugation, a single free cell is formed in each gamont by internal budding (Fig. 120). Each bud here is a gamete and the zygote is formed by union of the two in the parental brood chamber. 230 BIOLOGY OF THE PROTOZOA The phenomena of internal budding in the ameboid Myxosporidia of the Cnidosporidia, are still different in character and fate of the buds. Here in the endoplasm local islands of protoplasm are quite Fig. 119.— Endogenous budding in Gregarinida. A to D, Eleutheroschizon dubosqui and formation of endogenous agametes. (After Brasil.) E to G, Schizocystis sipunculi and similar formation of agametes. (After Dogiel.) separated from the surrounding protoplasm of the parent. Such islands, called pansporoblasts by Gurley (1893) or internal "cells" by Davis (1916), are specialized reproductive centers in each of REPRODUCTION 281 K L M Fig. 120. — Gamete formation and fertilization in Ophryocystis mesnili. A, two individuals attached by processes to ciliated cells of a Malpighian tubule of Tenebrio mollitor; B, union of gamonts in pseudoconj ligation; C, D, E, probable meiotie divisions of nuclei of the two gamonts; G to K, formation of two gametes and their union in fertilization; L to N, metagamic divisions resulting in eight sporozoites in the single sporoblast. (After Leger.) 232 BIOLOGY OF THE PROTOZOA which one or more sporoblasts are formed (see p. 545). In the same living parent organism internal buds in various stages of maturity may be present and in some cases the ameboid parent organism may Fig. 121. — Internal buds or "gemmules," b, of Sphaerospora dimorpha, a myxosporidian. (After Davis.) REPRODUCTION 233 ultimately become a mere cyst wall containing large numbers of encysted young. A quite different type of internal bud called a "gemmule" is formed in Sphaerospora dimorpha according to Davis (1916). These correspond to the agamont buds of the gregarines (Fig. 121). m. MULTIPLE DIVISION (SPORE FORMATION). In reproduction by multiple division the entire protoplasm breaks up simultaneously into a brood of minute young, a mere fragment with perhaps a residual nucleus, may be left unused. Although the end-product may be the same there is a difference in principle between rapidly following divisions of cells within a cyst (as in Colpoda cucullw) and the fragmentation of a cell into many minute cells. There is less difference between sporulation and multiple endogenous budding as in Schizocystis or Eleutheroschizon described above. Multiple division in many cases results in the formation of a brood of smaller cells which develop directly into organisms similar to the parent. In other cases the representatives of the brood are differentiated as gametes, and fertilization is necessary before devel- opment begins. We thus distinguish between sexual and asexual generations of spores, a distinction mainly characteristic of parasitic forms, but typical of many free-living types as well. In still other cases multiple division may follow immediately after fertilization, a phenomenon which is highly developed in the Sporozoa where the ultimate products of division — sporozoites have a renewed poten- tial of vitality. Multiple division or spore formation thus may occur either in the agamont (asexual) phase, or in the gamont and zygote phases (sexual) of the life cycle. Division, budding or sporulation in the asexual phase is called agamogony ( = schizogony) ; in the sexual phase gamogony ( = sporogony). In the great majority of Protozoa the two phases together in an alternation of generations, make up a complete life history. In Mastigophora sexual processes have in no case been safely established, multiple division when it occurs being agamogony. In animal flagellates, however, particularly the parasitic forms, a highly characteristic method of multiple division is widely dis- tributed. Here in certain phases or under conditions not yet well understood, trypanosomes, trichomonads, lophomonads and other parasitic flagellates undergo a process of asexual sporulation to which the specific term "somatella formation" has been applied. It is well described by Minchin and Thompson (1915) in the case of Trypanosoma lewisi (Fig. 122) as follows: "The parasites when taken up by the flea (Ceratophyllus fasciatus) 234 BIOLOGY OF THE PROTOZOA pass with the ingested food into the stomach (mid-gut) of the insect. In this part they multiply actively in a peculiar manner, not as yet described in the case of any other trypanosome in its invertebrate host; they penetrate into the cells of the epithelium, and in that situation they grow to a very large size, retaining their flagellum Fig. 122. — Trypanosoma lewisi. Cycle in the rat-flea Ceratophyllus fasciatus. 1, 2, blood trypanosomes entering the stomach; 3, 4, entering epithelial cells; 6-10, intracellular somatella formation; 11, 12, adult trypanosomes leaving cell; N, young trypanosomes repeating intracellular phase; C, Crithidial forms; H, haptomonads reproducing by division. (After Minchin and Thompson.) and undulating membrane, and exhibiting active metabolic changes in the form of the body, which in early stages of the growth is doubled on itself in the hinder region, thus becoming pear-shaped or like a tadpole in form, but later is more block-like or rounded. During growth the nuclei multiply, and the body when full-grown approaches a spherical form, and becomes divided up within its REPRODUCTION 235 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 (Fig. 122, 11). When this stage is reached, the flagellum, which hitherto had been performing active movements and causing the organism to rotate irregularly within the cell, u/Mdi^, i w imfm* 1 Fig. 123. — Polystomellina crispa. A zygote (A) develops into an organism with a microspherie type of shell (B) in which the nucleus divides by mitosis until many nuclei are present which form chromidia. The protoplasm fragments into reproduc- tive bodies or agametes, each having several granules of chromidia (C). Each agamete develops into an adult with a macrospheric type of shell (D, E) : when adult these fragment into hundreds of flagellated gametes (F) which fuse in fertilization and so complete the cycle. (From Lang and Schaudinn.) disappears altogether, and the metabolic movements cease; the body becomes almost perfectly spherical, and consists of the peri- plast envelope within which a number of daughter trypanosomes are wriggling very actively; the envelope becomes more and more tense, and finally bursts with explosive suddenness, setting free 236 BIOLOGY OF THE PROTOZOA the flagellates, usually about eight in number, within the host cell (Fig. 122, 12). 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." (loc. cit., p. 290). Similar multiple division phases have been described for Trypano- soma cruzi (Chagas, Hartmann), for Eutrichomastix seryentis, and Tetratrichomonas prowazeki (Kofoid and Swezy), Lophomonas blattae (Janicki) and others. In these cases, as in Trypanosoma lewisi, the number of individuals formed is usually eight. In Sarcodina there is a typical alternation of generations combined with multiple division best illustrated in the Foraminifera. Accord- ing to the independent observations of Schaudinn (1903) and Lister (1905) the zygote develops into an agamont characterized by an initial central chamber of relatively minute size (microspheric shell, Fig. 123, B). When fully grown the chromidia-laden protoplasm breaks up by multiple division into a great number of ameboid agametes (pseudopodiospores) each with a number of chromidial granules which fuse to form a nucleus. Each agamete develops into a gamont or individual of the sexual phase, characterized by a large initial central shell-chamber (macrospheric shell, Fig. 123, D, E). When these gamonts are mature, they also break up by multiple division into myriads of flagellated gametes (flagellispores, F). These are isogametes which fuse two-by-two forming zygotes, and these zygotes repeat the cycle by developing into microspheric individuals (Fig. 123, A). Similarly in Arcella vulgaris there is an alternation of generations which is even more complicated than that of the Foraminifera according to the descriptions of Swarczewsky (1908) and Elpatiewsky (1909). A zygote (amebula) develops into a typical adult Arcella agamont. This reproduces by agam- ogony in no less than four ways if these observers are correct. A first method is by exogenous budding whereby agametes (amebulae) are liberated to develop again into agamont s. Another method is by multiple endogenous budding whereby many agametes are formed each of which develops into an agamont. A third method involves the desertion of the parent shell and of the primary nuclei by the bulk of the protoplasm and secondary nuclei formed by chromidia, and breaking up of this mass into agametes which likewise develop into agamonts. Ultimately these agametes develop into gamonts which become either macrogametocytes or microgame- tocytes, or gamonts which conjugate as do the ciliates with an interchange of chromidia (chromidiogamy) . The macrogametocytes by multiple division give rise to macrogametes, and microgameto- cytes to microgametes. A macrogamete is fertilized by a micro- gamete, and the resulting zygote repeats the cycle. REPRODUCTION 237 Multiple division is safely established for a number of Radiolaria although it is not yet determined whether the products are agametes or gametes. In many cases the flagellated swarmers which are thus formed by one individual are large, while those formed from another individual are smaller. This has led to the view that the swarmers are anisogametes, but actual fertilization has not been safely established. They are formed from the materials of the cen- tral capsular protoplasm which, at first uninucleate, becomes multi- nucleate by repeated divisions of the nucleus. Comparatively little cytological work has been done on these forms which offer a promising field for further research. According to Brandt (1885) the nuclear material is distributed about the endoplasm in the form of many clumps of chromatin which later become vesicular nuclei and undergo mitotic divisions. Hertwig (1.879) describes the nucleus of Acanthometra as composed of a large endosome and a massive peripheral zone of chromatin which metamorphoses into a great number of small nuclei. In Aulacantha scolymantha accord- ing to Borgert (1900) the great primary nucleus gives off minute chromatin vesicles until the entire substance of the original nucleus is thus distributed in the endocapsular plasm and these become minute nuclei which now divide by mitosis. Ultimately the central capsule is dissolved, the pheodium disappears and the proto- plasm breaks up into many small spheres each with several nuclei. Differences in these spheres indicate the later differences in the resulting swarmers. A somewhat similar history has been described for the giant nucleus of Thalassicola, but despite the observations of Brandt (1885), Hartmann and Hammer (1909), Huth (1913), Moroff (1910) and others, the significance of the peculiar processes is not clear. A rather unusual phenomenon is described by Haecker (1907) in Oroscena regalis. Here the huge single nucleus of the central capsule divides into two nuclei of which one remains as a functional nucleus of the organism, the other is interpreted as giving rise to gametocyte nuclei. There is also some evidence, not con- clusive indeed, that an alternation of generations occurs, somewhat as in Foraminifera. Some types give rise by multiple division to isospores, c. g., Aulacantha, which are biflagellated cells with charac- teristic crystalloid structures interpreted by Brandt as the product of an asexual generation. Other individuals of the same species give rise to broods of anisospores which are interpreted as microgametes and macrogametes representing the sexual generation. In Mycetozoa multiple division is characteristic but complicated by the typical plasmodium nature of the organisms. Such Plas- modia are formed usually by the plastogamic union of amebae arising from spores, the nuclei remaining separate and thus forming a multinucleated protoplasmic aggregate. Many of these nuclei degenerate (Kranzlin, Jahn); some become active agents in the 238 BIOLOGY OF THE PROTOZOA formation of specialized structures of the fruiting bodies (elaters, etc., Kranzlin, 1907); others divide by mitosis to form nuclei of the spores contained with the elaters in the spaces of a meshwork formed by a special protective and supporting part of the fruiting bodies called the capillitium (Fig. 184, p. 447, see also p. 44(i). Multiple division in the Sporozoa is characteristic of practically all Coccidiomorpha, particularly in agamogony. The nuclei divide repeatedly by mitosis until many are formed, after which the body plasm breaks up into as many agametes as there are nuclei. In many cases a portion of the old cells is left unused or not included in the protoplasm of the offspring. Thus in Plasmodium vivax and other malaria organisms, the pigmented granules (melanin) are left behind when the agametes separate (Fig. 124) ; in many coccidia the agametes are oriented in respect to such residual products. Multiple division is also characteristic of the developing zygotes of gregarines and hemamebidae, the eight sporozoites of gregarines and the multitude of Sporozoites of Plasmodium being formed in this manner. A B C Fig. 124. — Malaria organisms. .4, Plasmodium vivax in blood corpuscle; B, same in agamete formation with distributed melanin (m). C, Plasmodium ?nalariae, agamete formation with concentrated melanin, c, red blood corpuscle; m, melanin; n, nuclei; /), parasite; v, vacuole. (After Calkins.) In the above account of the reproductive activities of the Protozoa no attempt has been made to give an exhaustive treatment, but other examples will be given in the following chapters on classi- fication. In many cases in the above description there is evidence of reorganization of the protoplasm and evidence that may be inter- preted as supporting Child's view of de-differentiation as an offset to the accumulation of products of metabolism which hamper further metabolic activities. Some of this evidence is given in connection with the phenomena of equal division, particularly in division of the ciliated forms and the conclusions reached are in agreement with Child's. Hartmann, also, comes to a similar REPRODUCTION 239 conclusion in connection with merotomy experiments on Amoeba polypodia (1924). In the latter an individual was cut in two frag- ments; the nucleated part regenerated, but instead of permitting it to divide it was cut again when fully grown. This process was repeated until the original ameba had been cut 32 times in forty- two days and without an intervening division. The control ame- bae from the same clone divided 15 times in the same period. This experiment would appear to confirm Child's argument that amputa- tion of a part of the differentiated protoplasm would effect a partial rejuvenescence, and Hartmann interprets it in this way: "Repro- duction," he says, may rightly be interpreted as a process of reju- venation. Our continued amputations in these experiments provide a substitute for the rejuvenating effect of reproduction (1924, p. 458). His further conclusion that his results "indicate experi- mentally, a potential immortality of the protozoan individual" (p. 456) can scarcely be allowed on the basis of forty-two days' experience. A single individual of Urolcptus mobiJis has lived for more than ninety days without dividing, and similar but younger individuals have been cut as in Hartmann's experiments, to find out if ciliates would sustain Child's conclusion. The results (not pub- lished) were invariably negative, although Uroleptus is an excellent type for this kind of work and invariably undergoes rejuvenescence after conjugation and after endomixis (see Chapter VIII). With unequal division by budding and multiple division there is further evidence of reorganization with reproduction. The small cells that are budded off contain none of the differentiated cellular elements of the parent organism. The spores are likewise provided with protoplasm whose activities are unhampered by accumulated products. This is clearly evident in the asexual reproduction of Plasmodium vivax (p. 238), and is well illustrated in forms where specialized structural elements are indications of the differentiations which the old protoplasm has undergone. Thus in Mycetozoa some of the hundreds of nuclei degenerate and give rise to spiral elaters which with their spiral walls are made up of microsomes and kinetic elements (Strasburger, Kranzlin), while parts of the proto- plasm become differentiated into encrusting peridia and supporting capillitia. All of these differentiations are left behind when the spores are formed and distributed. Analogous somatic structures are also characteristic of the spore-forming stages of some types of Gregarinida and Myxosporidia. In the former the spore-contain- ing organs are either relatively simple spore cysts as in Monocystis types (Fig. 213, p. 531) or more complicated structures— sporangia — of some polycystid gregarines (e. g., Echinomera hispid a or Gre- garina cuneata). In the former the spores are dispersed by the formation of gas which bursts the cyst membranes. In the latter, finger-formed tubes are developed from the peripheral protoplasm 240 BIOLOGY OF THE PROTOZOA of the cyst. These are formed from residual "chromidia" which collect in rings about the periphery and from which the finger- formed tubes grow into the mass of developing zygotes (Fig. 125). When the cysts are mature absorption of water causes the rupture of the cyst walls, the tubes are forced out and evaginated as an inturned glove finger may be blown out. The spores then are distributed through these hollow tubes or sporoducts. In Myxosporidia still more complicated structures recalling the capillitia of Mycetozoa, are characteristic of the spore-forming stages. In Syhaeromyxa sabrazesi according to Schroder (1907) and in Myxobolus pfeifferi according to Keysselitz (1908) the internal Fig. 125. — Gregarina cuneata. A, surface view of sporocyst with ripe sporoblasts issuing from sporoducts (e). B, C, sections of sporocyst with ripening spores and developing sporoduct (0- (From Calkins after Kuschakewitsch.) bud (pansporoblast) which is destined to form the spores, contains two nuclei, one of which is smaller than the other. These nuclei increase by division until there are 14 altogether; 2 of these degen- erate without further function, and the remaining 12 are divided into two groups of 6 each, the protoplasm dividing with them to form two protoplasmic multinucleated bodies which will develop into sporoblasts (Fig. 164, p. 325). Of the 6 nuclei in each cell, 2 are "somatic" and take part in the formation of the shell or cap- sule of the sporoblast; 2 others are also "somatic" and participate in the formation of the polar capsules and threads characteristic of the Cnidosporidia; the remaining 2 nuclei persist as germinal REPRODUCTION 241 nuclei which, according to observations of several different authori- ties, later fuse into one (p. 546). In all of these cases the specialized structures accompanying spore formation are formed only at one period in the life cycle and a period which comes at the end of long-continued metabolic activ- ity. They represent therefore, a differentiated protoplasm which is not evident in the protoplasmic make up of the progeny. What is true of these visible differentiations is also probably true of analogous differentiations which are not visible, and we have reason to believe that the products of unequal division and of multiple division are not encumbered by protoplasmic conditions which hamper vitality— in other words, that they have undergone reorgan- ization. Such young forms have again the potential of vitality of the genotype and are able to go through the series of differentia- tions which are characteristic of the life of the genotype. IV. DEVELOPMENT. In Metazoa, development starts with the fertilized egg and con- sists in the progressive formation of organs and organ systems by differentiations, and grouping of differentiated cells. A strict com- parison of Protozoa with Metazoa in development would involve the history of a fertilized cell through all phases of asexual reproduction (comparable with somatic cell division) to the gamont stage. Only by a fanciful interpretation, however, can the entire progeny of a single fertilized cell of Protozoa be regarded as an individual similar to a metazoon, although there are similar phases of vitality which may be indicated in common by the terms youth, maturity and age (see Chapter VII). The protozoan "individual," however, is a single cell and as usually seen is in the agamont stage. In the majority of Protozoa little or no development is necessary, the daughter cells being almost perfect individuals when formed and similar enough to the parent to be mistaken for nothing else. Here the only pro- cesses that can be regarded as development are those which have to do with the formation of shell structures, as in Coleps hirtus, etc., and the new development of anterior parts of posterior daughter cells and posterior parts of anterior cells. It is quite different, however, with the products of multiple bud- ding or of multiple division. Here the young forms are unlike the parent, and during growth undergo changes which may properly fall under the heading of development. In some cases, for example in Foraminifera, Mycetozoa, and Sporozoa, the small fragments produced by a parent may or may not require fertilization in order to develop. The zygote of Polystomellina crispa or of Trichosphaerium sieboldi, formed by the fusion of flagellated gametes (flagellispores) develops into the asexual generation by protoplasmic growth and 16 242 BIOLOGY OF THE PROTOZOA nuclear division, but without cell division, development of the former being indicated externally by the formation of a many-chambered shell. Similarly in the Mycetozoa the zygote formed by ameboid or flagellated gametes develops into a Plasmodium by cell fusions and nuclear divisions. In the Sporozoa the zygotes, formed by union of similar gametes (isogametes) or of dissimilar gametes (anisogametes) undergo a variable number of metagamic divisions, three in the majority of Gregarinida and two or more in the Coccidiomorpha. The end- result of such metagamic divisions is the formation of two or more similar sporozoites which are entirely different from the adult indi- viduals and undergo a more or less complex development. When they are introduced into a new host the sporozoites are liberated Fig. 126. — Development of a polycystic! gregarine (schematic) . n, nucleus of host cell ; p, parasite. (After Wasielewsky.) from their capsules, or introduced naked into the blood by some intermediate host. They make their way to the definitive site of parasitism, penetrate into cells and begin their development. In the simpler gregarines only the young stages are passed in such host cells and growth is not accompanied by any marked structural differentiations. In the polycystid gregarines the parasite never becomes entirely detached from its host cell until it is fully mature and de-differentiation begun by the loss of the attaching organ (epimerite). With its growth the body becomes differentiated into an anterior chamber (protomerite) and a nucleus-holding posterior chamber (deutomerite) and in the different species these three portions of the cell become variously ornamented and specialized. The epimerite particularly becomes modified in different ways that are useful for purposes of anchorage (see p. 536). It may be a mere REPRODUCTION 243 ball of protoplasm as in Gregarina longa; a spade-shaped structure as in Pileocephalus hern'; a long knobbed proboscis either simple or provided with spines as in Stylorhynchus longicollis or Gmiorhynchus monnieri; or there may be many finger-form processes as in Echino- mera hispida or thread-like processes as in Pterocephalus giardi. In Corycella armata it becomes a single crown of hooks; in Beloides firmus hooks combined with a lone spine. While these epimerites serve primary for attachment, they also serve, in some cases at least, as food-getting organs. In Pyxinia moebiuszi the epimerite forms a long haustoria-like process which extends through the epithelial cell of the gut and into the blood lacunae of the sub- mucosa (Fig. 103, p. 201) and in Stylorhynchus longicollis a canal is said to extend from the tip of the epimerite through the proto- merite and into the deutomerite of the parasite serving for the passage of food (Leger). The buds of Suctoria have a rather complicated developmental history, especially in forms whose "embryos" are parasitic in other Protozoa (Sphaerophrya species). The buds possess cilia which are arranged in different patterns in the various species, and by which they swim actively about until they finally settle down for develop- ment. They also possess, as a rule, some longer cilia at the anterior end which have been homologized with the adoral zone of the ciliated Infusoria, and at the posterior end they possess a sucking disc by means of which the buds attach themselves to some solid object either living or lifeless, and from which a stalk is developed. With growth of the stalk the cilia are absorbed and tentacles— suctorial, piercing or seizing— are developed. In the parasitic forms the cili- ated embryos may develop tentacles while in the motile condition, but on coming in contact with a quondam host, cilia and tentacles are absorbed and as an ectoparasite the young form makes a pit in the cortex of the host. It may then reproduce by cell division in this pit until as many as 50 or more are produced, and these escape through a slit-like birth opening of the improvised brood pouch. In some types of Protozoa finally, especially in the colonial flagellated forms, the single cell undergoes a series of cleavage stages the sequence of which is similar to that of many types of eggs of Metazoa. This is particulary striking in forms like Epistylis, Zoothamnium and other colonial filiates, which, as adults, consist of more or less definite numbers of cells arranged in definite patterns. CHAPTER VII. VITALITY. A normal active protozoon is a bit of protoplasm in which the vital activities are perfectly balanced, correlated and coordinated in response to internal and external stimuli. If the physiological balance is disturbed by abnormal activity or inactivity in one or other function the result is evident in the general vitality of the organism. The organization, however, is not rigidly fixed and undergoes adaptive changes in response to the new T conditions until activities are again coordinated. The Protozoa thus agree with all protoplasm in having the power of adaptation or ability of the pro- toplasmic substances to react within limits to unusual stimuli in such a way as to maintain perfect correlation and coordination under the new conditions. An interesting case of orderly response to unusual conditions was the fusion of two conjugating individuals of Uroleptus mobilis (Calkins, 1924). Instead of separating at the end of twenty-four to twenty-six hours as in ordinary conjugation, these two individu- als remained attached for six days during which time the usual reorganization processes occurred in each. On the seventh day they fused along the entire ventral side, forming a bilaterally symmetrical individual with two oppositely placed mouths and peristomes, two contractile vacuoles and two independent sets of macro- and micro- nuclei (Fig. 127). On the eighth day this remarkable creature divided three times, giving eight double individuals all similar to the original bilaterally symmetrical one from which they came. They continued to divide at the rate of approximately one division per day on the average for a period of four hundred and five days and through three hundred and sixty-seven divisions. The interest- ing fact here is the correlation of two distinct sets of structures and functions so as to act harmoniously and synchronously as one indi- vidual, and the setting up of an entirely new organization. Had the two individuals separated as in normal conjugation their meta- bolic processes would not have been synchronous, the periods of division would have been more or less similar but not identical. In the double individuals the two sets of eight macronuclei behaved differently in different individuals. In one case each set would fuse prior to division to form a single ellipsoidal macronucleus (Fig. 128), behaving thus like tw 7 o normal individuals when ready to divide VITALITY 245 (p. 218). In the other case the sixteen macronuclei would all fuse to form one single macronucleus which would divide and form two groups of eight each (Fig. 129). In the latter case there was not Fig. 127. — Uroleptus mobilis; origin of double individual. Above, two conju- gating cells; below, the double individual which was formed by the fusion of two such conjugating individuals. (Original.) only a definite adaptation to the new conditions but a further advance toward a composite animal of a new type and with a novel organization. The synchronous activities indicate that common 246 BIOLOGY OF THE PROTOZOA responses to common stimuli were operating and that a perfect equilibrium was established throughout. Vitality, as the sum total of all the protoplasmic activities set up in response to internal and external stimuli, is variable. Varia- tions due to external conditions may be readily seen in the effects of heat and cold. Increased temperature increases oxidation leading C Fig. 128. — Uroleptus mobilis. Division of double individual; type with two divi- sion nuclei. A, stages in the fusion of the two sets of macronuclei independently; B, two division nuclei and two new peristomes; C, division of the cell, each half with two sets of nuclei. (After Calkins.) to more rapid movements including food-taking activities, more active digestion, assimilation, growth and reproduction. It involves more waste and more active pulsation of the contractile vacuole. Conversely, decreased temperature slows up the entire series of activities and vitality is reduced. In like manner any condition of the environment which tends to quicken, to weaken, or to nullify VITALITY 247 any one link in the chain of vital activities will have its effect on the general vitality. It is not improbable that internal reorganization, or disorganiza- tion, with increase or decrease of activity in all or in some part of the protoplasmic make-up may bring about similar variations in vitality. Thus changes in organization may be effected by amphi- mixis or by long-continued metabolic functioning with correspond- ;/ # i Fig. 129. — Uroleptus mobilis. Division of double individual; type with one divi- sion nucleus. D, the single nucleus formed by fusion of the two independent sets of maeronuclei ; E, first division of the single nucleus; F, reconstruction after division with a new type of macronucleus formed from the single division nucleus. (After Calkins.) ing effects upon the general vitality. The chemical and physical make-up of the protoplasm of an individual may change with con- tinued metabolic activities and lead to a change from what is termed a labile condition when actions, reactions and interactions are per- fectly balanced and at a maximum of activity, to a more stable condition when these activities become increasingly unbalanced or cease altogether. 248 BIOLOGY OF THE PROTOZOA I. ISOLATION CULTURES. The study of protozoon protoplasm by the isolation culture methods has thrown considerable light on these problems of general vitality. If a bit of such protoplasm in the form of a single indi- vidual organism, and its progeny by division, is maintained under conditions of food and temperature as constant and uniform as possible, then variations in vitality may be measured and compared in relation to phenomena in the life cycle which are suspected of playing a role in connection with the lability of that protoplasm. In order to study protoplasm in this manner it is necessary to adopt some measure of vitality which will be an expression of the sum-total of all vital activities. Since every function is a link in the chain of vital activities any one function would do were it possible to measure it accurately, but the difficulty comes with the inability to measure excretion, or nutrition or irritability in any complete and definite manner. Reproduction, however, can be readily measured and being dependent upon the general functions of metaJbolism, becomes an excellent measure of vitality in a relative and comparative sense. In one way or another the division-rate has been used^as a measure of vitality ever since Maupas, in 1888, first attacked the problem of age and natural death in Protozoa by the isolation culture method. In practically any free-living form of Protozoa if proper condi- tions of food and temperature are provided, the general vitality or sum-total of functional activity as measured by the division-rate, continues more or less uniformly for long periods. The single individuals thus watched appear to be self-sufficient and able to continue their vital activities indefinitely. The question may be raised as it has been raised repeatedly, does the protoplasm of such an individual retain this constant potential of vitality indefinitely, or like a machine, does it wear out sooner or later, and will it ulti- mately stop altogether? The problem thus worded is only a partial restatement of the old problem concerning life and death of unicellular organisms which Weismann raised more than fifty years ago. He took the ground that Protozoa do not grow old and do not die a natural death, both of which are prevented by an individual dividing into two while in full vigor. The two young ones thus formed by division leave no parental corpse but share the old protoplasm between them and they in turn grow and similarly divide, so that old age is impossible and natural death inconceivable. Weismann further maintained that these fateful phenomena— age and death are penalties which the Metazoa must pay for their privilege of specialization and dif- ferentiation into somatic and germinal protoplasm. Protozoa he compared with the germinal protoplasm of Metazoa in common VITALITY 249 with which they have the potential of an indefinitely continued existence. The experiments of Maupas (1888) to determine by isolation cul- ture experiments whether Infusoria do actually grow old were not convincing. He found, indeed, that a bit of protoplasm in the form of a single infusorian cell if isolated in a suitable culture medium would live, grow and divide. One individual cell formed by such division, if similarly isolated, would repeat the process, and from its progeny another representative bit of protoplasm would con- tinue the race. Maupas found that, ultimately, such protoplasm Fig. 1.30. — Stylonychia pustulata, senile degeneration. B, C, degenerated individuals without micronuclei. (After Maupas.) would lose its vitality and the race would die after morphological and physiological evidences of degeneration (Fig. 130). In this manner he followed the history of Stylonychia pustulata through 316 generations by division when the race died. Another species, Stylonychia mytilus, died out after 319 generations; Leucophrys patula after approximately 060 generations, etc. The single indi- vidual was isolated in culture medium under a cover-glass and kept in a moist chamber. Here it divided repeatedly during a period of from two to six days until many individuals were present (in one case 935) all descendants of the original ore. One of these was then isolated and the process repeated. From these experiments 250 BIOLOGY OF THE PROTOZOA he concluded that Infusoria die a natural death after a typical life cycle and after a definite number of generations by division. The criticism was soon advanced that adverse conditions and bacterial products were responsible for death of his organisms, or, that instead of dying from old age they were slowly killed. There certainly was some justification for this criticism for not only was the covered medium abnormal but the accumulation of bacterial and protozoan products of metabolism might well have been detri- mental, particularly if certain types of bacteria gained supremacy. Woodruff (1911), furthermore, has shown that excretion products of Paramecium are detrimental to Paramecium, and Stylonychia products to Stylonychia, and the implication is that any type, if continued for long intervals in an unchanged medium, will slowly weaken in vitality and ultimately die. Such criticisms, continued even to the present time in connection with isolation culture work, do not minimize the value of the splendid contribution of Maupas in these pioneer studies on vitality. The present day scepticism in regard to his general conclusion is based upon diverse results obtained by various experimenters with mass cultures as compared with isolation cultures, the great majority of the latter giving results which confirm Maupas. In these the criticism that an unfit environment gradually killed the organisms has been met by the use of carefully prepared culture media and by daily transfers of the experimental organisms to freshly prepared media. In this manner the undue accumulation of bacteria and their products is prevented while the organisms under observation are never present in large numbers. By use of this method of study the life cycles of many different kinds of ciliates have been established and with the exception of the results obtained by Enriques (1913, 1915, 1916), Chatton (1923) and of Woodruff (1908-1921), they all agree in demonstrating a gradually waning vitality and ultimate death of the protoplasm under observation. The method now generally employed is to start with an ex-conjugant, or individual which has just emerged from conjugation and allow it to reproduce by division three times. Four (Woodruff) or five (Calkins) of the eight resulting individuals are then isolated and continued in daily isolation cultures as "pure lines," four or five pure lines to a " series." For vitality comparisons the daily division-rates of all lines of a series are averaged for periods of five days (Woodruff) or ten days (Calkins), and when the cycle is completed the consecutive five- or ten-day division-rates may be plotted to give a graph in which the ordinates represent the average rates of division, the abscissas the consecutive periods. By this method the history of the vitality of the protoplasm under obser- vation is summarized in a graphic and effective manner (Figs. 131, 132, 133). VITALITY 251 The above method was first used in connection with the life history of Paramecium caudatum (Calkins, 1904), and many other experiments of similar nature were made on this genus by later 1W n — 1 — - ... I— ... JO r >. — ... ~ — ... — _ - «i L ... ..j r- ._, — ._ [•■ 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 l'J 20 21 222324 25 26 27 28 2<> 30 31 323334 3536 37383940 41 Fig. 131. — Composite graph of vitality of twenty-three series of Uroleptus mobilis, each having vitality of more than 85 per rent (solid line). The ordinates represent the average numbers of divisions in ten-day periods. The dotted line is the vitality graph of the double organism. (After Calkins.) observers. It turned out to be an unfavorable subject in some respects for the study of this particular problem of vitality, for in 1914 Woodruff and Erdmann announced the discovery of a periodic ao- 25 ?.o 1.5 1.0 .5 J J ^ -1 1 -i Fig. 132. — Vitality graph of Pleurotricha lanceolata. (After Baitsell.) reorganization process without conjugation or encystment in Para- mecium aurelia which is exactly comparable with one type of parthenogenesis occurring in Metazoa (see p. 316). The discovery 252 BIOLOGY OF THE PROTOZOA of this reorganization process which they called "endomixis" was the culmination of Woodruff's brilliant and long-continued study of the life history of Paramecium aurelia which he began in 1907, and which had been generally hailed as giving positive proof of the correctness of Weismann's point of view. Parthogenesis, how- ever, has the same effect upon organization and upon vitality that conjugation has, and as Woodruff and Erdmann showed that "endomixis" occurs approximately once in thirty days in Para- mecium aurelia and about once in sixty days in Paramecium cau- datum, any experiments and observations on vitality are valuable only as they lie within these limits of time. For this reason many of the conclusions of Hertwig (1889), of Joukowsky (1898), of Calkins (1903, 1904, 1913) and of Jennings (1909, 1913) drawn from 4.0 3.5 o.O 2.5 2.0 1.5 1.0 .5 -"N ' /' /V V \ V '*s« A % V '* > \ \ \ \ \ \ < i ^ k -> ■ ^ /~ / N • * \ x > \ i i i Fig. 133.— Vitality graph of Spathidium spathula. (After Woodruff and Spencer.) observations on Paramecium are of questionable value, and should be used cautiously in connection with the present problem. In other forms, however, analogous reorganization processes occur during encystment and are thus advertized in cultures whereas Paramecium does not encyst under such conditions but continues with low vitality to live and move during such periods of depression when "endomixis" is taking place. While the list of recent experimenters with the Infusoria is rather a long one, the actual number of different organisms studied is comparatively small, but different experimenters working with the same species obtained strikingly similar results. Thus Pleurotricha lanceolata has been studied by Joukowsky (1898) and by Woodruff (1906), the former following out four series, three of which died out after approximately 220, 250 and -142 generations without conjuga- • VITALITY 2o3 tion while a fourth was abandoned after 458 generations. Woodruff, using the daily isolation method, found a gradually waning vitality with ultimate death. Baitsell (1914) also carried out isolation cul- tures with this organism, obtaining a vitality curve similar to that found by Woodruff (Fig. 132). Oxytricha fallax has been similarly studied by Enriques (1905), by Woodruff (1906) and by Baitsell (1914). The first gives no detailed account of his cultures but makes the general statement that this and other organisms cultivated by him are capable of multiplying asexually ad infinitum. Woodruff, however, finds a definite curve of vitality similar to that of Pleurotricha with a waning vitality and ultimate death after 860 generations by division, and Baitsell followed the history of three cultures all showing the typical life history, one dying out in the 131st generation, a second in the 159th, a third in the 150th, while a fourth culture in test-tubes lived for a longer period but it also finally died, none of these cultures approaching the long history of Woodruff's strain. Stylonychia pustulata also has been cultivated by Enriques (1905) and by Baitsell (1912), the former giving no statistical data but maintaining that division can go on indefinitely without degeneration or conjugation if the conditions are right. The latter follows out the history in isolation cultures and finds a typical curve of vitality with waning vitality ending in death, in the longest line after 572 generations. In other organisms Woodruff (1905) found waning vitality and death in Gastrostyla steinii after 288 generations, and Gregory (1909) a simi- lar result with Tillina magna after 548 generations, and ( "alkins (1912) a similar result with Blepharisma undulans after 224 gen- erations. In all the cases cited above the organisms under investigation are bacteria feeders, and despite the daily change of medium and care in maintaining the isolation cultures the old criticism of bac- terial poisoning or deleterious effects of the medium has been repeatedly advanced. Woodruff, however, has kept Paramecium aurelia continuously living for seventeen years on the same bac- teria diet, "endomixis" occurring at stated intervals and the same observer using the same methods has followed other organisms through periods of waning vitality and death. Metalnikov (1919) similarly has continuously cultivated Paramecium caudatum with- out conjugation. It seems highly probable, therefore, that the prevention of death has little to do with the environment in these experiments but lies in the organisms themselves— with Paramecium in the phenomenon of "endomixis." More direct evidence that bacteria contamination is not respon- sible for the ultimate death in isolation cultures is afforded by similar experiments with carnivorous ciliates. With these it is possible to use bacteria-free culture media in which the food organ- 254 BIOLOGY OF THE PROTOZOA isms are introduced with the experimental individual. Again in the majority of cases the ultimate result has been the same as with bacteria eaters. Thus Actinobolina radians was followed through 448 generations in isolation cultures in sterile spring water with Halteria grandinella as food (Calkins, 1912) and Spathidium spathula through 218 generations with Colpidium colpoda as food (Moody, 1912), the organisms finally dying in both cases. Further and very complete evidence that environmental condi- tions are not responsible in any direct way for waning vitality and death is afforded by a long-continued study of the protoplasm of Uroleptus mobilis, an hypotrichous ciliate (Calkins, 1918, 1919, 1920, etc.). This rare organism found and isolated in 1917 is a bacteria eater and was cultivated on a medium consisting of flour and timothy hay boiled in spring water and allowed to stand for twenty- four hours before using. Individuals were transferred daily to such fresh medium in order to avoid an excess of bacteria. For each series of five lines the division rates were figured in ten-day unit periods which were then averaged for sixty-day periods at ten-day intervals. The vitality history of twenty-three series averaged for sixty-day periods and the history of the double Uroleptus are shown in Fig. 131. The average division-rate here for the first sixty days was 15.4 divisions per ten days from which it descended regularly in successive sixty-day periods at ten-day intervals until death. A single series by itself would be no evidence that slow killing had not occurred. But when two of the progeny of a series are allowed to conjugate with one another at any time after the first 75 genera- tions, the ex-con jugants repeat the historv of the parent series but do not die when the parent series dies. In this maimer the proto- plasm of the original Uroleptus which was isolated November 17, 1917 was still under observation twelve years later, although any single series lived from ten months to a year only. The life of the progeny overlaps that of the parent; its progeny overlaps it, etc.; the daily treatment of parents and offspring was identical through- out; both were subject to the same deleterious conditions if present but parents died and offspring lived, a history which was repeated more than 140 times with as many series during a period of twelve years. From these clear-cut experimental results with Uroleptus mobilis the fact is obvious that under these experimental conditions a fairly uniform life cycle is the rule. The 140 completed life cycles upon which this conclusion is based were all characterized by the same phenomena, viz.: (1) A high initial vitality of the ex-conjugant lasting for a limited period; (2) gradually waning vitality ending in complete exhaustion and death; (3) a period of sexual "immatur- ity" lasting from the first thirty to ninety days during which encystment occurred if appropriate external conditions were pro- VITALITY 255 vided but conjugation did not occur; (4) a period of maturity beginning after the first thirty to ninety days approximately and lasting until the ultimate depression when conjugation, under ap- propriate external conditions did occur; and (5) a period of old age indicated by morphological degeneration with accumulating physiological depression which ended in death. The many different series studied furnish ample opportunity for the comparison of vitality in different series. In some there is a greater intensity of vitality, i. e., the average division-rate is higher throughout the cycle; in others the endurance factor is greater, i. c, the individuals live for longer inter-divisional periods without division and the cycle is correspondingly lengthened (see Chapter VIII). On the basis of such consistent experimental results one is tempted to generalize and to hold that all Protozoa pass through a similar life history. The temptation is increased by the confirmation of the main results in connection with an entirely different ciliate, Spathidium spat hula, in the hands of a no less competent observer than Woodruff (Woodruff and Spencer, 1924). Spathidium is car- nivorous and feeds normally on Colpidium colpoda. Woodruff and Spencer's isolation cultures were carried on in a basic medium of standardized beef extract to which a few individuals of Colpidium were added . The individuals were transferred daily to fresh medium and new food. Many complete series were followed from ex-con- jugants, four lines to a series until the protoplasm died a natural death. A typical example is illustrated in Fig. 133, representing the division-rate averaged for five-day periods (solid line) and one offspring series. "The data presented show that in the great majority of cases the cultures died out sooner or later after a some- what gradual decline in the division-rate" Qoc. cit. p. 178). Seventy- nine series ran synchronously with their parent series for at least fifteen days; some of these were then discarded but enough were followed through to afford a justifiable basis for conclusions. Here then we have again a large number of series carried on in isolation cultures, all derived from the same ancestral single ex-conjugant, and dying out "after a somewhat gradual decline in division-rate." Woodruff, however (loc. cit.), does not grant that the decrease in vitality is due to any intrinsic ageing tendency in the protoplasm, but believes that both in Uroleptus and in Spathidium the proper milieu for continued life was not provided in the culture methods used, and implies that when a series dies in the absence of conjuga- tion or of endomixis, it is ipso facto evidence of a faulty environment. The matter is important for, if Woodruff's conclusion is correct, it brings us to an impasse in the subject under discussion. He sup- ports his argument with the citation of cases on record in which there is no evident diminution in the division-rate under the condi- 256 BIOLOGY OF THE PROTOZOA tions of culture, and in such cases he believes that natural environ- mental conditions have been supplied. He obtained some cases of greater longevity in a few series of Spathidium, and although the methods and the culture medium supplied did not differ in any way from those used in the series that showed decline and death, he concludes that somehow the conditions were more suitable, and that when suitable the ciliate has the ability or potential for an indefinitely continued existence without the necessity of conjugation (fertilization) or of an equivalent process. Chatton (1921 ) shares this scepticism : " One may even conclude," he says, "that the more the facts accumulate, especially those of an experimental nature, the more nebulous does this conception of a life cycle (in filiates) become" (loc. cit. p. 128). The "facts" thus mentioned include the exceptional results with experimental culture methods by Woodruff as above, by Baitsell, Dawson, Enriques, Mast and others, these being the most prominent, in connection with the Infusoria. It is quite possible, as M. Robertson (1929) brings out, that conditions of the milieu are such that stimuli from the environment which ordinarily call forth adaptive changes in the organization are not developed. In a similar manner Dawson (1919) found that an amicronucleate race of Oxytricha hymenostoma presents a typical cyclical curve of vitality, and death follows a gradually decreasing vitality, if the organisms are cultivated in isolation cultures. If maintained in mass cultures they were found to live for a considerable period longer than the isolated forms, and Dawson concludes that if a suitable medium is provided an indefinite life is possible without conjugation, endomixis or encystment. It is conceivable that environmental media may induce different protoplasmic reactions at different periods of the life cycle, as shown by Gregory's (1925) experiments with Uroleptus, and that proper salts in the medium at appropriate periods would enable the protoplasm to maintain its youthful labile condition. Individuals might thus be "doctored" at intervals with a resulting repression of cumulative differentiations and a corresponding maintenance of youth. This was the under- lying principle of Woodruff's cultivation of Paramecium aurelia on a variable diet, the medium being changed at intervals but in this case without difference in his results. Austin (1927) likewise, sub- jecting Uroleptus mobilis to different media throughout entire cycles, was unable to alter the usual history. It is possible that old pro- toplasm might be reorganized by increasing the permeability and with proper interaction between protoplasm and medium, restored to its original labile condition. In other groups than the ciliates, exceptions to the type of life history shown by Uroleptus are true of the few cases known. In the animal flagellates for example there is no case of indubitable VITALITY 257 proof of fertilization in the entire group. On the other hand, there have been no successful attempts to cultivate such flagellates by the isolation culture method so that we are entirely uninformed as to the relative vitality in a life cycle. It is possible that processes analogous to endomixis in ciliates take place during encystment stages but as to this we are also ignorant. With these exceptional cases, therefore, we must wait for further information. Exceptional cases are increased through Belaf's observations on Actinoyhrys sol, a heliozoon (1924). A single line of his main culture was followed through 1244 generations by division during two years and eight months. Fertilizations were obtained from time to time in mass cultures, but these were prevented in the isolation cultures, the latter showing no indication of reduced vitality with continued life (Fig. 134). Belaf also concludes that, given proper conditions, the protoplasm of Act'vnophrys has the possibility of indefinitely continued life and reproduction by division. IJ 1 — s .0 l_ L 1 z i 4 5 6 T 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 26 29 30 31 32 33 34 35 36 37 Fig. 134. — Vitality graph of Actinophrys sol. (After Belaf.) In these exceptional cases we meet indeed with di