/ m D Dl c c II II E c [0 m IB m IB IB 3SS^S^^^^S^^^SE3E 13 J Marine Biological Laboratory Library \s\ Woods Hole, Mass. m ID ID Presented by Mrs, E. B. Harvey- Sept. 1, I960 3^^^^^^^^^^^^^^^ C ' THE BIOLOi; Y OF THE PROTOZOA BY GARY N. CALKINS, Ph.D. PROFESSOR OF PROTOZOOLOGY, COLUMBIA UNIVERSITY ILLUSTRATED WITH 238 ENGRAVINGS LEA & FEBIGER PHILADELPHIA AND NEW YORK 1926 COPYRIGHT LEA & FEBIGER 1926 PRINTED IN U. S. A. PREFACE. Protozoology as a branch of the biological sciences, has meant little more than the application of biological or zoological methods to a definite but limited group of organisms, the Protozoa. The taxonomical, morphological and cytological aspects are well devel- oped, and much progress has been made on the pathological side as well as on the side of general physiology, distribution and ecology. But there is no common aim, little common background and no common point of view through which these many aspects of Pro- tozoa-study are woven together in any definite way to make a science of Protozoology. In this respect Protozoology differs from other branches of the Biological sciences. Bacteriology, for example, deals with miscellaneous minute organisms, but the science is well-knit through special technical methods and by serological aims. Genetics has for material the whole world of living things, but is more definite in its ends than almost any other branch of the biological sciences. Knowledge concerning the Protozoa has developed upon one fundamental concept, viz.: that they are organisms of one cell. This, however, has not been a unifying conception; indeed, through sophistry, even this common ground is questioned by some. As a student of the Protozoa for many years, and as a teacher, it seems to me that what is most needed in protozoology at the present time is a common point of view from which we may compare and evaluate the vast annual output of observations and experiments. For such a common viewpoint it is necessary, however, to go beyond the conception of the cell to the underlying and more fundamental principles of biology. In the present work I have brought together the conclusions founded on thirty years of research on the Protozoa and on an equal number of years of teaching protozoology at Columbia University and recently at the Marine Biological Laboratory at Woods Hole. I venture to hope that the presentation may be a step towards the unification of the various phases of Protozoa-study and a suggestion of a common point of view in protozoology. The underlying biological principle in this presentation is the irritability of protoplasm, combined with protoplasmic organization. This organization is specific for each type of living things and is IV PREFACE present in fundamental form in spores, cysts and eggs. Each such oi"ganization under appropriate stimuli undergoes differentiation through which the derived or visible organization is developed from the fundamental organization. Through irritability' of protoplasm and reactions to internal stimuli arising through metabolic activities as well as through reactions to external stimuli, the fundamental organization is progressively changed. Such changes lead to reproduction by division whereby the changed organization is restored to the fundamental t^'pe. Other changes are cumulative and lead to special modifications of organization which we recognize as meiotic, gametic and zygotic phenomena, with accompanying processes of reorganization and restoration to the fundamental organization. Reorganization thus may be accomplished by divi- sion alone (for example animal flagellates) by parthenogenesis (endomixis in ciliates) , or by fertilization phenomena. Such changes are cyclical in character and differ from other changes in funda- mental organization (variation) which may be induced by permanent change in external environment, or by changed stimuli resulting from modifications of the germ plasm. This conception is fully developed in the following pages. The various t^'pes of visible organizations which form the basis of classi- fication are described and keys are introduced to aid in the placing of the more common genera. The fundamental vital f mictions are treated as manifestations of vitality. Life and vitality are treated as independent concepts— life as organization, and vitality as the activity of the organization. The various phases of vitality —youth, maturity, and old age— are consequences of changed or changing organization through continued metabolism. Death is disintegra- tion of the organization. To many friends and colleagues who have helped in the prepara- tion of this work I wish to express my grateful appreciation. The illustrations, testify to the artistic skill of many different assistants, among whom I am particularly indebted to INIiss Mabel L. Hedge (frontispiece and others), Mr. B. Manson Valentine, Mrs. Martha Clark Bennett and Miss R. Bowling. To the publishers, finally, I want to express my thanks and appreciation for their patience and good nature in waiting for a work long overdue, and for their coopera- tion and interest in the making of the book. Gary N. Calkins. Columbia University, 1926. /l^S^ ,v CONTENTS. ^^ The following table is a synopsis of the subjects treated and the order of treatment; for more detail subjects, genera, and authors cited, see the general index and bibliographj- at the end of the book. CHAPTER I. Introduction. Distribution of Protozoa 23 General Organization of the Protozoan Body 27 A. Form-relations of Protozoa 29 1. Protoplasmic Consistency 29 2. Membranes, Shells and Skeletons as Form-determining Factors 31 3. Mode of Life 33 4. Mode of Reproduction and Form 35 5. Inheritance 38 B. Protoplasmic Structure 39 C. Plastids of the Protozoa 46 1. Chromatin 46 2. Chromidia 48 3. Volutin Grains 49 4. Chondriosomes 49 5. Chromoplastids and Pj-renoids 50 D. Metaplastids of the Protozoa 50 Special Bibliography 55 CHAPTER II. Nuclei ant) Kinetic Elements. 1. The Nuclei of Protozoa 56 (a) Chromatin 58 (b) Linin 66 (c) Membrane 67 (d) Plastin 67 (e) Nuclear Sap or Enchylema 68 2. Multiple and Dimorphic Nuclei 68 3. Kinetic Elements 74 (a) Intranuclear Kinetic Elements (Endobasal Bodies) .... 75 1. Large Homogeneous Endobasal Bodies 76 2. Endobasal Bodies with Centrioles 76 3. Nuclei with Pole Plates and without Endobasal Bodies . 81 (b) Extranuclear (Cytoplasmic) Kinetic Elements 83 1. Blepharoplast, Basal Body and Centriole 84 2. Parabasal Body and Blepharoplast 92 3. Other Cj-toplasmic Kinetic Elements 101 4. Nuclear Division and the Problem of Chromosomes 112 (a) Cliromatin and Chromosomes 114 Special Bibliography 125 z/f^y VI CONTENTS CHAPTER III. Structural Differentiations. 1. Differentiations of the Cortex 127 (a) Cortical Differentiations for Support and Protection .... 128 (b) Motile Organoids 132 1. FlageUa 134 2. Pseudopodia 140 3. CiUa 144 4. Composite Motile Organs 147 (c) Other Organoids Adapted for Food-getting 153 (d) Oral and Anal Cortical Modifications 155 (e) Contractile Vacuoles 161 Special Bibliography 163 CHAPTER IV. General Physiology. Life, Organization, and Vitality 164 Functional Activities of the Individual 167 A. Excretion of ^Metabolic Waste 168 B. Irritability 171 C. Nutrition 175 1. Food-getting 176 2. Products of Assimilation 201 Special Bibliography 201 CHAPTER V. Reproduction. General Reproduction; All Reproduction Cell Division 203 I. Equal Division and Evidence of Reorganization 208 A. Division and Reorganization in Mastigophora .... 209 B. Division and Reorganization in the Sarcodina .... 213 C. Division and Reorganization in Infusoria 217 (a) Evidence of Nuclear Reorganization 218 (b) Evidence of Cytoplasmic Reorganization .... '223 II. Unequal Division (Budding or Gemmation) 227 A. Exogenous Budding 227 B. Endogenous Budding 231 III. Multiple Division (Spore-formation) 236 IV. Development 245 Special Bibliography 247 CHAPTER VI. Special Morphology and Taxonomy of the Mastigophora. Classification of the Phylum Protozoa 248 Sub-phylum Mastigophora, Diesing 250 Class I. Phytomastigoda, Dofiein 253 Amoeboid and Metabolic Types 254 Flagella 254 CONTENTS Vll Sub-phylum Mastigophora, Diesing — Class I. Phytomastigoda, Doflein— Chi'omatophores and Stigmata 255 Trichocysts 256 Nutrition 256 Order I. Clirysomonadida 258 Sub-order 1. Euchrysomonadina 261 Sub-order 2. Rhizochrysidina 264 Sub-order 3. Chrysocapsina 264 Order II. Cryptomonadida, Stein 265 Sub-order 1. Eucryptomonadina 266 Sub-order 2. Phaeocapsina 267 Order III. Dinoflagellida, Stein 267 Sub-order 1. Diniferina 275 Sub-order 2. Adinina 278 Sub-order 3. Cystoflagellina 278 Order IV. Phytomonadida, Blochmann 279 Order V. Euglenida, St«in 283 Order VI. Chloromonadida, Klebs 285 Class II. Zoomastigoda, Doflein 285 Order I. Pantastomatida, Minchin 286 Order II. Protomastigida 288 Order III. Polymastigida, Blochmann 292 Order IN. Hj-permastigida, Grassi 295 Class III. Key to Common Genera of ^Nlastigophora .... 298 Special Bibliography 314 CHAPTER VII. Special ^Morphology and Taxon'oiit of the Sarcodina . 315 Class I. Actinopoda, Calkins 318 Sub-class I. Heliozoa, Haeckel 319 Order 1. Aplu-othoraca, Hertwig and Lesser 320 Order 2. Chlamydophora, Hertwig and Lesser, 320 Order 3. Chalarothoraca, Hertwig and Lesser 321 Order 4. Desmothoraca, Hertwig and Lesser 321 Sub-class II. Radiolaria, Haeckel 321 Class II. Rhizopoda, von Siebold 323 Sub-class I. Proteomj'xa, Lankester 324 Sub-class II. Mycetozoa, de Bary 326 Order I. Acrasida, van Tieghem 329 Order II. Phytomyxida, Schroter 330 Order III. Euplasmodida, Lister 331 Sub-class III. Foraminifera, d'Orbigny 331 Sub-class IV. Amoebsea 335 Order 1. Amcebida 337 Order 2. Testacea " . . 339 Class III. Key to Genera of Actinopoda 341 Order I. Peripylea, Hertwig 343 Sub-order 1. Spha;rellaria, Haeckel 344 Sub-order 2. Polycyttaria, Haeckel 344 Sub-order 3. Collodaria, Haeckel em. Brandt and Haecker . . 344 Order II Actipylea, Hertwig 345 Order III. Monopylea, Hertwig 347 VlU CONTENTS Class III. Key to Genei'a of Actinopoda — Order IV. Tripylea, Hertwig 348 Sub-order 1. Phseocystina, Haeckel 348 Sub-order 2. Pha^osphseria, Haeckel 348 Sub-order 3. Phipocalpia, Haeckel ... 348 Sub-order 4. Phsogromia, Haeckel 349 Sub-order 5. Phseoconchiae, Haeckel em Haecker 349 Sub-order 6. Phseodendria, Haecker 349 Key to Common Genera of Rhizopoda 349 Special Bibliography 362 CHAPTER VIII. Special Morphology and Taxonomy of the Infusoria. Class I. Ciliata, Biitschli 376 Order I. Holotrichida, Stein 376 Sub-order 1. Astomina, Biitschli 377 Sub-order 2. Gymnostomina, Biitschli 377 Sub-order 3. Trichostomina, Biitschli 382 Order II. Heterotrichida, Stein 386 Order III. Oligotrichida, Biitschli ; .... 388 Order IV. Hypotrichida, Stein 389 Order V. Peritrichida, Stein 395 Class II. Suctoria, Biitschli 398 Key to Genera of Infusoria 401 Special Bibliography 414 CHAPTER IX. Special Morphology and Taxonomy of the Sporozoa. Class I. Telosporidia, Schaudinn 421 Sub-class I. Gregarinida 422 Order 1. Eugregarinida, Doflein Emend 428 Sub-order 1. Acephalina, Koelliker 428 Sub-order 2. Cephalina, Delage 429 Order 2. Schizogregarinida, Leger (1822) 433 Sub-class II. Coccidiomorpha, Doflein 435 Order 1. Coccidia, Leuckart 436 Order 2. Hiemospoi'idia, Danilewsky em Doflein 441 Class II. Neosporidia, Schaudinn 445 Sub-class I. Cnidosporidia, Doflein 448 Order 1. Myxosporidia, Biitschli 449 Sub-order 1. Eurysporea, Kudo (1919) 453 Sub-order 2. Sphseosporea, Kudo (1919) 453 ■ Sub-order 3. Platysporea, Kudo (1919) 454 Order 2. Microsporidia, Balbiani 455 Sub-order 1. Alonocnidea, Leger and Hesse 458 Sub-order 2. Dicnidea, Leger and Hesse 459 Order 3. Actinomyxida, Stolg 459 Sub-class II. Sarcosporidia 461 Class III. Questionable Protozoa, Chlamydozoa 462 Special Bibliography : . 464 CONTEXTS IX CHAPTER X. Vitality. I. Isolation Cultures 469 II. Organization and Differentiation 482 1. Interdivi.sional Differentiations 483 2. Cyclical Differentiations 488 A. Cyclical Differentiations Peculiar to Youth 489 B. Cyclical Differentiations Peculiar to Old Age 490 C. Cyclical Differentiations Peculiar to Matiu-ity .... 494 Sununary 505 Special Bibliography 508 CHAPTER XI. Phenomena Accompanying Fertiliz.\tion. I. The Environmental Conditions of Fertilization 509 (a) Ancestry 509 (b) Environment 510 II. Internal Conditions at the Period of Fertilization 514 III. The Process of Fertilization 516 A. Meiotic Phenomena 518 (a) Conjugant Meiosis 518 (b) Gametic Meiosis (Wilson, 1925) 530 (c) Zygotic Meiosis (Wilson) 531 B. Disorganization and Reorganization 534 (a) Phenomena of Disorganization 534 (b) Metagamic Activities and Reorganization 535 I^'. Parthenogenesis , . . 539 A. Endomixis 540 B. Autogamy 545 Special Bibliogi-aphy 551 CHAPTER XII. Effects of Reorganization and the Origin of Variations in the Protozoa. I. Effects of Reorganization on Vitality 552 1. Renewal of Vitality as a Result of Conjugation 558 2. Intensity of Vitalit\^ and Extent of Renewal 559 3. Relative \'itality of Different Series and Effect of Parents' Age on ^'itality of Offspring 563 4. Rejuvenescence After Parthenogene.sis (Endomixis) ... 564 II. Heredity and Variations in Protozoa 566 A. Uniparental Inheritance 567 B. Biparental Inheritance .... 575 Special Bibliography 583 Bibliogi-aphy 585 BIOLOGY OF THE PROTOZOA. CHAPTER I. IXTRODUCTIOxN. 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 kno^\ii as the life cycle. The maze of microscopic life to which the scientific world was first introduced by Anton von Leeuwenhoek in 1675 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 animalculcB and later under the more descriptive term Infusionsthiere of Ledenmiiller (1763). The correct zoological position of the higher types of ani- mals was recognized before the middle of the nineteenth century and the group of strictly unicellular forms was first definitely out- lined by von Siebold in 1S48 under the name Protozoa, a term sub- stituted by Goldfuss (1820) for Oken's suggestive Urthiere (1805), while the old name Infusoria has been retained for one of the sub- divisions 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 Alga^, and unicellular animals or Protozoa. It is no reflection on modern science that we are unable to clearly 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 2 18 BIOLOGY OF THE PROTOZOA which have been the stepping stones in this evohition 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 accurately distinguish either of these groups from the others. Haeckel (1866) undertook to avoid the difficulty by combining 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 an^' treatise on Protozoa, some types which with equal right should be included in works on lower plants or on Bacteria. 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 many cases these colonial aggregates resemble tissues of metazoa in their structural appearance, but tissue cells are dependent upon other parts of the animal for fulfilment of their vital activities while every cell of a colonial protozoon may be self-sufficient and independent, and differentiation among them is limited, at most, to reproducti\'e and somatic cells {e. g., Volvox globator, Pleodorina illinoisensis, and their close relatives). 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 INTRODUCTION 19 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" (1S93) 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 conceptions of a cell. In perhaps the majority of the Protozoa the fundamental vital activities are performed, as in the simpler i\.moebae or simple flagellates, by the protoplasm as a whole and without other visible specializations than nucleus and cell body. In other forms, 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). Bars of denser chitinous substance form an internal skeleton; special retractile fibers draw in a protrusible proboscis; similar fibers closing a dorsal and a \'entral 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 ^io^ i^ch in length. Analogous, if not so com- 20 BIOLOGY OF THE PROTOZOA plete intracellular differentiations are present in the majority of Infusoria, while many of the flagellates, notably the Trichonymph- idse, 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 Alar..- M ic--m c. v.- - - Fig. 2. — Diplodudum ecaudatum, a parasitic ciliate in cattle. A, anal canal and defecatory vacuole; C. F., one of the two contractile vacuole!?; M, motoriuni with fiber to circumpharyngeal ring; Mac, macronucleus ; Mic, micronucleus ; S, skeletal layer. (After Sharp.) of a metazoon. Compared with an Amoeba froteu.s or other simple rhizopod such complex organisms are highly specialized and show the extent to which intracellular differentiation may be carried. As Gurwitsch, Ilartmann, Dobell, and others have pointed out, the applicati(ni (jf the term cell which designates a structural unit with INTRODUCTION 21 Fig. 3. — Types of Protozoan colonies. A, B, front and side views of Platydorina caudata; C, Gonium pectorale. (A, B., from Doflein after Kofoid.) 22 BIOLOGY OF THE PROTOZOA specific physiological activity in Metazoa seems to be inappropriate, and as Whitman argued, inadequate. Cell aggregates or colonies are likewise highly variable in their functional specialization. While many of them consist of fortuitous groups of cells with dimensions A'arying with the number of indi- viduals joined together (e. g., Ophrydium versatile, Dinobryon sertularia, etc.), others are definite in form, number of cells, and in arrangement (e. g., Platydorina caudata, Kof.). Here the colony as such has a distinct individuality and in some cases {e. g., Gonium yectorale) undergoes a definite developmental cycle (Fig. 3). Again some colonies composed of otherwise independent cells do not react as separate individuals but the colony reacts as a coordinated whole. Thus Zudthamnium arhuscida, composed of many hundreds of indi- FiG. 4. — Types of Protozoa. A, Amoeba proteus, a rhizopod; B, Peranema tricho- phora, a flagellate; C, Stylonychia mytilis, a filiate; D, a polycystic! gregarine; E, Tokophrya quadri partita, a suctorian. {A, after Calkins, B, C, E, after Butschli; D, after Wasielewsky.) vidual 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 irri- tated (Fig. 210) . The entire aggregate contracts into a small ball, so minute that it is scarcely visible. The concerted action is due to the contraction of stalk myonemes which are continuous through- out the entire aggregate, like the coenosarc of some hydroid colonies. For such colonies of j^rotozoa, as for analogous colonies of hydroids, the expression "individual of a second order" has been applied. Between the limit's 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 subtli\'isions sim- plicity as well as extreme complexity of organization is represented, each subdivision including a series of representati\'e forms ranging from one extreme to the other. Differentiations in the different INTRODUCTION 23 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 foulest ditch, and adaptations to environments varying in char- acter from a mountain stream to the semifluid substance of an epi- thelial, 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 organisms which are invariably parasitic in mode of life and are essentially without motile organs (Fig. 4). 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 desiccation is fatal to the majority of Protozoa, although it is possible that some forms, like certain rotifers, may reab- sorb moisture and again become active. If the fluid protoplasm is surroiuided by impervious membranes evaporation is prevented 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 out of water (Fig. 5). Because of their light weight these C3'sts maA' be carried in the air and blown by the winds with dust, until surrounded 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 vegetation. In the infusions formed by soaking such dried vegetation in water various species of monads {Monas, Oicomonas, Bodo) and of 'ciliates (Colpoda, Oxyiricha, Stylonychia, Urostyla, Gastrosiyla , and Vorticella) and the rhizopod Amoeba 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 24 BIOLOGY OF THE PROTOZOA only 2| protozoon cysts per cubic millimeter of air and that these are limited 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 substances 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 In' land animals thus helping to maintain a common type of proto- £> Fig. 5. — Types of Protozoan cysts. A, oi Ochromonas s^p; B, oi Hydrurus foetidus; C, of Podophrya fixa; D, of Euglypha alveulata; E, of Chromuiina pascheri; F, of Vahl- kampfia Umax. {A, B, E, after Kiihn; C, D, F., after Calkins.) zoan fauna in pools and casual waters. Some forms to which Lauterborn (1901) has applied the term "sapropelic fauna," appear to be able to live without free oxygen. Thus Frontonia leucas, Prorodo7i oinwi, Spirosfomum amhigimm, Pchrmyxa pahfstris, P. hinucleata, etc., which usually live in relatively 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 INTRODUCTION 25 (Dactylochlamys yisciformis, Lauterb., Saprodinium dentatum, Laiiterb., Discomorpha pectinata, Levand., Pelodinium reniforme, Lauterb.). Doflein, following the suggestion made earlier by Bunge, believes that the anaerobic parasitic forms of the digestive tract may have had their initial start towards parasitism when living as such sapropelic forms. ^ 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 inimicable 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 water of roof or pail, and in damp moss. In many cases they are active for only an hour or more until their Tvorld dries up when they may again encyst, 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 Amoeha terrlcola being a typical case, while other genera and species are discovered from time to time (Bodo, Prowazekia, Spironema, Oicomonas, Cerco- monas, Ndgleria punctata and many others. While excessi\'e heat kills them, excessive cold does little harm beyond retarding vital activities and the melted ice of glaciers may teem with them, and some species are not harmed by exposure to liquid air. 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. The\' 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 symbionts or parasites. The common Protozoa of our own ponds and pools are exactly the same in genera and species as those found in similar places in • The suggestive experiments and conclusions of Avery and Morgan (1924) give reason for the beUef 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 Europe, Asia, Siberia, Africa, South America and Australia; they are cosmopohtan, and the temptation to describe new species because they happen to have been found in some hitherto unexplored local- ity has no justification from the facts of geographical distribution. This is particularly applicable to the fresh water forms but does not apply equally to the deep sea t\'pes. The littoral fauna of salt water like the fresh water forms, appear to have a cosmopolitan distribution according to the observations of Gourret and Roesser (1886), of Levander and of Hamburger and Buddenbrock in Europe, and in North America where 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 Foraminifera, are not found in the polar seas and vice versa. Some strictly pelagic forms on the other hand, notably Noctiluca jiiiliaris, 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 vertical distribution as well. Certain genera and species of Radio- laria and Foraminifera are present in the surface waters but are never found at the depth of from 600 to 3000 feet, while some fam- ilies, notably the Challengeridse and Tuscaroidee, 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, are 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 Dinofiagellata are present in salt than in fresh water. Ciliates are poorly represented in the deep sea, although one famil}'— 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 relati^'ely few salt water representatives. Many forms, especially the chlorophyll-bearing flagellates, are too sensitive to live vigorously in stagnant waters but thrive in the pure water of lakes and reservoirs, a predilection on their part which frequently leads to offensive odors and tastes in natural drinking waters (Uroglenoysis amcricona, Symira uxella). The distribution of parasitic forms belonging to all groups of the Protozoa, obviously follows the distribution of their hosts and we INTRODUCTION 27 know too little on this subject to generalize; where animals are segregated the opportunities for parasitism are enhanced while some climatic conditions are more ad^•antageolls than others for the spreading of germs. Thus the blood-dwelling parasites are more common in the tropics than elsewhere, the biological conditions favorable to the intermediate transmitting hosts being largely responsible for their numbers and variety. GENERAL ORGANIZATION OF THE PROTOZOAN BODY. Although Protozoa belong unquestionably to the microscopic world their sizes va^^' within wide limits. Some are large enough to be picked up with forceps {Porospora gigautea, up to 16 mm.) and many of the larger ciliates are easily visible to the unaided eye {Bursaria tnmcatella, Spirostoinum avibiguum) while many smaller types can be seen by the trained eye as mere white specks which, in some cases, may be identified by their characteristic movements {e. g., Paramecium, Frontonia, DUeptus, AmphUeptus, Loxophyllum, etc.). At the other extreme in size are types which are barely visible even with the most powerful lenses of the microscope. From 8 to 16 such forms have ample room for existence in a red blood corpuscle (Bahesia canis), or 200 to 300 may live simulta- neously in a single infected liver or spleen cell of man (Leishmania 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 2^^77-0 of an inch. Thus Leish- mania donovani measures from 2^t to 4/z, Paramecium caudatuni upward of 200^, Bursaria truncafeUa, 1500/i, 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 caudafum may measure from 175// to 250 At when fully grown and similar variations are characteristic of all species. Environmental conditions, espe- cially 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 synonyms by new names for the abnormal forms. Thus Dileptus anser 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 Vroleptus mohilis (see Fig. 1), in its adult vegetative condition, measures about 150yu, but immediately after conjugation not only is it reduced 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 28 BIOLOGY OF THE PROTOZOA B Fig. 6. — Dileptus anser, two sister cells. A, normal individual; B, individual starved for several days. (From Calkins.) Fig. 7. — Uroleptus mohilis Engelm. Old age specimens showing degeneration of macronucleus M and loss of micronuclei. See frontispiece. (After Calkins.) INTRODUCTION 29 frequently measures less than IbiJ. (Fig. 7); and has a different appearance from the more youthful stages. A. Form-relations of Protozoa.— The more important factors which determine form in Protozoa are: (1) The density or con- sistency of the protoplasm; (2) the presence of lifeless secretions and deposits in the form of membranes, shells and skeletons; (3) the mode of life; (4) the mode of reproduction; (5) inheritance. (1) Protoplasmic Consistency.— All protoplasm contains the same fundamental chemical elements, C, H, X, O, and P which are necessary for the performance of vital activities. With these fundamental elements are associated mineral elements of one kind or another, Xa, K, Ca, Mg, Fe, etc., usually as salts of different kinds and water. The ph\'sical properties vary with the composi- tion in different cases and some types are more fluid, some more dense, than others. As a jelly-fish or medusa is obviously more fluid than the closely related hydroids or sea anemones, so it is with Protozoa. Some types are remarkably watery in their make-up while others are dense and stiff; a Nuclearia delicatula is much more fluid than Amoeba proteiis, and the latter more fluid than a Pelomyxa palustris. These dift'erences in consistency of the protoplasm have much to do with the form assumed by Protozoa, and more fluid forms, if not confined by resistant cell membranes, readily change in form according to en\ironmental conditions, or b\' virtue of forces coming from metabolic activities within. Amoeba proteus and other species of Amoeba are amorphous and are constantly changing in shape, a characteristic phenomenon to which the term amoeboid movement is applied, and the same protoplasm ma\' be spherical in form, or flattened on the substratum, or extended in ^'arious ways. Many forms, under certain pressure conditions in the surrounding medium due to evaporation or reduced volume of water, will suddenly burst and disappear lea^•ing no trace whatsoever of their previous presence. This phenomenon has been repeatedlv' mentioned by earlier observ- ers in connection with t\pes 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 ordinar\' phases of acti\it\' but when the pressure from within becomes too great for the resistance of the membrane the latter collapses, the cell disap- pearing 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 caudatum, 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 30 BIOLOGY OF THE PROTOZOA than 1000 operations on Paramecium cavdatum have been success- ful, but the percentage is greatly increased hy preliminary treatment with neutral red. Other forms of ciliates on the other hand may be cut in any plane, Uronychia transfiiga and Vroleptus mobilis for example, reacting to such operations with all the physical prop- erties 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 Fig. 8. — Euglypha alveolata {A), and Cochliopodium sp. (fi). (After Calkins.) suspended water-dwelling types like Actinophyrs sol, ActlnosphoB- 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 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 of the most gener- alized representatives of all classes of Protozoa. INTRODUCTION 31 (2) Membranes, Shells and Skeletons as Form-determining Factors.— While density or consistency oi 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, ■¥ \<\ '^'^ 1 ^Hlll V, \^ \ r- I h : ^ ,: %. ^ ^*^- » -5- ■ --1* • - " A JP ' x> yf .-■ '/fiiiiir::.-. Fig. 9. — Allogromia oviforme, foramiiiiferon with chitinous monothalamous shell and reticulose pseudopodia. (D) a recently captured diatom; (S) chitinous shell. (From Calkins after M. Schultze.) and skeletons; by specialized protoplasmic differentiations; or by foreign bodies. Thus the density of the sluggish Pclomyxa pulustris is due to the enormous number of crystals of mud and sand, shells of diatoms and peculiar refractile bodies resembling glycogen in 32 BIOLOGY OF THE PROTOZOA make-up. Membranes of living substance, as in Cochliopodiym (Fig. 8) and the majority of flagellates and ciliates; of lifeless chitin as in Allogromia oviform e (Fig. 9) 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 Clathrulina elegans and the majority of Heliozoa retain their forms by virtue of the pro- tecting shells of lifeless materials deposited on a chitinous membrane. The protoplasmic bodies of many of the fresh water shelled rhizo- pods are relatively dense like that of the naked Amoeba verrucosa and B X '■^^Jv'""//! Fig. 10.- -Pscudodifflugia sp. circular mouth opening and mosaic shell {A). B, division stage. (Original.) are more or less globular or pyriform in shape. On such a proto- plasmic basis the shells of Difflugia species, Euglypha, Cyphoderia, Centropyxis, Arcella, etc., are deposited and these, once formed, are never changed (Fig. 10). Only rarely are these shelled rhizopods flattened or discoid as in Ilyalodiscus. The typical form in many shell-bearing or skeleton forming rhizo- 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 much evidence to show that the form and size of the elements making up the skeletal or shell parts depend. INTRODUCTION 33 upon the alveolar make-up of the protoplasm, the interalveolar deposits of silica, etc., taking the form of spicules as in Heliozoa and many Radiolaria, of bars, hexagons, rings, fenestrated capsules, etc. (Fig. 11). Fig. 11. -Schematic figure illustrating the modifications of skeletons according to mechanical principles of deposition. (After Dreyer.) (8) Mode of Life.— A third factor determining form is the mode of life. As we have seen floating forms are usually homaxonic or spherical; freely moving types on the other hand are usually mon- axonic. 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 usu- ally polyaxonic or radially symmetrical, the variations in form depending upon the nature of the attaching portion. Some for example are attached by the protoplasm of the posterior end of a cylindrical body {e. g., Cotlmrnia, VaginicoUa, 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 (Dinobryon species, Episiylis, Carchesium, Zoothamnmm, etc.). In the same individual the form may change with change in mode of life, well illustrated by Dimorpha mvtans (Fig. 12), by Nagleria gntheri or Trim astiga m oeha. Methods of food-getting and the nature of the food are also potent factors in determining form. ]\Iany of the diatom- and desmid-eating ciliates, whose food lies on the bottom, are characteristically flat- tened forms with the mouth on the under, or physiological ventral, surface (holotrichous ciliates belonging to the genera ChUodon, Orthodon, OpistJwdon, Chlamydodon, Loxophylhnn, etc., and the majority of the h\'potrichous ciliates). Special food-getting, or current-directing, organs frequently modify the form as in the collared flagellates (Choanoflagellates) and in t.\^es like Folliculina ampulla, Bursaria truncatcUa, cephalont gregarines, Pleuronema, etc. Shifting of the position of the mouth in response to different 3 34 BIOLOGY OF THE PROTOZOA food requirements has undoubtedly been the cause of some form changes as Biitschli has shown. Thus the proboscis-bearing species and the asymmetrical Chilodon types may owe their characteristic forms to such a shifting of the oral region (Fig. 13). The monaxonic types, while typically ellipsoidal in form, are usually characterized by a spiral twisting of the cell body, espe- cially in the rapidly moving forms. In some cases, notably in the •:^Z. Fig. 17. -Types of shells of Foraminifera. A, B, side and ventral aspects of Cornu- spira sp.; C, and D, types of Nodosaria. (After Carpenter.) inheritance. Fantastic types such as Discomorpha pectinata, Entodinivvi caudatum, or Phryocystis caudatiis are not uncommon. In its last anal\'sis form depends upon the chemical and physical make-up of the protoplasm and its polarity which signifies a specific protoplasmic organization and interaction of different protoplasmic substances. A minute fragment of Uroleptus mohilis 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 ultimate composition of protoplasm is dift'erent in difterent forms and specific for each species, and justify the view that there are as many kinds of protoplasm as there are species of Protozoa, Metazoa or living things generally. Considera- tions of this nature inevitablv lead us into the lines of thought INTRODUCTION 39 followed by Whitman, Gurwitsch, Dobell, and many others and to question again the adequacy of the cell theory m its application to Protozoa. Cgr '*-' ■ ^