an . ms =r, eee, Rae sep te ) ie Chess, ie! oF, re ox eS eS SPISF Fa Fe, ; Bere fee 4 WE nee % Mee. he A : ae aks St) ke tA, oe > 7 3 ees a] 125) LOLI ste ve fs PEEL oe rit Ore ie SEIIIEGI SED thf hie his Pe Whee A a te INA IP AE pF AS LEDEEER ev, y RE EEE REEL IN BA PE eae MPP Athi Lee : 6 “ ML PELL EL. Ss den Apne nngyicwl AA ne A ah bao ShA, wale LPL OETA LER IIRL ALLEN wi 16 1932 CORNELL UNIVERSITY THE Flower Veterinary Library FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 This Volume is the Gift of Dr. V. A. Moore Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http:/Awww.archive.org/details/cu31924001525314 PROTOZOOLOGY BY GARY N. CALKINS, Pu.D. PROFESSOR OF PROTOZOOLOGY IN COLUMBIA UNIVERSITY, NEW YORK Illustrated with 125 Engravings and 4 Colored Plates LEA & FEBIGER NEW YORK AND PHILADELPHIA 1909 Entered according to Act of Congress, in the year 1909, by LEA & FEBIGER In the Office of the Librarian of Congress. All rights reserved hn APS 4 PREFACE. Ir is my purpose, in the present volume, to discuss some of the old and some of the new problems in biology as illustrated by the lowest forms of animal life, the protozoa, the subject matter being founded on a course of Lowell Institute lectures given in the fall and winter of 1907. Interest in these organisms, of late, has centred mainly in the practical side until, to many biologists and to most medical men, protozodlogy implies the science dealing with pathogenic protozoa. Protozodlogy has a broader scope than this, and it is one purpose of this book to check, if possible, the limiting tendency and to point out again the important part that the protozoa play in the problems of modern biology. ‘This is the more necessary because, in my opinion, in the application of biological principles which underlie the vital phenomena of free and parasitic forms alike, will be found the most valuable data for the more practical sides of protozodlogy. Here in these mere specks of animated jelly, which rarely measure more than the hundredth part of an inch, we find, in their simplest forms, the manifold processes of the living organism. Digestion and assimilation; respiration, with its dual action of oxidation and renewal; excretion and secretion; irritability and fatigue; reproduction, together with the unfathomed mystery of fertilization and inheritance, all find expres- sion in these simple animals and raise the lowest protozoén immeasur- ably above the most complex of non-living substances. With such vital processes reduced to their lowest terms in these protozoa, we should expect to find a wealth of material for the study of life phenom- ena which in the higher animals are masked under a cloak of differ- entiated structures, and the study of these more general functions should form the basis for explanations or interpretations of the more specialized adaptations which are characteristic of pathogenic forms. This more comprehensive field, as I understand it, is the scope of modern protozoélogy. The researches of Louis Pasteur, in connection with fermentation, souring of wine, and the silkworm disease, led him to many reflections and conclusions as to the nature of various contagious and hereditary diseases. Perhaps more than any other single research, his investi- gations, begun in 1865, on the cause and ‘prevention of silkworm epidemics (to which De Quatrefages had given the name of pébrine, iv PREFACE because of the characteristic black spots), led him to the belief that many hutnan ills are similarly due to minute and microscopic forms of life, and so paved the way for the later generalization which now domi- nates medicine—the germ theory of disease. Ahead of his times in recognizing the present-day axiom that epidemics are ended by pre- vention rather than by individual treatment, Pasteur patiently advised and demonstrated, in connection with the silk industry, that perfect silkworms and moths would not develop from eggs having pébrine corpuscles on them. It is of no importance that these corpuscles were not recognized by him as the spores of a protozoén, but the important results which followed their discovery, and which led to increased length of human life, and to the mitigation of human and of animal suffering throughout the civilized world, make an increasingly sub- stantial monument to the patience, courage, and virility of this man of pure science, who, by the apotheosis of scientific method, proved these unknown corpuscles to be the cause of this silkworm disease. The recently opened chapter of the protozoan diseases of man might have been earlier studied had these observations of Pasteur upon the spores of Nosema bombycis been followed up. The parasitic protozoa were known and the free-living forms had been brought into prominence in scientific circles through the controversies over the cell theory and the theory of spontaneous generation, but more than thirty years were to elapse before general acceptance of the first human disease attributable to protozoa. The other minute organisms, bacteria and yeasts, whose presence Pasteur had demonstrated in his experiments on fermentation and spontaneous generation, were not neglected. In the hands of R. Koch the means of studying bacteria were perfected, and “culture” methods were introduced which soon raised bacterial research to the dignity of an independent branch of biological science. The ease with which bacteria could be studied, thanks to these methods, and the rapidly increasing list of bacterial diseases, seemed to divert the atten- tion of specialists from the pursuit of protozoan diseases and to confine it to research on those of bacterial origin. Attempts were repeatedly made, however, to cultivate protozoa as the bacteria are cultivated, on artificial media, but until the present decade such efforts, for the most part, were fruitless. The difficulties in applying the artificial culture method to the protozoa are due, essentially, to the differences in their mode of nutrition. Some of them, indeed, are similar to the bacteria in being saprophytic or saprozoic (to use Blanchard’s expressive term), absorbing liquid or dissolved proteid matter through the body wall. Such forms lend themselves to the culture method, and the success of Novy and MacNeal and others with trypanosomes, herpetomonads, etc., in artificial liquid media follows from this nutritional characteristic. Other forms of protozoa, as, for example, the parasitic amebee, may or PREFACE = may not lend themselves to the culture method, and then only upon the condition of having other living things as food. Many observers have found that intestinal amebz, and others that feed on bacteria, will thrive on solid culture media provided the latter are seeded with bacteria, and this fact is of the greatest importance in obtaining material for study. Other amebze cannot be cultivated in this way, and it is quite probable, as Liihe maintains, that many parasitic protozoa, especially the intracellular parasites, such as the coccidia, will never be successfully cultivated. There is need, furthermore, of caution in studying protozoa under such artificial conditions, for they are extremely sensitive to variations and are readily adapted to new conditions. The reactions, both morphological and physiological, of protozoa under such conditions of study require careful control. The study of protozoa, therefore, even when it is possible to apply bacteriological methods, is fundamentally different from the study of bacteria as at present carried on. The latter, dependent upon growth conditions, colony formation, reactions to media, etc., are essentially physiological and based upon the functions of the organisms. The study of protozoa, on the other hand, is essentially morphological, or based upon the structures of the protozoan cell, and involves the changes in cell structures which an individual undergoes during various phases of vitality. Hence it becomes necessary, first of all, to know the life history of the protozoén and the fundamental modifications which its protoplasm assumes. Modern protozodlogy, therefore, has demanded as a basis for genera and species of protozoa a knowledge of the complete life cycle, and as a basis for classification not the struc- tures of the single cells, but the structures which the protoplasm may assume throughout its entire life history from fertilization to death or until the next fertilization. The present volume, finally, does not aim at being an exhaustive treatise on the protozoa; it aims, rather, to give an introduction to the study of modern protozodlogy as seen from the author’s point of view; and for numerous omissions, incomplete references, etc., he can only plead the excuse of a large subject crowded into a limited space. G. N. C. New York, 1909 CONTENTS. CHAPTER I. GENERAL ORGANIZATION OF THE PROTOZOA. AvaGeneraliMorphologys 22°. 02 2 4 2. 4 8 en b&b Ge we Ge 8 Iprotoplasmicystructire=*) 94) 02 “a a ea Ge 2 eT Membranes, Shells, and Tests . . . . . . . . . . . 22 Plastids . . . ee eae ee ee ee ee) ee eee Vacuoles and their Munetions Pech etna uel cake meter Creer ge R401 Nuclei, Chromatin, and Chromidia Me te Cate mete et ee ee ets) Kinoplam . . TaD O28 Oh eo B. Organs of Locomotion and Glesiftetion EAE See the ce ee A a 2 Pay Pseudopodia and Classification of the Sarcodinga. . . . . .) . 85 Flagella and Classification of the Mastigophora . . . . . . . 42 Cilia and Classification of the Infusoria . . . . . . . . . 49 Parasites and Classification of the Sporozoa . . . . . . . O56 CHAPTER II. PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA. Hood=taking and Digestion . . . 2. «© 2 «© 6 mw ww ew te OL EXCretiOl Meant eC ry GES 2) Se eG Sa Oo sn ne ie BD Irritability . ... Re UR sb ge Pre ees Yo ah SI aan, ohn a Growth and Reproduction, ey ere era Nira) Cometh pte EST, Division Pees ky sy Lae) ee se Gave fl B34 Fe as SS LFS (Cl Clit Ca ee ge eas, Oy yO RY gy tag oe te 20. Sporulation MN Ried CIE een re Oe ee LM Aaah Gh Wf cie' oee eos’ nO: CHAPTER III. PROTOPLASMIC AGE OF PROTOZOA. AG Ry picalplitel@ycle mele fe se he oe oe oo Oe pouthwMatunty,and Age 9 2 te ee ew le le UCU LO hres Periocdote VOUbthmwerenbeet mn Nese en ke ee A DT GhancestatpMlatiritvaeme unt Sle hs ks ee et we es ald NcqiOChLOrn dite ee eeeRE toe rn CS ey ek 4 LS SexalWitierentiatione eee. 5 0. 4 & 8 wow 8 «2 2 » » « » 126 OBL ARS orgie es ee eee a ae acai tn viii CONTENTS CHAPTER IV. CONJUGATION, MATURATION, AND FERTILIZATION. Fertilization by Autogamy Fertilization by Endogamy Fertilization by Exogamy Parthenogenesis : Maturation in Protozoa Significance of Conjugation CHAPTER V. PARASITISM. Structural Modifications and Mode of Life of Protozoan Parasites . Reproduction and the Life Cycle Endogenous Cycle ; Exogenous Cycle and its Veracca Sporulation in Gregarines Sporulation in Coccidia Sporulation in Myxosporidia . : Exogenous Life of Protozoan Parasites . Air-borne Protozoa an Transmission by Inheritance . : Transmission by Intermediate Hosts : Effects of Protozoan Parasites on Their Hosts Protozoa and the Cancer Problem CHAPTER VI. THE PATHOGENIC FLAGELLATES. The Genus Spirocheta and its Allies . Spirocheta Balbianii The So-called Flagella of Spirochetes The Spirochete Nucleus He: Division of Spirochetes Form Changes and Life Econ Are Spirochetes Protozoa or Bacteria? . CHAPTER VII. THE PATHOGENIC FLAGELLATES (CONTINUED). The Genera Herpetomonas and Crithidia Herpetomonas Donovani and Kala Azar The Genus Crithidia 139 146 150 161 164 171 175 178 181 187 189 192 192 193 195 196 198 201 204 217 220 223 225 226 228 231 233 238 241 CONTENTS ix CHAPTER VIII. THE PATHOGENIC FLAGELLATES (CONTINUED). The Genus Trypanosoma . . ow (oe eee at mcd TM Sae Se dy Ae mE a oe © The Motile Apparatus of ficypanosoren bi Be, GAP” Fon al Gay exe os Ui The Trypanosome Nuclei Pe ee ten ee eee eh tay, ee ee AOD Form Changes of Trypanosomes. . . . . .. .. . . . . 257 Reproduction of Trypanosomes . . . . . . . . . . . . . 260 Agglomeration. . Nhe Aa in Ba ae Re ee ek cae wee Pea | Invertebrate Hosts Si Tite Gyele be eae A Ge On emere oe tel The Effects of Trypanosomes upon Neriobente Hosts Be ich coe PBS oth wee PARTE CHAPTER Ix. THE PATHOGENIC HEMOSPORIDIA. HbhewGenustBabesiammierese is tS 5 6) et ie he STO Structural Characteristics . . ie whte Mccntete ete eter. Sy eet a3 Transmission by Ticks and Life Gyele s Bape a eee ee da ae 477 MhetOrganismsyof:Malaria 2. 3; 2 2 te en a ee TD CHAPTER X. THE PATHOGENIC RHIZOPODA. Entameba and the Dysentery Problem. . . . . . . . . . . 294 Rabies and the Negri Bodies in, tis, Hog Ss CN CR ape te ere SATII) Smallpox and the Guarnieri Bodies. 2. . . . . . . 802 Other Protozoan Diseases of Obscure Etiology. . . . . . . . . 811 PROTOZOOLOGY. CHAPTER I. GENERAL ORGANIZATION OF THE PROTOZOA. A PROTOZOON is a primitive animal organism usually consisting of a single cell, whose protoplasm becomes distributed among many free living cells. These reproduce their kind by division, by budding, or by spore formation, the race thus formed passing through different form changes and the protoplasm through various stages of vitality collec- tively known as the life cycle.* Fic. 1 Types of protozoa. A, Ameba proteus, a rhizopod (after Calkins); B, Peranema trichoph- orum, a flagellate (after Biitschli); C, Stylonychia mytilis, a ciliate with specialized cilia (after Biitschli); £, Tokophrya quadripartita, a suctorian (after Biitschli); D, Pyxinia, sp., a poly- cystid gregarine with primite and deutomerite (after Wasielewsky); c, contractile vacuole; e, epithelial host cell; n, nucleus; v, food vacuole. It is quite impossible within the limits of a small volume to give a detailed or even adequate account of the many sides of interest of the unicellular animals. ‘The wide range in habitat, from the purest waters of lake or sea to the foulest ditch, or the adaptation to environ- ments varying in character from a mountain stream to the semifluid substance of an epithelial nerve or muscle cell, has brought about 1 Definition by Calkins, 1906. tw 18 GENERAL ORGANIZATION OF THE PROTOZOA manifold varieties of protozoén structure. To describe all of these modifications under one or a few headings, and to attempt to formulate general laws from the different and often highly complicated life histories, is out of the question. Nevertheless, in spite of the struc- tural modifications and special adaptations to particular modes of life, it is possible to group the different kinds of protozoa in four defi- nite types, first outlined by the French microscopist Felix Dujardin in 1841. Three of these types—sarcodina, mastigophora, and infu- soria—are based upon the form of the locomotor organs, pseudopodia, flagella, and cilia respectively, while the fourth type—sporozoa— including the gregarinida, first recognized as unicellular organisms by Kolliker in 1845, are devoid of motile organs, and are invariably parasitic in mode of life (Fig. 1). A. GENERAL MORPHOLOGY. While the different kinds of protozoa are undoubtedly the simplest animals known to us, they comprise at the same time some of the most complicated forms of cells, and the protoplasmic differentiations within these cells are frequently highly developed. In some cases these modifications are so highly evolved that we have little reason to regard such cells as units of structure comparable with the tissue cells of higher animals and plants, but should look upon them as composed of still more elementary vital units, and to this extent the cell theory, when applied to them, is inadequate. The wide distribution of the protozoa and their varied modes of life lead to the greatest possible differences between them and even within the limits of the same class. No one form is characteristic of any type, but in all cases where the body is plastic and subjected to an even environmental pressure, as in floating, or in intracellular, quies- cent forms, the body is spherical (homaxonic), readily changing, how- ever, into an elongate or monaxonic form when the organism moves or is subjected to a current. In all divisions, when for any reason the surrounding medium becomes unsuitable, or in some cases for pur- poses of digestion or reproduction, the organisms secrete a thick and resistant covering of chitin, and they remain thus “encysted’’ until conditions are again suitable, and such cysts are usually spherical. The size of protozoa likewise varies within wide limits. Some of them are on the very limits of vision, and some, apparently, are invisible, even when the eyes are assisted by the highest powers of the microscope. ‘Thus, the organism causing yellow fever, and thought to be a protozo6n, is so minute that it has never been seen, although its habitat and its general history are well known. Other protozoa, on the other hand, are relatively enormous single cells, a Pelomyxa palustris GENERAL MORPHOLOGY 19 or a Bursaria truncatella, reaching the size of 2 mm. (one-twelfth of an inch), while the parasitic gregarine Porospora gigantea of the lobster’s gut attains the length of 16 mm., or two-thirds of an inch. Unlike the majority of bacteria, the size of any given species of protozoa often varies within wide limits, and this in the same environ- ment. ‘The reasons for this difference are numerous, sometimes it is due to starvation, sometimes to developmental condition, and some- times to the variations in vitality at different periods in the life history. Thus, two cells from the same culture of dileptus species may be mis- taken for different species, the difference between them being so great, Fic. 2 Dileptus, sp. Two sister cells. A, normal individual with macronucleus in form of scattered chromatin granules (chromidia); B, individual starved for several days. From photographs taken with same magnification. and due solely to the lack of food in one case (Fig. 2). This divergence in size is particularly noticeable in the parasitic forms, where many factors influence the development of the cell. Many forms of protozoa, especially the flagellated types, have acquired the habit of association into colonies, and with such associa- tion have gained the economy which comes from division of labor, so that here in the colony forms may be found the first step in the differentiation of cell aggregates and the nearest approach of protozoa to the metazoa. Such colonies have been designated according to their mode of formation, gregaloid, spheroid, arboroid, and catenoid 20 GENERAL ORGANIZATION OF THE PROTOZOA colonies. A gregaloid colony arises by the adventitious union of previously separated cells. Thus, many of the so-called “agglomera- Uroglena americana, Calkins, a spheroid colony, consisting of monads embedded in a gelatinous matrix. Fie. 4 Codosiga cymosa Say. Kent, an arboroid colony of Choanoflagellates. (After Kent.) tions” of spirochetes and trypanosomes aré gregaloid colonies brought about by some adverse condition of the environment. A spheroid GENERAL MORPHOLOGY 21 colony is a more perfect compound individual in which the cells are embedded and held together in a common gelatinous matrix (Fig. 3). An arboroid colony is one formed by continuous division of cells which remain attached at some point, such colonies often being large dendritic branched aggregates (dinobryon, epistylis, carchesium, etc., Fig. 4). A catenoid colony, finally, is formed by the union of two or more cells end to end or side by side. (a) Protoplasmic Structure.—The body of a protozoén is made up of a somewhat gelatinous, diaphanous substance, to which Dujar- din, in 1835, gave the name “‘sarcode,” but which M. Schultze, in 1863, showed to be identical with the substance “protoplasm” of higher plants and animals, and named by von Mohl in 1846. The minute structure of this protozoén protoplasm appears to be little more than a fine network, the meshes of which are sometimes minute and narrow, as though compressed, and sometimes large and open. The substance of the walls of the meshwork appears to differ noticeably from that within its spaces, the former more dense and made up of fine granules (microsomes), the latter more fluid and containing granules of con- siderable size. Microchemical reactions show that these granules differ in chemical composition, and that some are reserve food par- ticles, others reserve matters for one use or other, and that still others are waste matters. This protoplasmic make-up, which Biitschli (92) compared with a foam structure (Schaumplasma), was described by him as consisting of fine drops of a liquid alveolar substance, enclosed within the meshes of a continuous interalveolar substance, also liquid but of a different density. Each alveolus he compared with a bubble ina foam structure; the air of the bubble corresponding to the alveolar, the walls to interalveolar, substance. While the inner protoplasm of all protozoa is probably alveolar in nature, there is considerable variation in structure due to the great variations in size of the alveoli and of the granules contained within them. In some forms (e. g., in the heliozobn actinospherium) the vacuoles are so large as to give a parenchymatous appearance to the cell, but in others they are so minute as to give a uniformly dense appearance; between these two typical cases fall the remainder of the types of protozoa. The granules within the walls of the alveoli are equally variable in size; in some cases they are very minute, corre- sponding, apparently, to the fine elementary granules which Altmann (94) regarded as the basis of all protoplasm, while in other cases they are obviously of different kinds. There is reason to believe that some of these interalveolar granules are endowed with a specific function, and that some of them underlie the various motor activities of the cell (“‘kinoplasm” of Strasburger; “ergastoplasm” of Prenant). It is certain that the protoplasmic alveoli tend to condense toward the periphery of the cell, the condensation due, apparently, to the loss of 22 GENERAL ORGANIZATION OF THE PROTOZOA the more fluid alveolar substance, while the specific kinetic elements, if present, are concentrated. Such an hypothesis might very well account for the contractility of the ectoplasm of an ameba or for the various locomotor appendages of flagellated and ciliated forms (see page 29). It is on the basis of these protoplasmic modifications that the pro- tozoa are grouped into classes, orders, and finer subdivisions, and the most important of these have to do with the changes undergone by the outer protoplasm. ‘This is the part of the cell that comes in contact with the surrounding medium, and this is the part, therefore, if any, which becomes changed by such contact. Being on the outside, it is the region of the cell for food ingestion, and we find it differentiated into mouth parts and into protoplasmic modifications for the procuring and directing of food. It is also the seat of motion, and may be differentiated into a great variety of motile organs which are so char- acteristic that classification is based mainly upon them. These motile organs, all of which may be traced back to a similar primitive type, may become modified into complex organs of the cells, while the function of locomotion is frequently changed into that of food getting, or into a sensory function of touch. It is an interesting point in this connec- tion that the sensory apparatus arises in the outer or cortical plasm as a response of protoplasm to the surrounding medium, and it is signifi- cant that in all higher animals the sensory and nervous systems arise from the outermost layer of cells, the ectoderm. In many protozoa, especially among the simpler rhizopods and some of the sporozoa, there may be no distinction between the inner and the outer protoplasm. Such cases, however, are exceptional, for in the majority of protozoa a well-marked ectoplasm can be distin- guished. In most cases the difference appears to be mainly in the presence or absence of granules, their distribution depending upon the density of the plasm. No great morphological value can be placed upon this regional difference, for it appears to be only an index of the physical condition of the protoplasm. In Ameba proteus, for example, the outer layer is dense and the granules of the alveoli are forced into the more fluid endoplasm, but in pelomyxa the protoplasm appears to be everywhere the same in density and the granules penetrate to the very periphery. In some of the rhizopods, especially the shelled forms, the distribution of granules according to density is so marked that several zones can be made out. In this connection it is significant that in the artificial mixtures which Biitschli so successfully made to imitate protoplasm, a similar regional differentiation into outer and inner structures could be distinguished, a result due in this case to surface tension. (b) Membranes, Shells, and Tests.—It is possibly due to such a tendency of protoplasm to stiffen under the influence of surface tension GENERAL MORPHOLOGY 23 in water that we may turn for an explanation, first pointed out by Gruber (’81), of the outer condensation of protoplasm resulting in the numerous types of membranes and tests of the rhizopods or of the outer coverings of the protozoa in general. The simplest form of membrane is an almost invisible cuticle of extreme delicacy, and it would be difficult to say whether such coverings are due to the physical change of the protoplasm or to secretion of a covering material which gradu- ally hardens in the water (as cysts are formed). In the ordinary forms of ameba, at any rate, the pellicula is merely a hardening or condensa- Fie. 5 A, Englypha alveolata; B, Cochliopodium. tion of the outer zone, and in the different species of ameba all grades may be distinguished up to the relatively thick membranes of Ameba tentaculata or Ameba actinophora. In other forms of protozoa there is a gradual increase in density from within outward and the body of the cell is covered by a living membrane which may become complicated by the addition of muscular fibrils (myonemes), sensory or tactile organs (cirri), or various protective structures like hooks, spines, and tentacles (Figs. 5 and 6). Like many of the cells which constitute the tissues of higher animals, 24 GENERAL ORGANIZATION OF THE PROTOZOA the protozoén has the power of manufacturing by chemical processes, over and above those which are devoted to nutrition, various products which are secreted just within or outside the peripheral protoplasm, where they may form a protective armor in the shape of shells, or tests. The materials thus formed within the cell body may be chitin (as in the case of Arcella vulgaris or in any other shelled rhizopod where the shell material is always laid down upon a chitin base); cellulose (as Fic. 6 Ceratium tripos, a dinoflagellate. (After Stein.) in the dinoflagellates); calcium carbonate (as in the foraminifera) ; or silica (as in the radiolaria). The secretions may take the form of definite plates, as in dinoflagellates, of continuous deposits, or of symmetrical skeletons which are often very complex. When the deposit is regular and continuous the shell material is added to the chitin membrane, the walls growing thicker with age of the organism; but when the material is deposited at one time (dictyotic moment), GENERAL MORPHOLOGY 25 as in the radiolaria, the deposit follows the contour of the protoplasmic alveoli and gives rise to skeletons often of extreme beauty (Fig. 8). In a number of fresh-water rhizopods the bulk of the shell material is not secreted, but the test is composed of foreign particles, such as Fic. 7 Types of marine rhizopod shells (Reticulariide, Carpenter). diatom shells, sand, mud, or detritus of any kind, all fused together and to a chitinous substratum by means of a mucilaginous cement secreted by the inner protoplasm. These shells and skeletons after death of the organisms sink to the bottom of ponds, lakes, or seas, where they may form thick beds of Fic. 8 Schematic figure illustrating the modifications of skeletons according to mechanical principles of deposition. (After Dreyer.) calcium carbonate (as in globigerina ooze), or silica (as in radiolaria ooze). Such beds have been thrown up from time to time in the past by volcanic upheavals, forming more or less extensive areas of proto- zoan land in which foraminifera or radiolaria may be easily identified. 26 GENERAL ORGANIZATION OF THE PROTOZOA (c) Plastids.—In addition to the basic substances making up the fluid protoplasm there are larger or smaller granules of different kinds embedded in the alveolar or interalveolar material; these granules may be food particles ready for assimilation, waste particles waiting for excretion, metaplasmic particles like oil drops, pigment grains, and the like, or foreign particles like sand grains, calcium, silica, etc., to be used in building shells or stalks. The plastids that are formed in a great many protozoa, especially in those types which lie on the boundary line between the lower plants and the protozoa, may have a considerable economic importance. Many of them are starchy in nature, 7. e., formed products to be used Fic. 9 4 A complex polythalamous shell (schematic) of Operculina. (After Carpenter.) The shell is represented as cut in different planes to show the distribution of the canals (a’,a’’,a’”’); c, ¢, c, the outer chambers with double walls (d, d, d), one of which is shown in section (g). The chambers communicate by apertures at the inner ends of the septa (e), and by minute pores (f). The outside (6) of the shell is marked by the radial septa. as food; others are starch-forming centres or pyrenoids, which are usually embedded in plastids of large size, called chromatophores from the color they possess. ‘These colors, due to some form of chlorophyl, may be bright green like the foliage of higher plants, or red, orange, yellow, brown, or black, according to the nature of the materials which combine with the chlorophyl. When great numbers of these color- bearing protozoa are massed together the result is a brilliantly colored area; red snow, for example, being due to aggregates of hematococ- cus, the red coming from the color of the minute chromatophore in each small cell. Similarly, great patches on the sea may be colored orange by the presence of noctiluca, or red by peridinium, while GENERAL MORPHOLOGY 27 drinking waters are not infrequently made unsightly because of the red coloring matters of Euglena sanguinea, or of the yellow coloring matters of dinobryon or uroglena. In some cases the pigment is due to collections of waste materials stored up in the cell, products of proteid metabolism held in reserve for some useful purpose, or to be voided to the outside. The black pigment of metopus or of tillina is a waste product of this nature, while the yellow to brown pigment of some of the colony forms is utilized in building the stalk. The fats, oils, and other metaplasmic products, stored up in these minute cells, minute as they are in the individual, are, collectively, a great nuisance, or, in some parasitic forms, may be a menace to the life of the host. Potable waters are frequently rendered unfit to drink because of the odors and tastes due to these products of protozoan vitality. Such odors are rarely due to putrefaction of the organisms, but rather to the liberation of the minute drops of oil upon disintegra- tion of the cell bodies. As crushing a geranium leaf causes minute drops of oil to be thrown into the air, giving the fragrant perfume of the plant, so disintegration of a uroglena colony, crushed by the pressure in pumps and mains, liberates the minute oil drops stored up in the inner protoplasm, but the cod-liver oil smell which they give to the water is far from fragrant. Such water is harmless so far as the health is concerned, but very offensive to the esthetic sense. So characteristic are these metaplasmic products, that many kinds of protozoa can be recognized in drinking waters simply by the odors they impart. The oils, which in the majority of cases, like fat, are probably a reserve store of nutriment, may, in some cases, become useful for purposes of protection. An interesting case of a possible protecting function is that of noctiluca, where the particles of oily matter are rapidly oxidized upon exposure to the air, resulting in a brilliant flash of light, and giving one great source of the phosphorescence in the sea. The possibility of a protective function comes from the fact that the fatty material is thrown out of the body upon irritation, and the flash of light may scare away small enemies. Other plastids that are used for purposes of protection are tricho- cysts and trichites. These are minute structures derived from the nucleus (Mitrophanow, 1904) and arranged radially about the entire periphery, as in paramecium, frontonia, etc., or in certain regions only, as in dileptus or chilodon. When the organism is irritated the contents of the capsules are thrown out with considerable force, and the poison which they contain is strong enough to paralyze any single-celled opponent, or, possibly, as Mast (’09) suggests, they form, after their discharge, a dense protective envelope which cannot be penetrated by small enemies. Sometimes they are used as weapons * 28 GENERAL ORGANIZATION OF THE PROTOZOA of offence as well as protective organs, and the minute hunters stalk about with them in search of prey (see page 77). (d) Vacuoles.—The other formed structures of the inner proto- zoan body are the vacuoles. These for the most part are mere fluid- filled spaces, but in many cases they possess a definite and permanent form and are frequently complicated in structure. The vacuoles are either storage or contractile vacuoles. The former are minute improvised stomachs, and in them the food matters are digested. The latter are the more complex structurally, varying from simple spaces, which fill with fluid and empty to the outside in rhythmic periods, to great branching canal systems with storage reservoirs and contractile vesicles, the excretory system permeating the entire inner protoplasm with a network of vessels. (e) Nuclei, Chromatin, and Chromidia.—aAt the present time no one who has made a careful study of protozoan cells accepts Haeckel’s view (’66) that some forms of unicellular animals are without nuclei (Monera). Itis, indeed, truethat there aremany forms in which nuclei, in a morphological sense, are not permanently retained, but the essen- tial part of the morphological nucleus—the chromatin—is invariably present. Sometimes this chromatin is distributed uniformly through- out the cell (the “distributed nucleus” of tetramitus, dileptus, etc.), but usually it is concentrated about a central body (division centre) having some of the attributes of a centrosome, or it is confined within a firm nuclear membrane. Within the last four years there has developed an ever-growing tendency to recognize in protozoa two distinct types of nuclei. These are distinguished from one another in the majority of cases not by any structural characteristics, but by their functions in the cell. One type, the trophonucleus, has to do with the ordinary vegetative func- tions of metabolism. The other type, which may be designated the karyogonad, or simply the gonad nucleus, has no function in ordinary metabolism, but is the source of chromatin forming the nuclei of con- jugating gametes. In a broad sense, therefore, the karyogonad repre- sents the germ plasm of protozoa. The forms assumed by the chromatin in these two types of nuclei vary within wide limits. In many cases both are included within one common nuclear membrane, and are separated from one another only at periods of maturation in preparation for fertilization (most gregarines, coccidia, and many flagellates). In other cases the gonad nucleus becomes separated from the trophonucleus at an earlier period in the life history of the individual, and appears in the cytoplasm in the form of distributed chromatin granules (idiochromidia of many different genera, ‘“‘chromidialnetz,” etc.) or as compact and homogeneous nuclei (micronuclei of infusoria, “secondary” or gametic nuclei of sarcodina). GENERAL MORPHOLOGY 29 The trophonuclei also may be permanently distributed in the form of chromatin granules, or, under certain conditions of the environ- ment, may assume this condition (chromidia formation). ‘The former is characteristic of the vegetative nucleus of some infusoria (e. g., dileptus, Fig. 2), the latter as a result of starvation or overfeeding, or other abnormal environmental condition (e. g., “chromidia”’ formation in actinospherium, Hertwig). (For further discussion of the significance of chromidia formation, see page 115.) In addition to the chromatin elements which enter into the make-up of nuclei, there are specific materials of the cell which apparently underlie the kinetic functions of protozoa. In some cases these are aggregated into definite nucleus-like bodies to which the name kineto- nucleus (Woodcock) has been applied (e. g., in trypanosoma and other flagellates). Such organs of the cell will be considered at greater length in the following section. ({) Kinoplasm.—The question as to a specific motor or kinetic substance in the cell has been repeatedly raised in general cytology and is still unsettled. Strasburger has long maintained that the plant cell possesses such a specific kinetic substance, which he termed “kinoplasm” and which enters into the formation of mitotic figures, flagella, cilia, and the peripheral zone of protoplasm. It is, according to him, a substance which forms all of the motor organs and underlies all of the physical activities of the cell. Similarly for animal cells, Boveri (88) early pointed out that the astrospheres and other parts of the spindle figure are composed of a substance apparently quite different from the rest of the protoplasm, and suggested the term “archoplasm” for it. Subsequent observers have amplified this view and some, notably Prenant, have endeavored to show that archo- plasm, or, in a larger sense, kinoplasm, is not only specific, but a kind of “superior” protoplasm, self-perpetuating and distinct. Wilson (00), summing up the evidence for and against such a view in relation to metazoan cells, comes to the conclusion that such substances, if they exist in the cell, represent a more or less persistent but not permanent phase, or product, of cellular metabolism. (The Cell, page 323.) Prenant’s point of view is probably the most satisfactory in con- nection with the protozoan cell, for here the specific substances are more persistent than in the higher animal cells, and in most cases they assume the form of definite, active, kinetic bodies closely associated with the mechanism of nuclear division and of locomotion. To this body of the protozoan cell, whether within or without the nucleus, the non-committal term ‘‘division centre’? has been applied (Calkins, 1898). In many different kinds of protozoa this division centre remains inside the nucleus, giving rise to what Boveri has called the “centro- nucleus” type. It is almost universally found among the represen- 30 GENERAL ORGANIZATION OF THE PROTOZOA tatives of the flagellated and ciliated protozoa, and a characteristic form is found in Euglena viridis and its allies (Fig. 10). Here a definite intranuclear body is surrounded by chromatin granules, and when the cell is ready to divide, this division centre, like a centrosome, divides first and the chromatin elements are separated into two equal groups, each half following one ofthe centres. In this case, and in some of the infusoria (e. g., Paramecium aurelia [caudatum]) the division centre seems to be formed from a specific substance, and it appears tobe a permanent body in the cell, retaining its individuality from generation to generation. Fic. 10 A Mitosis in Euglena. (From Wilson after Keuten.) A, preparing for division; the nucleus contains a division centre surrounded by chromatin granules; B, formation of an intranuclear “central spindle;” C, later anaphase, and D, telophase stage. Much more enlightening, however, are the conditions in the heliozoa. Here, in many cases, there is a central granule in the geometrical centre of the cell, which was early noted by Grenacher (’69) and Schultze and called by the former the “Centralkorn.” The axial filaments of the pseudopodia centre in this granule, which divides like a centrosome prior to division of the cell, while the axial filaments radiate out on all sides like the astral fibers of a mitotic figure. Biit- schli (92) was the first to compare this body with a centrosome, and the view was quickly accepted by cytologists, while the most complete GENERAL MORPHOLOGY 3l observations regarding its history have been made by Schaudinn (96) in the case of acanthocystis and spherastrum (Fig. 11). This central granule or division centre, while thus apparently per- manent in the adult forms of heliozoa, must be regarded as a product of protoplasmic changes which have their seat in the nucleus. This is clearly shown by the formation of the central body in small cells of the above organisms that have been produced by budding. Schaudinn has shown that in the formation of these buds the nucleus divides by amitosis, after which the daughter nuclei migrate to the periphery of the cell, where they are budded off with a small amount of cytoplasm. Fie, 11 RSS F G Nuclear division and budding in Heliozoa. (After Schaudinn.) A, vegetative cell of Spherastrum with the axial filaments focussed in a central granule (centrosome); B, D, division of nucleus in Acanthocystis; EL, F, flagellated and ameboid buds of Acanthocystis; G, exit of the centrosome from the nucleus. ue In some cases as many as twenty-four buds are thus formed by the same animal, although this is an unusual number. The history of these buds is somewhat different in different cases. In the simplest ones the bud merely drops off of the parent and remains on the bottom for some days, where it moves about by ameboid motion. ‘These buds contain no portion of the original division centre, nor does a new division centre arise in them until about five days after their formation, when in each bud a new division centre makes its appearance inside the nucleus, from which it migrates to the cytoplasm, where it takes up its position in the geometrical centre of the cell and gives rise to the 32 GENERAL ORGANIZATION OF THE PROTOZOA axial filaments, and with their formation the young organism for the first time assumes the appearance of a heliozo6n (Fig. 11, F, G). These structures of the protozoa certainly justify, if any do, the use of the term kinoplasm. Not only are they connected with the activity of the cell in division, but they are also closely identified with the motile organization of the cell. In heliozoa, as already pointed out, they are the centres for the formation of the axial rays of the pseudo- podia, which vary in motile power from practically quiescent append- ages in forms like Actinophrys sol, through a slight elasticity in forms like acanthocystis to vigorously vibratile appendages in artodiscus, which cause the minute organism to dance about the field on the tips Fic. 12 Dimorpha mutans. (After Schoutedan.) Two flagella and radiating axial filaments centring in the extranuclear division centre. of its pseudopodia. The similarity between these axial filaments and flagella of the flagellated organisms is well shown in the case of Dimor- pha mutans, in which the majority of the axial filaments are similar to those of other heliozoa, but two of them remain uncovered by stream- ing protoplasm and whip about in the surrounding water like the vibratile lashes of the flagellates. One of these flagella, according to Schoutedan (’07) serves to anchor the animal, while the other provides a food current (Fig. 12). In such cases the close connection of these axiopodia with flagella is clearly shown and may well help to point out the course of evolution of heliozoa and flagellates, perhaps the former from the latter. The actual participation of such division centres in the formation GENERAL MORPHOLOGY 33 of the more active motile organs is well shown in the flagellated pro- tozoa. In the majority of cases where the morphology has been minutely studied, the flagellum has been traced either to such a basal body or to the nucleus, while in some forms, notably in trypanosoma, the materials of the vibratile or undulating membrane, of the flagellum, which forms its edge and continues beyond the cell as a free whip, and of the contractile myonemes are all derived from such a division centre called by Woodcock the ‘“‘kinetonucleus,’’ which, in, some cases at least, has some of the attributes of a morphological nucleus. In many cases this active substance of the division centre is confined to the nucleus, where it may be in the form of a definite and permanent body, as in euglena and its allies, or it may be diffused throughout the nucleus as in actinophrys and actinospherium. The substance of the axial filaments of such forms is derived from the nucleus by a nuclear secrétion, as Schaudinn has clearly shown in the case of Camptonema nutans. All of these, however, are characteristically quiet forms, and the activity of the division centre is shown only in the process of nuclear division. In Actinophrys sol a typical spindle with centrosomes and fibers is formed as in the metazoa and all from the substance of the nucleus. There seems to be unmistakable evidence, therefore, that the sub- stance of the division centre is formed within the nucleus and that a definite body, or condensation of this substance, occurs at certain periods of vitality and has a more or less continuous existence as such. This body makes its appearance in the bud of acanthocystis, during mitosis of actinophrys and during reorganization of the cell after fertilization in trypanosoma and its allies. It divides as do the nuclei, and like a centrosome has a certain individuality in the cell. In certain other types of protozoa the substance of the division centre may be permanently outside of the nucleus. This is the case in the rhizopod parameba and in the flagellate noctiluca, while in the latter there is good evidence to show that the material is diffused throughout the cell body during vegetative phases. It is not too imagi- native to think of a diffusion of this material throughout protozoan cells generally, as it may be diffused through the nucleus, and it is conceivable that the basal bodies of cilia, the substance of the con- tractile centres of flagella and myonemes are, like the basal bodies of flagella or the Centralkorn of the heliozoa, only local condensations of such kinoplasm, which, in the long run, must be traced back to the nucleus. 34 GENERAL ORGANIZATION OF THE PROTOZOA B. ORGANS OF LOCOMOTION OF PROTOZOA, AND CLASSI- FICATION. As Dujardin (’41) early pointed out, the motile organs of protozoa offer a natural basis for classification, which, with proper subdivisions, is quite adequate to satisfy all of the requirements of a natural system. Within the last year or so some confusion has arisen because ‘of the different forms an organism may assume at different periods of its life history. Herpetomonas (Leish mania) donovani, the cause of kala azar, for example, has an intracellular non-motile phase in addition to a free-living, flagellated phase, and in such a form it is conceivable that some difficulty might arise as to whether the organism should be classified as a sporozo6n or as a flagellate. Such exceptions, however, do not offer insuperable difficulties, and may, indeed, serve a useful purpose in pointing out the path of evolution which the organisms in question have undergone. They do not in any way destroy the value of the motile apparatus as a basis for classification. Dujardin outlined three of the four great divisions of the protozoa, while the fourth, the Sporozoa, was named by Leuckart in 1879. The first group of protozoa was characterized by Dujardin as “animals provided with variable processes” (pseudopodia); the second as “animals provided with one or several flagelliform filaments” (flagella) ; and the third as “ciliated animals.” Gregarinida, belonging to the fourth group, were the first protozoa to be regarded as single cells, Kolliker (45) regarding them as such. The finer subdivisions of these several groups are made chiefly according to the variations in the structure of the motile organs, the Sarcodina, for example, are here subdivided into two classes, the Rhizopoda and the Actinopoda, according as the pseudopodia are amorphous or ray-like. ‘These classes in turn are divided into sub- classes, the former into Reticulosa, Mycetozoa, Foraminifera, and Amebea, the latter into Heliozoa and Radiolaria. Some subdivisions of the protozoa deserve especial mention because the organisms included, occupy an anomalous position in the scale of living things. One such group, the Mycetozoa, is sometimes placed as a group of rhizopods, sometimes as fungi. In their simplest forms these organisms are minute cells with lobose pseudopodia, which are soft and miscible and fuse upon coming together. Such fusions result in great accumulations of protoplasm known as plasmodia, which may assume a variety of shapes and may become so highly differentiated as to resemble higher metaphytes much more than single celled protozoa. Agowier such group, the Phytoflagellida, have long been the subject of academic wrangling as to the ‘boundary line berweee animals and plants. Similarly, the Spirilloflagellata are today the ORGANS OF LOCOMOTION OF PROTOZOA 35 subjects of contention between bacteriologists and protozodlogists. Little satisfaction, however, comes from such wrangling, and there is little practical value in connection with these hypothetical boundary lines beyond setting the limits to text-book or monograph. Pseudopodia, and Classification of the Sarcodina.—In many respects pseudopodia are the simplest forms of motile organs. They are merely prolongations or outflowings of the cell protoplasm, the external expressions of internal physical forces which biologists have tried in vain to analyze. In the inner protoplasm of nearly all kinds of protozoa, the almost fluid cell contents with granules of various kinds, food more or less digested, and with waste products, are in a constant movement or cyclosis. In the more highly differentiated forms of protozoa, this flow is quite confined to the inner protoplasm, the firm cell membrane preventing an outward manifestation of the forces which cause the flow. In the shell-less Sarcodina, however, there is no firm outer covering, and the peripheral protoplasm gives way at the points of least resistance and an outward flow of protoplasmic stuff is the result, this flow ceasing with the exhaustion of the particular force which caused it, while a new point of rupture gives rise to a new pseudopodium. ‘Thus the motile organs of these low types are incon- stant, endlessly changing centres of ‘protoplasmic energy, which have defied the physicist, the chemist, and the biologist. Not all pseudopodia are of this simple type, however, and some of them have a permanent form with supporting skeletal elements. ‘The former, transitory kind, are characteristic of the ordinary rhizopods such as ameba, arcella, difflugia, etc., which are familiar to the novice as “the lowest forms of animal life,” and they appear and disappear again with an ever- fascinating, inexplicable regularity. ‘These are the so-called lobose, “lobopodia,”’ or finger-form pseudopodia. The second, more permanent kind of pseudopodia, are sometimes called axiopodia, because of the presence of a stiff axial filament, com- posed of condensed protoplasm similar to acanthin or chitin, which runs through the axis of the pseudopodium. ‘These pseudopodia, characteristic of the class Actinopoda, stand out, ray-like, from all sides of the usually spherical animal, and give a peculiar radiating appearance which led the early students of the group to call them the sun-animals, a name which Haeckel, with characteristic felicity, turned into Heliozoa. In these the protoplasmic flow leads to no change in configuration of the motile organ, but courses outward on one side of the pseudopodium and backward on another. The central axis belonging, as shown above, to the category of kino- plasmic substances, has a certain amount of elasticity, and may bend and straighten again with considerable force, and thus the pseudo- podium becomes a more or less vigorous organ of locomotion, an acanthocystis rolling over and over with a slow vibration of the elastic 36 GENERAL ORGANIZATION OF THE PROTOZOA filaments, while an artodiscus dances about the field with an energetic, but erratic movement due to the springiness of the tips of its axiopodia. Fic. 13 fr Lichnaspis giltochii, Haeck. One of the Actipylea. (After Haeckel.) The spines are arranged in accordance with the Miillerian law as follows: a, a, a, a, northern polar spines; b, b, b, b, northern tropical spines; c, c, ce, — equatorial spines; d, d, d, d, southern tropical spines; and e, e, e, southern polar spines. In some forms both flagella and these pseudopodia exist at the same time, as in dimorpha or in myriophrys, while in the former the one may change into the other. These axiopodia, therefore, are of con- ORGANS OF LOCOMOTION OF PROTOZOA 37 siderable interest from a theoretical point of view, and indicate a possible line of evolution which the protozoa may have followed in the past (Fig. 14). The Heliozoa possessing these axiopodia are not very numerous nor are there many species; they are never parasitic and are mainly confined to fresh water, only a few being found in the sea. Another group, however, closely allied to the Heliozoa, the Radiolaria, are exclusively marine. More than four thousand species of these marine forms are known, and they are provided for the most part with the same kind of pseudopodia as those of the Heliozoa, while the great majority of them possess supporting skeletons of acanthin or silica, often exquisitely designed (Fig. 13). Fie. 14 Myriophrys paradoxa, Pénard. (From Lang after Pénard.) Heliozoén with axiopodia and flagelliform cilia. Still another type of pseudopodia which may be considered inter- mediate between the lobose and the filose types is the reticulose type, so called from the side streams of protoplasm which start from the central streams and fuse or anastomose with other pseudopodia form- ing a network or reticulum of protoplasm. The calcareous shells of these forms are usually perforated, so that their pseudopodia have easy access to the surrounding medium. Such perforations gave rise to the term foramen- or window-bearing, and under the name Fora- minifera these rhizopods have been known ever since D’Orbigny gave the name in 1826. In addition to the function of locomotion, the pseudopodia of these forms become a trap for diatoms, other protozoa or larval stages of higher forms, the sticky protoplasm making escape very difficult, while the struggles of the prey stimulate an additional flow of protoplasmic secretions by which digestion takes place. 38 GENERAL ORGANIZATION OF THE PROTOZOA PHYLUM PROTOZOA.* Subphylum SARCODINA. Protozoa showing no connections with the bacteria, usually of simple structure and characterized mainly by motile organs in the form of changeable protoplasmic processes—the pseudopodia. Class I. RHIZOPODA. Sarcodina without axial filaments in the pseudopodia, which may be lobose, filose, or reticulose. Subclass 1. Proteomyxa. Minute organisms with soft, miscible pseudopodia, which anastomose upon touching; the cells unite at times to form plasmodia; frequently parasitic. Typical genera: Gymnophrys, Cienkowsky, 1876; Pontomyxa, Topsent, 1893; Vampyrella, Cienkowsky, 1876; Pseudospora, Cienkowsky, 1876; Plasmo- diophora, Woronin, 1878; Nuclearia, Cienkowsky, 1876. Subclass 2. Mycetozoa. Pseudopodia-forming single cells which fuse to form plasmodia, the latter often of great complexity. There are so many charac- teristics of the fungi in the organisms of this group that their systematic posi- tion is unsettled; botanists include them with the fungi as a primitive group under the name Myxomycetes or slime moulds. Order 1. Acrasi®. The single cells unite to form a common mass, but the cells do not fuse, hence a pseudoplasmodium is formed which is enclosed in a gelatinous mantle. Typical genera: Copromyxa, Zopf, 1885; Acrasis, Van Tieghem, 1880; Dic- tyostelium, Brefeldt, 1869. Order 2. Fitoptasmop1a. The aggregated cells are not firmly united, but remain connected for the most part by delicate threads of protoplasm. Typical genera: Labyrinthula, Cienkowsky, 1876; Chlamydomyxa, Archer, 1875. Order 3. Myxomycetes. The aggregation of the cells is here complete and often results in the formation of complex fructifications in which hygroscopic threads. play an important part in scattering the often flagellated spores. Typical genera: Fuligo, Haller, 1768; Craterium, ‘Trentepol, 1797; Stemonitis, Gleditsch, 1753; Didymium, Schrader, 1797. Subclass 3. Foraminifera. Rhizopoda with fine branching and anastomosing pseudopodia which form an irregular network around the entire body or parts of it. Shells, when present, are calcareous, provided with many pores (Per- forina) or without pores (Imperforina), and consist of one chamber (Mono- thalamous) or of many chambers (Polythalamous). Rigid diagnoses are here impossible, for the limits of the orders are ill-defined, and in some cases it is. difficult to accurately place organisms which are sometimes grouped as fora- minifera, sometimes as test-bearing amebe. The classification adopted here is that of Lister, 1903. Order 1. Gromimpa. (Fresh-water test-bearing forms removed.) The cell cover- ing is simple and for the most part without calcareous deposits; chitinous and single chambered. Typical genera: Gromia, Dujardin, 1835; Microgromia, Hertwig, 1874; Diplo- phrys, Barker, Shepheardella, Siddall, 1880; Platoum, F. E. Sch., 1877. Order 2. Asrroruizipa. Lister recognizes four families. Here the test is com- posite, large, and monothalamous; the walls are formed of chitin with firmly attached particles of sand, mud, sponge spicules, etc. 1 The classification adopted for a group of animals or plants in which life histories are but little known and relationships obscure must be of a tentative nature, and the one here sug- gested, while indicating relationships as they appear with our present knowledge, is only a snap shot, as it were, of a growing subject and makes no claim of finality. PHYLUM PROTOZOA 39 Typical genera: Astrorhiza, Sandahl, 1857; Syringammina, Brady, 1884; Pilu- lina, Carpenter, 1862; Saccammina, Sars, 1868; Rhabdammina, Sars, 1868; Haliphysema, Bowerbk., 1862; Marsipella, Norman, 1878. Order 3. Lrrvotma. Lister recognizes four families. Here the test is arenaceous, usually regular, mono- or polythalamous. Lister notes that it comprises sandy isomorphs of certain types of hyaline or porcellanous forms. Typical genera: Lituola, Lamarck, 1801; Rheophax, Montfort, 1808; Haplo- phragmium, Reuss, 1860; Hippocrepina, Parker, 1870; Polyphragma, Reuss, 1860; Cyclammina, Brady, 1884; Loftusia, Brady, 1884; Parkeria, Carpenter, 1862. Order 4. Mitioripa. Lister recognizes six families. Here the test is typically calcareous and hyaline, but may be covered with sand or detritus. Typical genera: Cornuspira, M. Sch., 1854; Spiroloculina, D’Orb., 1826; Tri- loculina D’Orb., 1826; Vertebralina, D’Orb., 1826; Articulina, D’Orb., 1826; Peneroplis, Montfort, 1810; Orbiculina, Lamarck, 1801; Orbitolites, Lamarck, 1801; Alveolina, D’Orb., 1826; Keramosphera, Brady, 1884; Nubecularia, Defrance. Order 5. TexTuLaripa. Lister recognizes three families. Here the chambers are arranged in one or two series, which may be alternate, spiral, or irregular; arenaceous and with or without a perforated calcareous basis. Typical genera: Textularia, Defrance, 1824; Valvulina, D’Orb., 1826; Virgulina, D’Orb., 1826. Order 6. CHILOSTOMELLIDA. Lister has three genera. The test is calcareous, polythalamous and finely perforated. Typical genera: Chilostomella, Reuss, 1860; Allomorphina, Reuss, 1860. Order 7. Lacenrpa. Lister recognizes four families. Here the test is similar to the last save for the monothalamous shell, which, however, may be compound by the union of chambers end to end in a straight or curved series. Canals and canalicular skeleton wanting. Typical genera: Lagena, Walker, and Boys, 1784; Nodosaria, Lam., 1801; Poly- morphina, D’Orb., 1826; Ramulina, R. Jones, 1875. Order 8. GLosicERINIDA. Not divided into families. The test is perforated and calcareous, with few chambers arranged in a spiral. Canals and canal system absent. Typical genera: Globigerina, D’Orb., 1826; Orbulina, D’Orb., 1826. Order 9. Rotatipa. Lister recognizes three families. The test is calcareous and perforated, with all of the chambers visible from one aspect, and arranged in a spiral; some of the more highly developed forms with canal system. Typical genera: Spirillina, Ehr., 1841; Discorbina, Parker and Jones, 1862; Calearina, D’Orb., 1826; Rotalia, Lamarck, 1801; Tinoporus, Carpenter, 1857; Carpenteria, Gray, 1858. Order 10. Nummuitipa. Lister recognizes three families. Here the test is calcareous, filled with tubules, and bilaterally symmetrical (except Amphis- tegina), and with canal system in the higher forms. Typical genera: Fusulina, Fischer, 1829; Polystomella, Lamarck, 1822; Oper- culina, D’Orb., 1826; Nummulites, Lamarck, 1801; Orbitoides, D’Orb., 1826 (Fig. 9, p. 26). Subclass 4. Amebea. Here are included the more common forms of rhizopods with blunt or lobose pseudopodia which do not anastomose on touching one another, a physiological character which indicates a well-marked difference in the different types of rhizopods. The protoplasmic body may bear shells or not. Order 1. Gymnamesipa. Here the body is uncovered, although there is, in many cases, a tendency of the peripheral plasm to harden into a denser, mem- brane-like zone which approaches the simpler forms of tests. 40 GENERAL ORGANIZATION OF THE PROTOZOA Typical genera: Ameba auct. Parameba, Schaudinn, 1896; Trichospherium, Schneider; Hyalodiscus, Hert. and Lesser, 1874; Chromatella, Frenzel, 1892; Pelomyxa, Greeff, 1874; Dactylosphera, Hert. and Lesser, 1874; Nucleophaga, Dangeard, 1895.1 Order 2. Testacea. The ameboid organisms here are covered by definite mem- branes or tests composed of different materials cemented to a chitinous base. The pseudopodia are protruded through the single opening of the shell and may be simply lobose or branched, but do not anastomose. Typical genera: Arcella, Ehr., 1838; Cochliopodium, Hert. and Lesser, 1874; Hyalospheria, Stein, 1857; Quadrula, F. E. Sch., 1875; Difflugia, Leclerc, 1815; Euglypha, Dujardin, 1841; Trinema, Dujardin, 1836; Campascus, Leidy, 1877. Class 2. ACTINOPODA. Sarcodina provided with fine, ray-like pseudopodia which are supported by a central axial filament corresponding to the kinetic material of flagella. Subclass 1. Heliozoa. Typically fresh-water forms of actinate protozoa in which there is no trace of a chitinous central capsule separating ectoplasm and endoplasm. Order 1. ApHRoTHoRAcA. Naked forms of heliozoa (except during encystment). Typical genera: Actinophrys, Ebr., 1830; Myxastrum, Haeckel, 1870; Actino- spherium, Stein, 1857; Actinolophus, F. E. Sch., 1874. Order 2. CaLamyDopHoRA. Heliozoa with a soft gelatinous or felted fibrous covering. Typical genera: Heterophrys, Archer, 1865; Spherastrum, Greeff, 1873. Order 8. CHaLaraTHoraca. Heliozoa with a silicious covering made up of separate or loosely connected spicules. Typical genera: Pompholyxophrys, Archer, 1869; Raphidiophrys, Archer, 1870; Pinacocystis, Hert. and Lesser, 1874; Acanthocystis, Carter, 1863; Diplo- cystis, Pénard, 1890. Order 4. DesMorHoraca. Heliozoa with a covering of one piece perforated by numerous openings. Typical genus: Clathrulina, Cienk., 1867. Subclass 2. Radiolaria. Actinopoda in which the inner protoplasm is separated from the outer by a firm chitinous “central capsule” perforated in different ways for the intercommunication of inner and outer parts. Exclusively salt- water forms, living at the surface, suspended at various depths, or near the bottom. Classification based upon Haeckel’s magnificent monograph in the Challenger reports. Division A. Porulosa. Spherical (homaxonic) organisms with spherical central capsule perforated by numerous scattered pores of minute size. Legion 1. Peripylea (Spumellaria). The central capsule is perforated by evenly scattered pores; a skeleton is usually present consisting of scattered silicious spicules, fused spicules, or a latticed network. Order 1. Cottipa (following Brandt, 1902). Solitary forms with or without skeletogenous spicules. Typical genera: Thalassicolla, Huxley, 1851; Actissa, Haeckel, 1887. Order 2. SPHEROZOEA (Brandt). Colony building forms with or without skele- togenous spicules. Typical genera: Collozoum, Haeckel, 1862; Collosphera, J. Miill, 1855. Order 3. SpHrrorpA. Skeleton present as one or several concentric spherical latticed or reticulate structures. 1In this group I would place, provisionally, the organisms of smallpox (Cytoryctes variole), of rabies (Neuroryctes hydrophobiz), and the allied organisms which Prowazek (1908) includes in his group Chlamydozoa. PHYLUM PROTOZOA 4] Typical genera: Haliomma, Ehr., 1838; Actinomma, Haeckel, 1862. Order 4. Prunoma. Haeckel recognizes seven families. With spheroidal, ellipsoidal to cylindrical skeleton, single or concentric, sometimes constricted. Typical genera: Ellipsidium, Haeck., 1887; Druppula, Haeck., 1887. Order 5. Discoma. Haeckel recognizes six families. The skeleton and central capsule are discoidal to lenticular. Typical genera: Cenodiscus, Haeck., 1887; Heliodiscus, Haeck., 1887. Order 6. Larcora. Haeckel recognizes nine families. The skeleton is ellip- soidal with asymmetrical axes, in some cases forming almost a spiral. Typical genera: Larcarium, Haeck., 1887, Pylonium, Haeck., 1881. Order 7. SPHEROPYLIDA (Dreyer). Peripylea having in addition to the distrib- uted pores one basal or a basal and an apical opening to the central capsule. Typical genus: Spheropyle, Dreyer, 1888. Legion 2. Actipylea (Acantharia). Porulose forms in which the pores are aggre- gated in definite areas; the skeleton usually consists of twenty spines of acanthin radiating from the centre of the organism in a regular order (Miil- lerian law). Branches from these spines may unite to form a latticed shell. Order 8. AcTINELLIDA. Haeckel recognizes three families. The radial spines are more numerous than twenty. Typical genus: Xiphacantha, Haeckel, 1862. Order 9. AcantHoNIDAs. Haeckel recognizes three families. The twenty spines are arranged in regular order (four equatorial, eight tropical, and eight polar), all are equal in size. Typical genus: Acanthometron, Miiller, 1855. Order 10. SpHEROPHRACTA. Haeckel recognizes three families. With twenty equal, quadrangular spines and a complete fenestrated shell. Typical genus: Dorataspis, Haeckel, 1860. Order 11. PrunopHracta. Haeckel recognizes three families. The twenty radial spines are unequal, and an ellipsoidal, lenticular, or doubly conical shell is present. Typical genus: Thoracaspis, Haeck., 1860. Division B. Osculosa. Radiolaria with monaxonic form and with the pores of the central capsule limited to an area on the base, or to one such primary basal area and two secondary, apical areas; these perforated areas of the central capsule are termed oscula. Legion 3. Monopylea (Nassellaria). The central capsule is subspherical to ovoid, consists of a single layer of chitin, and is perforated only at one pole. The skeleton is silicious. Order 12. Nassorpa. Haeckel recognizes only one family. Skeleton absent. Typical genus: Nassella, Haeck., 1887. Order 13. Piectorpa. Haeckel recognizes two families. A complete latticed shell is never formed, the skeleton consisting of three or more spines radiating from one point below the central capsule or from a central rod. Typical genus: Triplecta, Haeck., 1881. Order 14. StepHorpa. Haeckel recognizes four families. The skeleton consists of fused spines forming one or more rings. Typical genus: Lithocircus, Miiller, 1856. Order 15. Spyroma. Haeckel recognizes four families. The skeleton consists of a sagittal ring and a latticed shell furrowed in the sagittal plane; in some cases a lower chamber is added to the shell. Typical genus: Dictyospiris, Ehr., 1847. Order 16. Borryompa. Haeckel recognizes three families. Skeleton similar to the preceding, but having in addition one more wing-like process or lobe and one or more additional chambers. Typical genus: Lithobotrys, Ehr., 1844. 42 GENERAL ORGANIZATION OF THE PROTOZOA Order 17. Cyrrora. Haeckel recognizes twelve families. Skeleton similar to the preceding, but minus lobes or furrows. Typical genus: Theoconus, Haeckel, 1887. Legion 4. Cannopylea (Pheodaria). The chitinous central capsule is double, with a spout-like main opening at one pole and frequently with one or more accessory openings at the opposite pole. The skeleton is silicious and the spicules or bars are often hollow. The extracapsular protoplasm contains an accumulation of dark pigment granules (pheodium). Order 18. PHrocystiva. Haeckel recognizes three families. The skeleton con- sists of distinct spicules or is absent altogether; the central capsule is in the centre of the spherical body. Typical genus: Aulactinium, Haeckel, 1887. Order 19. PHEosPHERIA. Haeckel recognizes four families. The skeleton is a simple or double latticed sphere, and the central capsule is in the geometrical centre. Typical genus: Oroscena, Haeck., 1887. Order 20. PHrocromia. Haeckel recognizes five families. The skeleton is a simple latticed shell with a large opening at one pole; the central capsule is excentric, lying in the aboral half of the cell. Typical genera: Pharyngella, Haeckel, 1887; Tuscarora, Murray, 1876; Haeck- eliana, Murray, 1879. Order 21. Puroconcuta. Haeckel recognizes three families. The skeleton consists of two valves opening in the same plane as the three openings of the central capsule. Typical genus: Concharium, Haeck., 1879. Flagella and Classification of the Mastigophora.—Flagella do not present as many striking variations in form as do pseudopodia. Nevertheless, several different types exist. ‘The simplest form assumed is a slight, tapering filament broadest at the base and ending in an invisibly fine tip. It moves constantly, the tip forming a circle, while undulations or waves pass from base to extremity. In other types of flagella the tip alone moves, while the base is a conspicuous filament without undulation, the whole flagellum resembling a whip stock with lash. It is a remarkable sight to see a peranema, for example, with its stiff whip base, dragged along by the propelling movement of the tip end of the slender lash. In some forms of mastigophora the flagellum appears to be flattened out until it is quite band form. This is the case in some species of peridinium, where the band is drawn out to a pointed end, or in other cases it retains the same width throughout. In many of the flagellates there is but one flagellum attached at one end of the cell as in peranema or euglena. In other cases there are two, and these may be of similar or dissimilar length. In bodo and in most of the colony forming flagellates like dinobryon, synura, uroglena, etc., one is much shorter than the other. In many forms of bodo the longer flagellum trails along on the substratum so that the cell has the appearance of sliding along on a runner (Fig. 15). In some forms, especially the parasitic flagellates, this sliding flagellum has apparently fused with the cell membrane, projecting outward from PHYLUM PROTOZOA 43 one end as a trailing flagellum and forming a definite seam down one side of the cell body (trypanoplasma). ‘This seam in T'rypanophis grobbent becomes an undulating membrane, while in trypanosoma Fie. 15 Free-living flagellates with trailing flagellum. (After Calkins.) A, C, D, Bodo caudatus, Stein; B, Bodo globosus, Stein; #, Anisonema vitrea, Duj. 44 GENERAL ORGANIZATION OF THE PROTOZOA the anterior flagellum has disappeared apparently, leaving only the undulating membrane and the distal flagellum as motile organs. Finally, in spirocheta, especially in Spirocheta balbcanii, both free flagella have disappeared, leaving only the undulating membrane, while in some species of spirocheta even this remnant ‘of the motile apparatus has disappeared, leaving the organism with no visible means of locomotion. As such forms of spirocheta move with great freedom, it is not incredible that the remnant of the Raabecalls ele- ment is still retained within the membrane of the cell. In a number of forms the flagella are numerous and distributed uniformly around the body. Many of these types are of doubtful systematic position and are placed by some students of the group in the class ciliata while others regard them as flagellates. The nature of the flagellum in such cases justifies the mastigophora affinities, for they are long and undulating and have the characteristic flagellum movement. Such is the case in multicilia, actinobolus, myriophrys, etc., and in parasitic forms like trichonympha, pyrsonympha, ete. Other features of the cell body, however, such as the nuclei, tricho- cysts, ete., indicate relationship with the infusoria, and to classify such questionable forms as one or the other type shows the artificial character of even the best system of classification. The difficulty is one that is constantly met with by systematists, and in this case it serves a useful purpose by indicating the very close connection between the ciliated and the flagellated protozoa. The single flagellum is usually inserted deep within the substance of the body, sometimes, as in euglena, at the base of an opening at the end of the body; this opening, known as the flagellum fissure, is the means of exit of the waste matters of the cell, thrown out by the contraction of the contractile vacuole. ‘The flagellum originates deep within the substances of the protoplasm and usually in the vicinity of the nuclear membrane. ‘The energy constantly freed by protoplasmic oxidation is here concentrated, apparently, in the constantly moving material of the flagella. The contractile material, formed within the nucleus or at its periphery, as in the case of Camptonema mutans, is of similar nature to the material of the heliozoén axial filaments as shown in the case of dimorpha, and is associated in some way with the material of the mitotic figure or division centre, as shown by its origin from the blepharoplast in herpetomonas, crithidia, trypanosoma, and trypanoplasma. ‘The flagellum, therefore, is an element of the cell formed from the active or kinetic substances that are intimately associated with the nucleus. It is not merely a periplastic or mem- brane prolongation which may arise at any point on the cell periphery, but is much more deeply involved in the protoplasmic make- -up. The real flagellum is permanent, thrown off and reproduced again, only at times af cell division. . This point has importance in view of the ques- PHYLUM PROTOZOA 45 tionable nature of the so-called flagella of certain parasites belonging to the genus spirocheta, many of which are said to have flagella. These so-called flagella are apparently variable structures, for in Fic. 16 Trypanosoma raise. (After Robertson.) Forms observed in the digestive tract of the leech Pontobdella muricata. A, mature specimen from blood of skate; B to F, stages in the development of the flagellum from the kinetonucleus, and change in position of the latter in relation to the nucleus. many cases as “diffuse flagella” they appear not only at the ends of the cell, but at different points about the periphery, and there seems to be no uniformity about their distribution. ‘This is said to be the case in 46 GENERAL ORGANIZATION OF THE PROTOZOA Spirocheta duttont and in Spirocheta gallinarum. It is not improbable that such diffuse and variable filaments, and with them perhaps the so-called flagella of some bacteria, are mere transitory structures of the cell, which, like the filaments sometimes seen on the outer side of a diatom’s shell, owe their origin not to any formed structural element of the cell, but to some unformed exudation of a gelatinous nature, or to disintegration of the cell membrane, or to some other fortuitous cause. Whatever future research may show them to be, the so-called flagella of these forms are as yet much too indefinite and too uncertain to be taken as a basis for specific differences (see p. 223, and Fig. 88). The various modes of origin of true flagella, as distinguished from these transitory filaments, have recently been studied by Dobell (08), who makes out four distinct types, as follows: One, in which the flagellum arises directly from the nucleus (cf. axiopodia of actino- phrys or dimorpha); a second, in which the flagellum base is united to the nucleus by a connecting filament, the “zygoplast,” as in monas; a third, in which the flagellum arises from a basal granule which is independent of the nucleus, as in copromonas, herpetomonas, etc. ; and a fourth, in which the flagellum arises from a special “motor” nucleus, the “kinetonucleus,” as in trypanosoma (Fig. 16). CLASSIFICATION OF THE MASTIGOPHORA. Subphylum. MASTIGOPHORA. Protozoa in which the kinoplasm is concentrated in the form of one or more vibratile or undulating motile processes, called flagella, or in a kinetonucleus which may lie inside or outside of the tropho- nucleus. Simplest forms closely related to bacteria. Class 1. ZOOMASTIGOPHORA. Flagellated forms in which animal characteris- tics are predominant. Subclass. Lissoflagellata. “Smooth” flagellates, 7. ¢., without protoplasmic collars. Order 1. SprrocHETIDA. Organisms, often pathogenic, of somewhat uncertain position because of incomplete knowledge of flagella and life history; spiral in form, the turns of the spiral more or less plastic; nuclei unknown or dis- tributed as in bacteria; division either transverse or longitudinal, sometimes both. Typical genera: Spirocheta Ehr., 1833; (?) Treponema, Schaudinn, 1905; (?) Spiroschaudinnia, Sambon, 1907. Order 2. Monapipa. Organisms of simple structure, the body being often plastic or even ameboid and with one or more flagella at one end (so-called “anterior” end); there is no distinct mouth opening, ‘the food materials being ingested by a soft area of protoplasm at the base of the flagellum; in some cases the organisms are saprozoites. Family Rhizomastigide: Simple organisms with one or two flagella and with an ameboid body capable of forming pseudopodia which may be lobose, as in rhizopods, or axial, as in heliozoa; food taking is assisted by flagellum and pseudopodia. Typical genera: Mastigameba, Schultze, 1875; Dimorpha, Gruber, 1881: Actino- monas, Kent, 1880; Mastigophrys, Frenzel, 1891. CLASSIFICATION OF THE MASTIGOPHORA 47 Family Cercomonadide: The organisms are frequently plastic and changeable in form, but unable to form pseudopodia; there is but one flagellum with a flagellum-fissure at the base; nutrition is holozoic, saprozoic, or parasitic. Typical genera: Cercomonas, Dujardin, 1841 (a very uncertain genus); Herpe- tomonas, Kent, 1880, (‘including Donovan-Leishman bodies”); Crithidia, Léger, 1904; Oikomonas, Kent, 1880; Copromonas, Dobell, 1908. Family Codonecide: Small colorless monads which secrete and remain in gelati- nous or membranous cups. Typical genera: Codoneca, James-Clark, 1866; Platytheca, Stein, 1878. Family Bikecide: Minute organisms of peculiar shape, the basal broader portion bearing a tentacle-like process; nutrition is holozoic; the individuals single or colony forming. Typical genera: Bicoseca, James-Clark, 1867; Poteriodendron, Stein, 1878. Family Heteromonadide: Small colorless monads possessing one or more accessory flagella in addition to the primary one; they frequently form large but delicate colonies upon a common stalk. Typical genera: Monas, Stein, 1878; Dendromonas, Stein, 1878; Anthophysa, St. Vincent, 1824; Rhipidodendron, Stein, 1878. Order 3. HerERomasticips. A small group comprising various kinds of flagel- lated forms which are sometimes naked and plastic, sometimes provided with a highly differentiated membrane. The essential morphological characteristic is the possession of two or more flagella, one or two of which are directed downward and backward, while the other is directed forward and used in locomotion. Typical genera: Bodo, Stein, 1878; Phyllomitus, Stein, 1878; Oxyrrhus, Dujar- din, 1841; Anisonema, Dujardin, 1841; Trimastix, Kent, 1881. Order 4. TrypaNosomatipa. Organisms of elongate, usually pointed form, and of parasitic mode of life; with one or two flagella arising from a special “motor’’ nucleus, and with an undulating membrane provided with myo- nemes running from the kinetonucleus to the extremity of the cell; one of the flagella is attached to the edge of this membrane throughout its length, and may terminate with the membrane or be continued beyond the body as a free lash.* Typical genera: Trypanosoma, Gruby, 1841; Trypanoplasma, Lay. and Mesnil, 1904; Trypanophis, Keysselitz, 1904. Order 5. Potymasticipa. Organisms characterized by numerous flagella, frequently arranged in groups, and with one or many mouth openings usually at the bases of the flagella. Tribe 1. Astomea. Organisms with many flagella uniformly distributed, and without special mouth openings. Typical genera: Multicilia, Cienk., 1881; Grassia, Fisch., 1885. Tribe 2. Monostomea. Organisms with mouth opening at the base of the group of from four to six flagella. Typical genera: Collodictyon, Carter, 1865; Trichomonas, Donné, 1837; Megas- toma, Grassi, 1881; Tetramitus, Perty, 1852. Tribe 3. Distomea. Organisms with two mouth openings at the bases of the two groups of flagella. Typical genera: Hexamitus, Dujardin, 1838; Trepomonas, Dujardin, 1839; Spi- ronema, Klebs, 1893; Urophagus, Klebs, 1893. 1The conclusions of Novy, MacNeal, and Torrey (1907) that herpetomonas, crithidia, and trypanosoma are synonyms cannot be accepted on the basis of cultural methods alone; when the life history of these parasitic forms is known in detail will be time enough to speak of synonyms, and as the important structural characteristic which the membrane represents far outweighs the cultural characteristics, it is better to hold to the older view and thus to prevent further complications in what is already almost a hopelessly complicated group. 48 GENERAL ORGANIZATION OF THE PROTOZOA Tribe 4. Trichonymphinea. Parasitic forms of the digestive tract covered with a coating of long flagella. : E Typical genera: Trichonympha, Leidy, 1877; Pyrsonympha, Leidy, 1877; Jenia, Grassi, 1885. Order 6. EucLenrpa. Large forms of flagellates possessing one or two flagella, a contractile often complicated body wall, a mouth and pharyngeal opening at the base of the flagellum through which the contractile vacuole opens to the outside; chromatophores are often present and colony forms are not uncommon. Family Euglenide: The organisms are elongate with more or less pointed ends and usually with one flagellum. The membrane is marked with spiral stripings indicating the course of the myonemes. Red eye spots, and green chromato- phores are usually present. Pyrenoids and paramylum granules usually present in abundance. Typical genera: Euglena, Ehr., 1830; Trachelomonas, Ehr., 1833; Phacus, Nitsch, 1816. Family Astastide: The body is elongate and usually provided with a striped membrane and otherwise similar to Euglena, but there are no eye spots and the body is always colorless. Typical genera: Astasia, Ehr., 1838; Rhabdomonas, Fres., 1858. Family Peranemide: The body is either stiff or plastic, and is usually symmetrical. Typical genera: Peranema, Dujardin, 1841; Petalomonas, Stein, 1859. Order 7. SILICOFLAGELLIDA. Organisms with a peculiar lattice-like skeleton of silica, one flagellum, and simple structure. Parasitic on radiolaria. Typical genus: Distephanus, Stohr, 1881. Subclass 2. Choanoflagellata. Simple flagellated protozoa with a well-defined and characteristic protoplasmic collar surrounding the base of the flagellum. They frequently form colonies in which the cells are embedded in a gelatinous or a chitinous matrix. Typical genera: Monosiga, Kent, 1880; Codosiga, James-Clark, 1867; Pro- terospongia, Kent, 1880; Diplosiga (with two collars), Frenzel, 1891; Phalan- sterium, Cienk., 1870. Class I]. PHYTOMASTIGOPHORA. Flagellated forms in which the plant char- acteristics, if not predominant, are clearly marked. Here are classified the majority of complex colony forming types, but the single cells are invariably of simple structure, possessing eye spots, pyrenoids, and yellow, green, or brown chromatophores. Subclass 1. Phytoflagellata. In this group the organisms have yellow or green chromatophores. Order 1. CHRYSOFLAGELLIDA. With yellow chromatophores. Typical genera: Chromulina, Cienk., 1870; Dinobryon, Ehr., 1838; Hyalobryon, Lauterborn, 1899; Mallomonas, Perty, 1876; Synura, Ehr., 1833; Uroglena, Ehr., 1833;, Chrysospherella, Lauterb., 1899; Cryptomonas, Ehr., 1831; Chilomonas, Ehr., 1831 (without chromatophores). Order 2. CHLOROFLAGELLIDA. With green chromatophores. Typical genera: Chlorogonium, Ehr., 1835; Polytoma, Ehr., 1838; Hemato- coccus, Agardh., 1828; Phacotus, Perty, 1852; Gonium, O. F. Miiller, 1773; Pandorina, St. Vincent, 1824; Eudorina, Ehr., 1831; Pleodorina, Shaw, 1894; Platydorina, Kofoid, 1899. Subclass 2, Dinoflagellata. Organisms with yellow or brown pigment, two or more flagella, and an outer shell of cellulose secreted in the form of plates. The body is usually cut by furrows, of which the transverse is the more important; one flagellum lies in this furrow, while the other is extended in advance of the organism. The two flagella combine to give a rotation and forward movement at the same time. CLASSIFICATION OF THE MASTIGOPHORA 49 Order 1. Apinipa. Dinoflagellates without furrows, the two flagella free in the water, the transverse with movement the same as though the furrow were present. Typical genera: Prorocentrum, Ehr., 1833; Exuviella, Cienk., 1882. Order 2. Dinireripa. Dinoflagellates with furrows, one transverse, the other longitudinal. Family 1. Peridinide. The transverse furrow is without wide ledges and the shell may be absent. Typical genera: Peridinium, Ehr., 1832; Ceratium, Schrank, 1793; Gleno- dinium, Ehr., 1835; Gymnodinium, Stein, 1878. Family 2. Dinophyside. ‘The borders of the cross furrow are developed into great ledges, making a deep furrow for the flagellum. Typwcal genera: Dinophysis, Ehr., 1839; Cithiristes, Stein, 1883; Amphidinium, Clap. and Lach., 1859; Ceratocorys, Stein, 1883; Triposolenia, Kofoid, 1906. Order 3. Potypinipa. The order consists of but one genus, Polykrikos, Biitschli, 1873, which is characterized by a naked body, by several transverse furrows and flagella, by macro- and micronuclei, and nematocysts. Subclass 3. Cystoflagellata. Marine protozoa, which are plant-like in having a highly parenchymatous body, a single nucleus and a firm membrane. The young forms pass through a dinoflagellate stage in development. Three genera: Noctiluca, Suriray, 1836; Leptodiscus, Hertwig, 1877; Craspe- dotella, Kofoid, 1905. Cilia, and Classification of the Infusoria.—Cilia are quite differ- ent from flagella, being shorter and moving with a sharp stroke in one direction and with a slower, non-forceful recovery in the opposite direction. Like the flagellum, the cilium is thicker at the base and tapers to a fine point, while it owes its contractility to the presence of a Fic, 17 Aspidisca hexeris, Quen. An hypotrichous ciliate with brushes of fused cilia. (After Calkins.) filament of kinetic granules placed along one edge of the cilium, the contraction of this ‘thread furnishing the power ‘of the cilium, while the synchronous contraction of thousands of similar cilia furnishes the motive power of the organism. In some forms, as in dileptus or paramecium, and the majority of 4 50 GENERAL ORGANIZATION OF THE PROTOZOA the largest forms of protozoa, the cilia are distributed evenly over the entire cell body. But in some cases they are limited to one-half of the body, as in halteria; in others to the ventral surface only, as in gastrostyla, oxytricha, and the hypotrichida in general, while in others they are reduced to a single girdle of cilia about the mouth, as in vorticella and its allies. An interesting feature in the comparative anatomy of infusoria is the fusion of simple cilia into motile organs of a more complicated type. Sometimes a bundle of cilia are grouped together in a small brush-like organ, as in aspidisca, where the constituent elements of the bundle can still be made out (Fig. 17). In other forms, as oxytricha, the bundles are more tightly fused to form compact motile organs, which are sometimes used for walking and running, or some- times they are differentiated for feeling, and so constitute an elemen- Fic. 18 Pleuronema chrysalis, Ehr., with well-developed undulating membrane. (After Calkins.) tary sensory apparatus. Again, the cilia are fused into continuous sheets, or membranes, which provide currents for bringing food toward the mouth, as in pleuronema or lembus (Fig. 18). Rows of small membranes, called membranelles, are found in three of the four orders of ciliata. These are always placed around the oral or peristomial cavity, and their synchronous beating brings a constant food-bearing current toward the mouth. In some cases, as the vor- ticella group, the cilia have quite disappeared, leaving, under ordinary vegetative conditions, only this row of membranelles. In one subdivision of the infusoria, the suctoria, the cilia disappear after a short embryonic life of the individual, and their place is taken by protoplasmic prolongations called tentacles. Some of these ten- tacles are hollow and provided with a suction cap, so that food may be drawn through them into the inner protoplasm. Others are sharp CLASSIFICATION OF THE MASTIGOPHORA 51 pointed and are used by the animal as piercing needles for pene- trating the membranes of the victims that are caught for food. The more than superficial resemblance of these suctoria to the heliozoa gives a clue to the possible evolution of the infusoria from sarcodina. We have seen that in forms like myriophrys, cilia and pseudopodia are equally distributed around the body. We have also seen that the central axis of such pseudopodia and flagella are of the same type, and are probably homologous structures; furthermore, we have seen that in actinobolus, projectile tentacles armed with trichocysts can be thrown out at any point on the periphery. These facts indicate the possibility of a common ancestry of the infusoria Fic. 19 Cilia and myonemes of infusoria: a, b and e after Johnson; c, d, f and g after Biitschli. The surface view of Stentor ceruleus (c, e) shows rows of cilia inserted on the borders of canal-like markings, each of which contains a myoneme (d). These are more clearly shown in the optical section (f). In Holophyra discolor (g) the canals and myonemes are inserted deeper in the cortical plasm. a, the membrane of Stentor ceruleus under pressure. from a heliozodn-like ancestral race, represented in present-day forms by types like myriophrys, hypocoma, ileonema, and mesodinium, which have both tentacles and cilia. From such an ancestral group the ciliata may have arisen by losing the tentacles and adapting the cilia to the various needs of the cell, while the suctoria may have arisen by loss of the cilia and development of the tentacles to meet all of the needs of the cell, the cilia appearing in the embryos of the suctoria as reminiscences of the earlier ciliated condition of the race. These motile organs of the protozoa, with the exception of the flagella, are products of the cortical protoplasm, the flagella retaining 52 GENERAL ORGANIZATION OF THE PROTOZOA the same origin from the nucleus that the axial rays of the filose pseudopodia ‘have. Cilia, however, arise from small basal bodies called microsomes, which have a nuclear origin and belong apparently in the same category of kinetic stuffs as the “substance of flagella. In many of the infusoria these granules are arranged in definite lines or rows, forming threads of contractile substance which lie immediately below the cuticle. These threads, called myonemes, are in reality primitive muscle elements, and their sudden contraction resembles the action of the complicated muscle bundles of the metazoa (Fig. 19). Subphylum INFUSORIA. Protozoa in which the motor apparatus is in the form of cilia, either simple or united into membranes, membranelles, or cirri. The cilia may be permanent or limited to the young stages. With two kinds of nuclei, macronucleus and micronucleus. Reproduction is effected by simple transverse division or by budding. Nutrition is holozoic or parasitic. Class I. CILIATA. Infusoria provided with cilia during all stages. Reproduction is brought about typically by simple transverse division. Mouth and anus are usually present. The contractile vacuole is often connected with a com- plicated canal system. Order 1. Holotrichida. Ciliata in which the cilia are similar and distributed all over the body, with, however, a tendency to lengthen in the vicinity of the mouth. Trichocysts are always present, either distributed about the body or limited to a special region. Suborder 1. Gymnosromina. Holotrichida without an undulating membrane about the mouth, which remains closed except during food-taking intervals. Family 1. Enchelinide. The mouth is always terminal or subterminal, and is usually round or oval in outline. Food taking is usually a process of swal- lowin Typical nee: Holophrya, Ehr., 1831; Urotricha, Clap; and Lach., 1858; Enchelys, Hill, 1752, Ehr., 1838; Spathidium, Duj., 1841; Chenia, Quen- nerstadt, 1868; Prorodon, Ehr., 1833; Dinophrya, Batschii, 1888; Lacry- maria, Ehr., 1830; Trachelocerca, Ehr. , 1833; Actinobolus, Stein, 1867; . Ileonema, Stokes, 1884; Plagiopogon, Stein, 1859; Coleps, Nitsch, 1827; Tiarina, Bergh, 1879; Stephanopogon, Entz, 1884; Didinium, Stein, 1859; Mesodinium, Stein, 1862; Biitschlia, Schuberg, 1886. Family 2. Trachelinide. The body is distinctly bilateral or asymmetrical, with one side, the dorsal, slightly arched. The mouth may be terminal or sub- terminal, or the entire mouth region may be drawn out into a long proboscis. An esophagus or gullet may or may not be present; w hen present, ‘it is usually supported by a sj pecialized framework. Typical genera: Aranielesigy Ehr., 1830; Lionotus, Wrzesniowski, 1870; Loxo- phyllum, Duj., 1841; Trachelius, Schrank, 1903; Dileptus, Duj., 1841; Loxodes, Ehr., 1830. Family 3. Chlamydodontide. The general form is oval or kidney-shaped. The mouth is almost always in the posterior region. The pharynx is supported by a rod-apparatus or a smooth, firm tube. Subfamily 1. Nassulinw. Ciliation is complete. Typical genera: Nassula, Ehr., 1833. Subfamily 2. Chilodontinw. The body is generally flattened, and the cilia are stronger on the dorsal side, or are confined to that region. Typical genera: Orthodon, Gruber, 1884; Chilodon, Ehr. -» 1833; Chlamydodon, Ehr., 1835; Opisthodon, Stein, 1859; Phascolodon, Stein, 1857; Seaphidio- don, Stein, 1857. CLASSIFICATION OF THE INFUSORIA 53 Subfamily 3. Erviliine. The cilia are confined to the ventral surface or to a por- tion of it. The posterior end invariably possesses a movable style arising from the posterior ventral surface. Typical genera: Egyria, Clap and Lach., 1858; Onychodactylus, Entz., 1884; Trochilia, Duj., 1841; Dysteria, Huxley, 1857. Suborder 2. TricHosromina. In addition to the general coating of cilia there is an undulating membrane or membranes at the edge of the mouth or in the pharynx. The mouth is always open. Family 1. Chiliferide. The mouth is in the anterior half of the body or close to the middle. The pharynx when present is short. The so-called “peristome area” leading to the mouth is absent or only slightly developed. Typical genera: Leucophrys, Ehr., 1830; Glaucoma, Ehr., 1830; Dallasia, Stokes, 1886; Frontonia, Ehr., 1838; Ophryoglena, Ehr., 1831; Colpidium, Stein, 1860; Chasmatostoma, Engelmann, 1862; Uronema, Duj., 1841; Urozona, Schewiakoff (Biitschli), 1888; Loxocephalus, Kent, 1881; Colpoda, Miiller, 1773. Family 2. Urocentride. The mouth, with a long, tubular pharynx, is in the centre of the ventral side. The cilia are confined to two broad zones around the body at each end. Typical genera: Urocentrum, Nitsch, 1827. Family 3. Microthoracide. Small asymmetrical forms, with the mouth invariably in the hinder portion. The cilia are always more or less dispersed, sometimes limited to the oral region. There may be one or two undulating membranes. Typical genera: Cinetochilum, Perty, 1849; Microthorax, Engelmann, 1862; Ptychostomum, Stein, 1860; Ancistrum, Maupas, 1883; Drepanomonas, Fresenius, 1858. Family 4. Paramecide. The mouth is sometimes in the anterior, sometimes in the posterior, half of the body, and is accompanied by a large, triangular ““peristome area,” running from the left anterior edge of the body to the mouth. Typical genera: Paramecium, Stein, 1860. Family 5. Pleuronemide. The mouth is at the end of a long peristome, which runs along the ventral side; the body is dorsoventrally or laterally com- pressed. The entire left edge of the peristome is provided with an undulating membrane which occasionally runs around the posterior end of the peristome to form a pocket leading to the mouth. The right edge of the peristome is provided with a less developed membrane. There may or may not be a well- developed pharynx. Typical genera: Lembadion, Perty, 1849; Pleuronema, Duj., 1841; Cyclidium, Ehr., 1838, a subgenus of the preceding; Calyptotricha, Phillips, 1882; Lembus, Cohn, 1866. Family 6. Isotrichide. The body is more or less plastic, but not contractile. The cuticle is thick and provided with evenly distributed cilia. The mouth is posterior and accompanied by a distinct pharynx. They are parasites in the digestive tract of ruminants. Typical genera: Isotricha, Stein, 1859; Dasytricha, Schuberg, 1888. Family 7. Opalinide. The form is oval, and the body may be short or drawn out to resemble a worm, They are characterized mainly by the absence of mouth and pharynx. Typical genera: Anoplophrya, Stein, 1860; Hoplitophrya, Stein, 1860; Disco- phrya, Stein, 1860; Opalinopsis, Feettinger, 1881; Opalina, Purkinje and Valentin, 1835; Monodontophrya, Vejdowsky, 1892. Order 2. Heterotrichida. Ciliata characterized by the possession of a uniform covering of cilia and an adoral zone, consisting of short cilia fused together into membranelles. 54 GENERAL ORGANIZATION OF THE PROTOZOA Suborder 1. Potyrricuina. Heterotrichous ciliates provided with a uniform coating of cilia. Family 1. Plagiotomide. The peristome is a narrow furrow, which begins, as a tule, close to the anterior end, and runs backward along the ventral side to the mouth, which is usually placed between the middle of the body and the pos- terior end. A well-developed adoral zone stretches along the left side of the peristome, and it is usually straight. Typical genera: re Stein, 1861; Plagiotoma, Duj., 1841; Nycto- therus, Leidy, 1849, a subgenus; pee “Perty, 1849; Metopus, Clap. and Lach., 1858; Spirostomum, Ehr., 1835. Family 2. Bursaride. The body is usually short and pocket-like, but may be elongate. The chief characteristic is the peristome, which is not a furrow, but a broad triangular area, deeply insunk, and ending in a point at the mouth. The adoral zone is usually confined to the left peristome edge, or it may cross over to the right anterior edge. Typical genera: Balantidium, Stein, 1867; Balantidiopsis, Biitschli, 1888; Con- dylostoma, Duj., 1841; Bursaria, O. F. Miiller, 1773; Thylakidium, Sche- wiakoff, 1892. Family 3. Stentoride. The peristome is relatively short and limited to the front end of the animal, so that its plane is nearly at right angles to that of the longitudinal axis of the body. ‘The adoral zone of cilia either passes entirely around the peristome edge, or ends at the right-hand edge. The surface of the peristome is spirally striated and provided w vith cilia. U ndulating membranes are absent. Typical genera: Climacostomum, Stein, 1859; Stentor, Oken, 1815; Folliculina, Lamarck, 1816. Genera incerte sedis: Cenomorpha (Gyrocorys, Stein), Perty, 1852; Maryna, Gruber, 1879. Suborder 2. OxicotricHina. Heterotrichous ciliates characterized by the reduced cilia, which are limited to certain localized areas. Family 1. Lieberkiihnide. This name was given by Biitschli for certain little- known forms, which were at first considered young Stentors. Family 2. Halteriide. The peristome has no cilia, and only a few scattered ones can be found on the ventral and dorsal surfaces. Typical genera: Strombidium, Clap. and Lach., 1858; Halteria, Duj., 1841. Family 3. Tintinnide. The body is attached by a stalk to a theca. Inside of the adoral zone of membranelles is a ring of cilia (paroral cilia). Typical genera: Tintinnus, Fol., 1889; Tintinnidium, Kent, 1881; Tintinnopsis, Stein, 1867; Codonella, Haeckel, 1873; Dictyocysta, Ehr., 1854. Family 4. Ophryoscolecide. Heterotrichous ciliates characterized by a_ thick cuticle and deep funnel-like peristome. The posterior end is provided with distinct spine-like processes, while the terminal anus is provided with a well- defined anal tube. Typical genera: Ophryoscolex, Stein, 1859; Entodinium, Stein, 1859; Diplo- dinium, Schuberg, 1888. Order 3. Hypotrichida. Ciliata in which the cilia are limited to the ventral surface of a dorsoventrally flattened body; they are frequently fused to form larger appendages, the cirri, and an adoral zone of membranelles. The dorsal sur- face is frequently provided with bristles. A pharynx may be absent or but slightly developed. Family 1. ees The peristome is but slightly marked off from the remaining frontal area. The cilia on the ventral surface are uniform in size and arrangement, and are not differentiated into cirri. Typical genera: Peritromus, Stein, 1862. Family 2. Oxytrichida. The peristome is not always distinctly marked off from the frontal area. In the most primitive forms the ciliation on the ventral sur- CLASSIFICATION OF THE INFUSORIA 55 face is similar to that of the preceding family. Almost invariably in these primi- tive forms some of the anterior and some of the posterior cilia are fused into large and more powerful appendages, the cirri, which are distinguished as the frontal and anal cirri, respectively. In the majority of forms all of the cilia are thus differentiated; strong marginal cirri are formed in perfect rows, and ventral cirri in imperfect rows. In addition to the adoral zone of membra- nelles, there is an undulating membrane on the right side of the peristome, and, in some cases, a row of cilia between the membrane and the adoral zone. These are the paroral cilia, and they form the paroral zone. Typical genera: Trichogaster, Sterki, 1878; Urostyla, Ehr., 1830; Kerona, Ehr., 1838; Epiclintes, Stein, 1862; Stichotricha, Perty, 1849; Strongylidium, Sterki, 1878; Amphisia, Sterki, 1878; Uroleptus, Stein, 1859; Sparotricha, Entz, 1879; Onychodromus, Stein, 1859; Pleurotricha, Stein, 1859; Gas- trostyla, Engelmann, 1862; Gonostomum, Sterki, 1878; Urosoma, Kowalew- sky, 1882; Oxytricha, Ehr., 1830; Stylonychia, Stein, 1859; Actinotricha, Cohn, 1866; Balladina, Kowalewsky, 1882; Psilotricha, Stein, 1859; Tetra- styla, Schewiakoff, 1892; Holosticha, Wrzesniowski, 1877. Family 3. Euplotede. Hypotrichous ciliates, which are characterized mainly by the considerable reduction of the cilia, frontal, marginal, and ventral cirri; the anal cirri, on the other hand, are always present. The macronucleus is band-formed. Typical genera: Euplotes, Stein, 1859; Certesia, Fabre-Domergue, 1885; Dio- phrys, Duj., 1841; Uronychia, Stein, 1857; Aspidisca, Ehr., 1830. Order 4. Peritrichida. Ciliata usually of cylindrical or cup-like form, in which the cilia are reduced, as a rule, to those which form the adoral zone, but sec- ondary rings of cilia may be present. Family 1. Spirochonide. Peritrichous ciliates in which the peristome is drawn out into a curious funnel-like process, either simple or rolled. They are parasitic forms in which reproduction by budding is characteristic. Typrcal genera: Spirochona, Stein, 1851; Kentrochona, Rémpel, 1894; Kentro- chonopsis, Doflein, 1897. Family 2. Lichnophoride. In addition to the adoral zone, there is a secondary circlet of cilia around the opposite end. The adoral zone is a left-wound spiral. A single genus Lichnophora, Claparéde, 1867, which is parasitic on various marine arthropods. Family 3. Vorticellide. Attached or unattached forms of peritrichous ciliates, in which the adoral zone, seen from above, forms a right-wound spiral (dexio- tropic). A secondary circlet of cilia around the under end may be present either permanently or periodically. Subfamily 1. Urceolarine. Vorticellide having a permanent secondary circlet of cilia which incloses an adhesive disk, and without a peristome fold. Typical genera: Trichodina, Stein, 1854; Cyclocheta, Jackson, 1875; Tricho- dinopsis, Clap. and Lach., 1858. Subfamily 2. Vorticellidinw. Peritrichous forms without a permanent secondary circlet of cilia, and provided with a peristome fold which can be contracted sphincter-like to inclose the peristome. Typical genera: Scyphidia, Lachmann, 1856; Gerda, Clap. and Lach., 1858; Astylozoén, Engelmann, 1862; Vorticella, Ehr., 1838; Carchesium, Ehr. 1830; Zoothamnium, Stein, 1854; Glossatella, Biitschli, 1888; Epistylis, Ebr., 1830; Rhabdostyla, Kent, 1882; Opercularia, Stein, 1854; Ophrydium, Ehr., 1838; Cothurnia, Clap. and Lach., 1858; Vaginicola, Clap. and Lach., 1858; Lagenophrys, Stein, 1851. Subclass 2. Suctoria. Infusoria having no cilia during the adult stages, but provided with them during the embryonic period. In a few cases the cilia are retained. They have tentacles of various kinds, some adopted for sucking, some for piercing. 56 GENERAL ORGANIZATION OF THE PROTOZOA Family 1. Hypocomide. These are unattached forms of Suctoria with a perma- nently ciliated ventral surface, and with one suctorial tentacle. Reproduction is effected by cross-division. A single genus, Hypocoma, Gruber, 1884. Family 2. Urnulide. A family of small attached forms, with or without a cup or theca; with one or two, rarely more, simple tentacles. Swarm-spores holo- trichous. Typical genera: Rhyncheta, Zenker, 1866; Urnula, Clap. and Lach., 1858. Family 3. Metacinetida. Thecate forms; the base of the cup is drawn out into a long stalk, and the walls are perforated for the exit of the tentacles. A single genus, Metacineta, Biitschli, 1888. Family 4. Podophryide. Stalked or unstalked forms of more or less globular shape. The tentacles are numerous and distributed about the entire surface or limited to the apical region; some of them are knobbed, others pointed and have a prehensile function. Typical genera: Spherophrya, Clap. and Lach., 1858; Endospheera, Engelmann, 1876; Podophrya, Ehr., 1838; Ephelota, Str. Wright, 1858; Podocyathus, Kent, 1881. Family 5. Acinetide. The individuals are naked and stalked, or thecate and stalked or unstalked. The tentacles are numerous, usually knobbed and all alike. Reproduction is effected by inner or endogenous budding, which may be simple or multiple. The swarm spores are usually peritrichous, but may be holotrichous or hypotrichous. Typical genera: Tokophrya, Biitschli, 1888; Acineta, Ehr., 1833; Solenophrya, Clap. and Lach., 1858; Suctorella, Frenzel, 1891. Family 6. Dendrosomide. Suctoria without stalks or theca. The tentacles are numerous, all alike, and knobbed and grouped in distinct tufts; they may be simple or branched. Reproduction by endogenous division; the swarm spores are peritrichous. Typical genera: Trichophrya, Clap. and Lach., 1858; Dendrosoma, Ehr., 1838; Staurophrya, Zacharias, 1893. Family 7. Dendrocometide. Sessile suctoria resting upon the entire basal surface or upon a portion of it raised as a stalk. The numerous tentacles are short and knobbed, and distributed over the entire apical surface or localized upon branched arms. Spore formation is endogenous; the swarm spores peri- trichous. Typical genera: Dendrocometes, Stein, 1867; Stylocometes, Stein, 1867. Family 8. Ophryodendridew. Stalked or sessile forms possessing numerous long, rarely knobbed tentacles, which are supported upon proboscis-like processes of the apical side. Reproduction is brought abot by endogenous budding. The swarm spores are peritrichous. Typical genera: Ophryodendron, Clap. and Lach., 1858. PROTOZOA WITHOUT MOTILE ORGANS, AND CLASSIFICATION OF THE SPOROZOA. To state that the sporozoa are without motile organs is not strictly accurate, for many of them have well-developed myonemes (gregarines) and move with a vermiform motion. Others have, at times, the power of progressing by means of pseudopodia (many of the neosporidia). Nor is the method of reproduction (spore formation) any less equi- vocal, for many forms reproduce by simple division as well as by spore formation (schizogregarinida). ‘This division, therefore, more than ‘PROTOZOA WITHOUT MOTILE ORGANS 57 any other of the unicellular animals must be regarded as provisional only and comprising numerous heterogeneous groups of organisms which can be more accurately classified only after the full life histories are made out. Some of these groups are obviously related to the mastigophora through the blood-dwelling flagellates, and others are equally related to the sarcodina. Two divisions only, the gregarinida and the coccidiidia, may be accepted as sufficiently definite to constitute an acceptable division of the protozoa. At the present time, Schau- dinn’s grouping into telosporidia and neosporidia cannot be bettered, although evidence is accumulating to show that the latter group is entirely artificial. SUBPHYLUM SPOROZOA.—Parasitic protozoa without motile organs, but capable of moving from place to place by structural mod- fications of one kind or other. Reproduction either simple or multiple, but mainly by spore formation, which is either asexual (schizogony) or sexual (sporogony). The following classification of sporozoa is based upon Labbé’s “sporozoa,” and upon “‘sporozoa”’ in Lankester’s Treatise on Zodlogy, Part I, Introduction and Protozoa. Second fascicle, with additions and changes necessary for the present work and to bring the classifi- cation up to date. Subphylum SPOROZOA. Class I. TELOSPORIDIA, Schaudinn. Sporozoa in which sporulation ends the life of the individual. Order 1. Gregarinida. Ccelozoic telosporidia reproducing usually by spore forma- tion alone, and after the fertilizing union of but slightly different gametes. Suborder 1. ScaizocrEcarin.©. Gregarines reproducing by division or by multiple budding in addition to spore formation. This interesting group, which is continually being added to by various obser- vers, was until quite recently represented by only those supposedly ameboid forms known as the Amebosporidia. The investigations begun by Léger and carried on by Léger, Dubosq, Dogiel, Brasil, and others of recent date have shown that the supposed ameboid processes are actually unchangeable, serving more as attaching organs and for the purpose of absorbing food than for the purposes of locomotion. There is no question that these forms are gregarines, and from the very characteristic types included here there is some hope of ultimately getting light upon the closer relationships of the entire group of sporozoa to other groups of protozoa. Genus 1. Schizocystis, Léger, 1900. Type species S. gregarinoides, Léger, from the intestine of larva of Ceratopogen sp. The trophozoites are somewhat similar to Monocystis, but differ in reproducing by the formation of a group of internal buds, which, as merozoites, leave the parent cell and grow into new trophozoites; these finally couple up, fertilization and sporulation result, and octozoic spores are finally formed, as in Monocystis (Fig. 76). Genus 2. Ophryocystis, A. Sch., 1884. Many species are known, most of which are parasites in the Malpighian tubules of beetles. The organisms have char- acteristic pseudopodia-like processes for purposes of attachment, and the trophozoites reproduce by simple division or by multiple division. Sporula- tion ultimately takes place, the process differing in different cases (Fig. 80). 58 GENERAL ORGANIZATION OF THE PROTOZOA Genus 3. Selenidium, Giard, 1884; emend Caullery et Mesnil, 1899. The body is attenuated and worm- -like, and marked externally by longitudinal striee due to the ectoplasmic myonemes. Epimerite conical and slender. Parasites of polychetes and numerous species are recorded. Suborder 2. Everecarry.®, Leger. Reproduction here is limited apparently to sporulation, division occurring, if at all, within the host cell and during the young stages. Tribe I. Acephaline, Kolliker. Eugregarines in which there is no division into chambers and in which at no stage is there an epimerite. Genus 4. Monocystis, Stein, 1848. The trophozoites are often highly contractile owing to the peristalsis brought about by the contractions of ectoplasmic myonemes. Spores boat-shaped and octozoic. Many species from worms and entomostraca, a typical species, M. agilis may be found almost invariably in the seminal reservoirs of the common earthworm, and excellent stages in sporulation and fertilization may be easily obtained. Genus 5. Zygocystis, Stein, 1848. The trophozoites are usually found in pairs or groups of three. Typical species, Z. cometa, Stein, found in the seminal vesicles and body cavity of the earthworm Lumbricus agricola. Genus 6. Zygosoma, Labbé, 1899. The trophozoite has typical and characteristic finger-like processes and is usually found in couples. Sporulation unknown. Typical species, Z. gibbosum, Greeff, 1880, in the gut of Echiurus pallassii. Genus 7. Pterospora, Racovitza and Labbé, 1896. The piriform trophozoites are always associated in couples. The spores have dissimilar poles and the epispore is drawn out into lateral processes. One species, P. maldaneorum, R. and L., from the celomic cavity of maldanid worms. Genus 8. Cystobia, Mingazzini, 1891. The trophozoites are large and irregular in form and usually have two nuclei due to the early fusion of two individuals. The spores are heteropolar, and the epispore is drawn out into chimney-like projections at one pole. One species, C. holothuriz, A. Sch., from the blood- vessels and body cavity of holothurians. Genus 9. Lithocystis, Giard, 1876. The trophozoite is characterized by an endo- plasm filled with crystals of calcium oxalate. The epispore has long pro- cesses. A single species from the coelomic cavities of various echinids. Genus 10. Ceratospora, Léger, 1892. The trophozoites fuse by their truncated ends and give rise to spores without encysting. The spores are character- ized by long spinous processes (Fig. 20). A single species, C. mirabilis, Léger, from ‘the body cavity of Glycera. Genus 11. U rospora, A. Schn., 1875. The spores are characterized by the presence of a long caudal filament at one pole. Several species from the body cavities of oligochetes, nemertines, sipunculids, and other marine invertebrates. Genus 12. Gonospora, A. Schn., 1875. The trophozoites are quite variable in form and give rise to heteropolar spores bearing from one to several tooth-like processes at one pole, and rounded at the other. Four species from the body cavities of polychetous worms. Genus 13. Syncystis, A. Schn., 1886. The spores are ovoid or boat-shaped, with spines or processes at each extremity. One species, S. mirabilis, A. Schn., from fat body.and ccelom of geo of Nepa. Genus 14. Diplocystis, Kunstler, 1887. The trophozoites fuse precociously to form spherical masses of gregarines in the body cavity of crickets and cock- roaches. The spores are either spherical or oblong. Genus 15. Lankesteria, Mingazzini, 1891. The spores are more or less flattened or spatulate, oval in outline, and octozoic. Type species, L. ascidie, Lank, from the gut of Ciona intestinalis. Genus 16. Callyntrochlamys, Frenzel, 1885. The trophozoites have a central constriction but no septum dividing the body into protomerite and deuto- PROTOZOA WITHOUT MOTILE ORGANS 59 merite; they are covered by a fur-like fringe of processes resembling cilia. The spores are unknown. ‘Type species, C. phronime, Frenz., from the gut of Phronima sedentaria. Genus 17. Ancora, Labbé, 1899. The trophozoite has a peculiar anchor-like form by reason of two lateral bulgings of the body. Spores unknown. Species, A. sagittata, Leuck, from the gut of Capitella capitata. Other genera provisionally placed here are: Pleurozyga, Mingazzini, 1891, from ascidians; Ophioidina, Mingazzini, 1891, from Bonellia; Kollikerella, Labbé, 1899, from Staurocephalus; Lobianchella, Mingazzini, 1891, from Alciope. Tribe II. Cephaline, Delage. Eugregarines possessing an epimerite at some stage of the life history, either in the adult phase or in the temporary young phases. The body is usually divided by a septum into protomerite and deutomerite, and the trophozoites are frequently associated in couples arranged tandem, each couple consisting of primite and satellite. The tribe consists mainly of parasites of the gut of various forms of arthropods. Legion A. Gymnosporea, Léger. The sporoblast mother cells give rise directly i sporozoites which do not form in sporocysts or specially protected sporo- lasts. Family 1. Aggregatide, Labbé. With sporozoites grouped irregularly about a number of residual masses. Genus 18. Aggregata, Frenzel, 1885. With the characteristics of the family. Several species from various crustacean hosts. Family 2. Porosporide, Labbé. Special centres of sporozoite formation are present (sporoblast centres), but they lack the protective sporocysts. Genus 19. Porospora, A. Schn., 1875. Trophozoite with small button-like epi- merite; cells very large (up to 16 mm.) and usually solitary. One species, P. gigantea, Van Beneden, from gut of the lobster. Legion B. Angiosporea, Léger. The sporocysts are well developed and usually double coated to form endospore and epispore. Family 3. Gregarinide, Labbé. Trophozoites with simple epimerites; sporo- cysts with or without sporoducts. Spores oval or barrel-shaped, and united in strings in species with sporoducts. Genus 20. Gregarina, Dufour, 1828. Cysts with sporoducts; epimerite small, conical, or knobbed (see Fig. 81, p. 191). Many species widely distributed in digestive tracts of various insects. Genus 21. Gamocystis, Léger, 1892. The trophozoite has a temporary epimerite. Cyst with sporoducts. Spores cylindrical and elongated. From gut of cock- roach and other insects. Genus 22. Evermocystis, Léger, 1892. The sporonts unite to form aggregates of several individuals. ‘The spores are ellipsoidal. Cysts without sporoducts. One species, E. polymorpha, Léger, from the gut of insects. Genus 23. Hyalospora, A. Schn., 1875. Cysts without sporoducts. Spores pointed at each end and bulging in middle. Gut of Petrobius sp. Genus 24. Euspora, A. Schn., 1875. Spores prismatic, cysts without sporoducts. One species, E. fallax, from gut of Rhizotrogus estivus. Genus 25. Spherocystis, Léger, 1892. Body spherical, protomerite temporary, cysts without sporoducts, spores oval. One species, 5. simplex, Léger, from the gut of Xyphon pallidus larva. Genus 26. Cnemidospora, A. Schn., 1882. The epimerite is large and lancet- shaped; sporonts solitary with globular protomerites. No sporoducts. Spores ellipsoidal, with thick spore cysts. One species, C. lutea, A. Schn., from the gut of Glomeris. Genus 27. Stenophora, Labbé, 1899. Sporonts large, with small protomerite. Cyst without sporoducts: spores fusiform with dark sutural line. One species, S. juli, Franz, from gut of species of millipedes. 60 GENERAL ORGANIZATION OF THE PROTOZOA Family 4. Didymophyide, Léger. The sporonts always associated in pairs, the protomerite of the satellite disappearing, thus giving the appearance of an organism with three chambers and two nuclei. _ Genus 28. Didymophyes, Stein, 1848. The epimerite has the form of a spike. Cysts open by simple rupture liberating the oval spores. Four species. Family 5. Dactylophoride, Leger. The epimerite is asymmetrical and irregular, with digitiform processes. Sporocysts open by simple rupture or by the swelling of a residual mass of plasm termed a ‘‘ pseudocyst.” Genus 29. Rhopalonia, Léger, 1893. The epimerite is irregular and asymmet- rical, bearing finger-formed prolongations. The trophozoite is solitary and with traces only of a protomerite. One species, R. geophili, Léger, from gut of geophilus sp. Genus 30. Echinomera, Labbé, 1899. The trophozoite massive and oval in outline; epimerite persistent and spiked, the point bearing small transitory digitiform processes. Cysts open by simple rupture. One species, E. hispida, A. Schn., from gut of Lithobius forficatus. Genus 31. Trichorhynchus, A. Schn., 1882. Protomerite truncated with an elongated and conical top. Cysts with oblong, wart-like protuberances. Cysts open by the swelling of laterally placed pseudocysts. Spores not in strings. One species, T. pulcher, A. Schn., from the gut of Scutigera. Genus 32. Pterocephalus A. Schn., 1887. Protomerite extends beyond the deuto- merite on the two sides and is divided into two lobes by a constriction; the two lobes are provided with sharp papillee, and are united on one side and so curved as to form a coiled horn. The spores are oval and associated obliquely in strings. One species, P. nobilis, A. Schn., from gut of Scolo- pendra. Genus 33. Dactylophorus, Balb., 1889. The protomerite is dilated excentrically and bears epimerite with digitiform processes. Sporonts are solitary and elongated; cysts spherical and spores cylindrical; cysts open by swelling of lateral pseudocyst. One species, D. robustus, Léger, from the gut of Cryp- tops hortensis. Family 6. Actinocephalide, Leger. Sporonts always solitary with simple, sym- metrical, or irregular appendages. Cysts open by simple rupture. Spores biconical, cylindrical, or navicular. Parasitic usually in the gut of carnivorous arthropods. Group A. Sciadiophorine, Labbé, 1899. Protomerite umbrella-shaped, and with radiating ridges. Spores biconical and with central] swellings, the opening at the equator by simple dehiscence, while the endospore opens terminally. Genus 34. Sciadiophora, Labbé, 1899. The epimerite is large and flattened and with the characteristics of the group. Three species from digestive tracts of phalangide. Group B. Anthorhynchine, Labbé, 1899. Spores ovoid with pointed ends; joined in strings; equatorial opening. Genus 35. Anthorhynchus, Labbé, 1899. Epimerite in form of a large grooved knob or button. One species from gut of Phalangium opilio. Group C. Pileocephaline, Labbé, 1899. Epimerite simple and regular; cysts open by simple rupture; spores usually biconical. Genus 36. Pileocephalus, A. Schn., 1875. Epimerite simple and regular and somewhat lance-like. Cysts open by simple rupture, spores biconical. Genus 37. Amphoroides, Labbé, 1899. Epimerite spiked or rounded; proto- merite very short and cup-like. Spores biconical. One species, A. polydesmi, Leger, from the gut of Polydesmus. Genus 388. Discorhynchus, Labbé, 1899. Epimerite large and discoid, with a distinct rim; protomerite larger than the deutomerite, which is regularly cylindrical and truncated posteriorly. Cysts spherical, spores biconical and slightly bent. One species, D. truncatus, Léger, from gut of Sericostoma sp. PROTOZOA WITHOUT MOTILE ORGANS 61 Group D. Stictosporine, Labbé, 1899. Spores biconical, with points slightly in- curved and with papille on the endospore. Genus 39. Stictospora, Léger, 1893. Epimerite with globular head depressed ventrally, and covered with ribs which project posteriorly as spikes. Spores biconical. One species, S. provincialis, Léger, from the gut of Melolontha and Rhizotrogus larve. Group E. Actinocephaline, Labbé, 1899. Epimerite always with appendages. Spores regular, navicular or subnavicular, biconical or cylindrical. Genus 40. Schneideria, Léger, 1892. Sporont has but one chamber; epimerite a thick plate bordered by rib-like thickenings. Spores somewhat thickened and biconical. Two species, S. mucronata, Léger, from gut of larvee of Bibio marci, and S. caudata from gut of larva of Sciara nitidicollis. Genus 41. Asterophora, Léger, 1892. The epimerite is a circular ridge with ribs surrounding a prominent central papilla. The protomerite is as large or larger than the deutomerite. Sporonts solitary; spores cylindrical with conical extremities. Two species, A. mucronata, L., and A. elegans, L., from the intestines of larvee of insects. 7 Genus 42. Stephanophora, Leger, 1892. Epimerite large and in form of a convex disk with a crown of digitiform processes. Spores cylindrical with conical ends. One species, 5. lucani, Stein, from gut of Dorcus sp. Genus 43. Bothriopsis, A. Schn., 1875. Epimerite in form of a large lens-shaped knob with non-motile processes. Sporonts highly developed and_ very motile. Spores biconical and thickened. One species, B. histrio, A. Schn., 1875, from the gut of Hydaticus sp. Genus 44. Coleorhynchus, Labbé, 1899. Sporont with sucker-like protomerite extending over deutomerite. The convex septum projects into the proto- merite. Cysts open by simple rupture; spores navicular. One species, C. heros, A. Schn., from gut of Nepa cinerea. Genus 45. Légeria, Labbé, 1899. Protomerite enlarged and club-like, with invading septum, as above. Spores with thick sporocysts and subnavicular inform. One species, L. agilis, A. Schn., from gut of Colymbetes sp. Genus 46. Phialoides, Labbé, 1899. Complex epimerite consisting of a discoid retractile cap surrounded by a circular ridge with collar-like membrane, with ridges ending in triangular teeth. Sporonts solitary, massive; spores biconical] and thickened. One species, P. ornata, Léger, from the gut of Hydrophilus larvee. Genus 47. Geniorhynchus, A. Schn., 1875. Epimerite in the form of a disk which bears fine pointed teeth and is carried on a long neck. Spores subnavicular. One species, G. monnieri, A. Schn., from intestines of nymphs of libellulidce. Genus 48. Actinocephalus, Stein, 1848. Epimerite sessile or borne on neck-like process, and is provided with hooks and spines. Spores biconical. Several species from digestive tracts of beetles. Genus 49. Py«inia, Hammerschmidt, 1838. Epimerite in the form of a cup with rim surrounding a central spine. Many species (Fig. 73). Genus 50. Beloides, Labbé, 1899. Epimerite in the form of a disk or knob and bearing about ten teeth in addition to a long spike. Spores navicular or oval. Two species parasitic in the gut of species of Dermestes. Genus 51. Stylocystis, Léger, 1899. Trophozoite non-septate; epimerite in the form of a long spine which is usually curved. Sporonts solitary with biconical spores. One species, S. precox, Léger, from the intestine of the larva of Tanypus sp. Family 7. Acanthosporide, Léger, 1892. Sporonts always solitary; epimerite simple or with appendages; cysts open by simple rupture; spores ornamented with bristles at the poles or at the equator. Parasites of carnivorous insects. Genus 52. Corycella, Léger, 1892. Protomerite spherical and somewhat dilated. 62 GENERAL ORGANIZATION OF THE PROTOZOA Epimerite a knob with a crown of eight large and recurved hooks. One species, C. armata, Léger, from the gut of Gyrinus natator. Genus 53. Acanthospora, Léger, 1892. Sporonts solitary and of elongate oval form. Epimerite a conical obtuse knob; spores oval with four bristles at each end and a circlet of spines about the equator. Three species, A. pileata, Léger, from the gut of larva of Omoplus, a typical species. Genus 54. Ancyrophora, Léger, 1892. Sporonts solitary; posterior part pointed. Epimerite a knob with appendages in the form of recurved hooks. Spores biconical with polar tufts and six equatorial bristles. Two or more species from carnivorous beetles. Genus 55. Cometoides, Labbé, 1899. Epimerite a spherical knob flattened cen- trally and bearing a circlet of flexible filaments. Spores with a bunch of bristles at each pole and two circlets of bristles about the equator. Two or more species from the larve of beetles. Family 8. Menosporide, Léger, 1892. Sporonts solitary, epimerite symmetrical, with appendages and connected with the protomerite by a long neck. Cysts spherical, opening by simple rupture. Spores in form of crescents more or less curved. Genus 56. Menospora, Léger, 1892. Epimerite cup-like and bordered by hooks. One species, M. polyacantha, Léger, 1892, from gut of Agrion puella. Genus 57. Hoplorhynchus, Carus, 1839. Epimerite in the form of a disk with sharp teeth. One species, H. oligacanthus, Sieb., from the gut of Calopteryx virgo, larva. Family 9. Stylorhynchide, A. Schn., 1886. Epimerite symmetrical with or without appendages. Cysts with two envelopes and pseudocyst. Brown or black- colored spores in strings. Genus 58. Lophocephalus, Labbé, 1899. Epimerite sessile, cup-like, with fringe of vesicular appendages. Protomerite compressed. Cysts irregular, sub- spherical. One species, L. insignis, A. Schn., in gut of Helops striatus. Genus 59. Cystocephalus, A. Schn., 1886. Epimerite vesicular, with short neck. One species, C. algerianus, A. Schn., from gut of Pimelia sp. Genus 60. Oocephalus, A. Schn., 1886. Epimerite a rounded knob on a short neck. One species, O. hispanus, A. Schn., from the gut of Morica sp. Genus 61. Spherorhynchus, Labbé, 1899. Epimerite small, spherical or oval, and carried on a long cylindrical neck constricted deeply below the epimerite. One species, S. ophioides, A. Schn., from the gut of Acis sp. Genus 62. Stylorhynchus, Stein, 1848. Epimerite smal] and knob-like, borne on an elongated neck of the protomerite. Deutomerite of the sporont much elongated; protomerite rounded. Two or three species, the most typical being S. longicollis, Stein, from the gut of Blaps mortisaga. Family 10. Doliocystide, Labbé, 1899. Epimerite regular and simple; no trace of aseptum. Spores oval with a polar thickening. Marine annelids. Genus 63. Doliocystis, Léger, 1893. No trace of septum; oval spores, and sporo- cysts with polar thickenings. Two or three species, the most typical D. pellucida, Kélliker, from the gut of Nereis sp. Other genera referred to this division by Labbé, Minchin, and other systematists are: Nematoides, Mingazzini, 1891, from the gut of cirrhipedes; Ulivina, Mingazzini, 1891. from the gut of Audouinia filigera: Sycia, Léger, 1892, from gut of same. Order 2. Coceidiidia. Cell-infesting sporozoa which usually reproduce by schizog- ony and by sporogony, thus giving a life cycle with an alternation of asexual and sexual generations. After fertilization the odsphere forms sporoblasts which may or may not (asporocystea) be covered by a sporocyst membrane, and which may each become transformed into one or several sporozoites. Suborder 1. Asporocystrnga. Coccidiidia in which the sporoblasts have no PROTOZOA WITHOUT MOTILE ORGANS 63 sporocysts. Here, if we were to be strictly consistent, we would advise, with Minchin, the inclusion of the malaria-causing organisms, and group the other hemosporidia with the genera included under the Sporocystinea. But it does not seem opportune at the present time to give up the old group Hemo- sporidia, at least not until the questionable ‘‘binucleate’” forms have been worked out in complete detail. Following Minchin, in naming the families according to the more char- acteristic of the contained genera, we have the following: Family 1. Eimeride (Asporocystide, Léger). Sporocysts absent, the sporozoites being naked in the parent cell (gymnospores). Genus 1. Eimeria, A. Schn., 1875. (Syn., Légerella Mesnil.) With the characters of the family. One species, E. nova, A. Schn., from the Malpighian tubules of Glomeris. Family 2. Isosporide (Disporocystide, Léger). The odsphere forms two sporo- blasts each with sporocysts (chlamydospores). Genus 2. Cyclospora, A. Sch., 1881. Each sporoblast forms two sporozoites. C. glomericola, A. Schn., 1881, intestine of Glomeris sp., and C. caryolytica, Schaudinn, from the intestine of moles. Genus 3. Diplospora, Labbé, 1893. Spores tetrazoic; many species occurring in birds, snakes, lizards, and frogs. Genus 4. Isospora, A. Schn., 1881. Spores polyzoic (?). I. rara, A. Schn., from the black slug, Limax cinereo niger. Family 3. Coccidiide, (Tetrasporocystide, Léger). The fertilized cell produces four sporoblasts with sporocysts (chlamydospores). Genus 5. Coccidiwm, Leuckart, 1879. The dizoic spores are spherical or oval. Many species almost entirely limited to vertebrate hosts, and found in nearly allorders. Here, also, belong some questionable forms, such as Paracoccidium prevoti, Lav. and Mes., from the frog. Genus 6. Crystallospora, Labbé, 1896. The spores are dizoic and the sporocysts in the form of a double pyramid placed base to base. One species, Cr. crys- talloides, Thél., from the intestine of Motella tricirrata of Roscoff (Fig. 20, L). Family 4. Klosside, (Polysporocystide, Léger). The fertilized cell contains many sporoblasts (chlamydospores). Genus 7. Barroussia, A. Schn., 1885. Spores spherical and monozoic; sporocyst smooth. Many species, a good type being B. ornata, A. Schn., from the gut of Nepa cinerea (Fig. 20, C). Genus 8. Echinospora, Léger, 1897. Spores monozoic, oval, and with spinous sporocyst. Typical species, E. labbei, Léger, from gut of Lithobius mutabilis. Genus 9. Diaspora, Léger, 1898. Spores, as above, but sporocysts not bivalve and with micropyle at one end. D. hydatidea, Léger, from gut of Polydesmus. Genus 10. Adelea, A. Schn., 1875. Spores dizoic with smooth, spherical or flattened sporocyst. Many species, a typical one, A. ovata, A. Sch., from gut of Litho- bius. Genus 11. Minchinia, Labbé, 1896. Spores dizoic, with oval sporocysts drawn out into long polar filaments. M. chitonis, Lankester, 1896. Genus 12. Eucoccidium (“Benedenia’’), Liithe, 1902. Spores trizoic, schizogony absent. E. eberthi, Labbé, from Sepia. Genus 13. Klossia, A.Schn., 1875. Spores tetrazoic or polyzoic, and with spherical sporocysts. Genus 14. Caryotropha, Siedlecki, 1902. Twenty, morc or less, sporoblasts, with twelve sporozoites in each. Sporocysts spherical. One species, C. mesnili, Sied., from the spermatogonia of Polymnia nebulosa. Genus 15. Klossiella, Smith and Johnson, 1902. Sporoblasts polyzoic, sporo- cysts subspherical thirty to thirty-four sporozoites. One species, K. muris, S. and J., from the kidney of the mouse. Types of spores. (After Wasielewsky, A. Schneider, Thélohan.) PROTOZOA WITHOUT MOTILE ORGANS 65 Questionable genera of coccidiida are the following: Hyaloklossia, Labbé, 1896, from the frog. Goussia, Labbe, 1896, from various species of fish. Usually classed as Coccidium species (Fig. 20, M, N). pee Labbé, 1895, from the gut of Lithobius. Usually classed with Coc- cidium., : = Rhabdospora, Laguesse, 1895; Gonobia, Mingazzini, 1892; Pfeifferella, Labbé, 1899; Molybdis, Pachinger, 1886; Cretya, Mingazzini, 1892; Gymnospora, Moniez, 1886, are all probably species of Coccidium. Order 3. Hemosporidia, Danilewsky. Blood dwelling sporozoa cytozoic or celo- zoic in mode of life in the blood constituents, and with or without alternation of hosts. A somewhat heterogeneous collection of parasitic protozoa with obscure affinities, pointing in part toward the flagellates, in part toward the coccidia. For convenience, and purely as a temporary matter, we follow Minchin in dividing the order into two suborders, Acytosporea and Hemo- sporea, the former including those blood-dwelling forms which seem to bear some relationship to Crithidia and Herpetomonas, the latter including the more Coccidia-like forms. x Suborder A. Acyrosporea. The trophozoite is an intracellular or intracorpus- cular parasite which usually completes its schizogony within the host cell. The sexual cycle is completed in the digestive tract or body cavity of some intermediate host—in all known cases some species of blood-sucking arthro- pod, usually an insect or arachnid. Genus 1. Plasmodium, Marchiafava and Celli, 1885. The organisms of human malaria are all referred to this genus. The characteristic morphological features are the presence of melanin pigment, oval merozoites grouped around a central residua] body, and spherical or crescentic gametes. Sporogony in the gut and body cavity of mosquitoes of the genus Anopheles. Three species generally recognized P. vivax, Grassi and Feletti, 1892, the cause of tertian fever, with schizogony every forty-eight hours. P. malaria, Lav., 1880, the cause of quartan fever, with schizogony every seventy-two hours. P. immacu- latum Gr. and Fel., 1892, the cause of pernicious malaria, with subspecies according to Craig and others, exhibiting quartan and tertian characteristics. This last species is generally held to be a distinct genus under the name Laverania, Gr. and Fel., 1890, but Schaudinn’s contention that crescentic instead of spherical gametocytes is an insufficient distinction for generic difference is rapidly gaining ground, and we follow it here. Minchin’s remark (footnote, p. 267, 1903), that the popular names given to the malaria- causing parasites (‘‘tertian,” ‘‘quartan,” and ‘“‘pernicious’’) are more intel- ligible and less misleading than the so-called scientific names, is confirmed by Liihe, but it seems to us that such confusion is only further aggravated by their retention of the generic name Laverania. In addition to the species of Plasmodium causing human malaria, Laveran described a species from the blood of apes under the name of P. kochi, and Liihe places in the same species the blood parasites of chimpanzees from Kamerun. Subgenus. Hemoproteus, Kruse, 1890. The cause of bird malaria. Merozoites and schizogony as in the preceding, sporogony in the digestive tract and body cavity of mosquitoes of the genus Culex. Gametocytes bean-shaped. The various species of this genus are now commonly referred to the genus Plas- modium. Common in birds. Genus 2. Babesia, Starcovici, 1893. (Syn., Pyrosoma, Smith and kilb.; Piro- plasma, Patton.) An intracorpuscular parasite of mammalian blood. Tro- phozoites usually piriform, without pigment, and reproducing by simple division or by budding within the blood corpuscle. Transmission by ticks and sporogony in the latter’s gut. 5 66 GENERAL ORGANIZATION OF THE PROTOZOA Many species: In man, B. hominis, Manson, the disputed cause of Rocky Mountain Spotted Fever; in cattle, B. bovis, Babes, 1888, and B. bigemina, Smith and Kilbourne, 1893, and B. parvum, Theiler, 1904; in sheep, B. ovis, Babes, 1892; in horses, asses, and mules, B. equi, Laveran, 1901; and in dogs, B. canis, Piana and Galli-Valerio, 1895. Some genera of questionable taxonomic value are referred to this suborder. Among them Polychromophilus, Dionisi, 1900, and Achromaticus, Dionisi, 1900, from the blood of bats of the genera Vespertilio and Vesperugo, must be temporarily placed. The former is characterized by the presence of pigment similar to that of Plasmodium, while in the latter such pigment is absent. Suborder B. Hemosporra, Minchin, 1903. Intracellular blood parasites usually called Hemogregarines, which become free in the blood. Alternation of hosts in some cases, but apparently not in all. Parasites mainly in cold-blooded animals. Genus 3. Lankesterella, Labbé, 1899. (Syn., Drepanidium Lank., 1882.) ‘The parasite is not more than three-quarters of the length of the blood cell of the frog in which it lives. Many species in many different species of frogs and toads. Life history not yet satisfactorily worked out; according to Hintze, it is completed in the frog’s blood and digestive tract; according to Billet, it involves a trypanosome phase analogous to that of Halteridium, as described by Schaudinn (Hemoproteus). Further observations are much needed. Genus 4. Hemogregarina, Danilewsky, 1885. The body of the parasite exceeds the length of the blood cells of reptiles which it infests, and is bent in the form of the letter U. Life history unknown, although varied observations have been recorded in connection with the ten or more species that have been described (see Liihe). Genus 5. Hepatozoin, Miller, 1908. A liver cell, and blood parasite of rats. Schizogony in liver cells, engulfing and encapsulation in leukocytes, dissolu- tion of capsule and copulation of gametes in the digestive tract of the inter- mediate host (a gamasid mite, Lelaps echidninus); sporulation in the body cavity of the mite, ingestion of the mite and its parasites by rat, penetration of gut wall by sporozoites and new infection of liver cells. One species, H. perniciosum, Miller, 1908 (Fig. 106, p. 271). Class II. NEOSPORIDIA, Schaudinn. Sporulation of the ameboid parasites takes place during the activity of the parent cell and without interfering with the vegetative processes. Celozoic, histozoic, or cytozoic parasites, mainly of vertebrate hosts, and especially of fish. Order 1. Myxosporidia, Biitschli. Relatively large neosporidia reproducing by pansporoblast formation, the spores provided with polar capsules containing more or less easily seen threads. Suborder 1. Disporea, Doflein, 1901. One pansporoblast containing two spores, produced by each trophozoite. Spores wider than long. Trophozoites float- ing freely in the fluids of various organs of fish hosts and frog hosts. Family 1. Ceratomyxide, Doflein. With the characters of the suborder. Genus 1. Ceratomyxa, Thélohan, 1892. The two valves of the spore produced into long attenuated processes. About nine species, mostly from the gall- bladders of fishes (Fig. 20, @). Genus 2. Leptotheca, Thélohan, 1895. Valves of the spore not drawn out into long processes. The sporoplasm completely fills the spore membranes. About six species from the gall-bladders of fishes and the kidneys of frogs (Fig. 20, J). Suborder 2. Porysporra, Doflein. More than two spores, usually a great num- ber, produced in each pansporoblast. The spores are longer than wide. PROTOZOA WITHOUT MOTILE ORGANS 67 (The characteristics distinguishing these two suborders are not very definite, and some more natural system should be worked out with further knowledge of the group. Under the polysporous forms, for example, the genus Sphero- spora is exceptional in having at least one disporous species and in having nearly spherical spores.) Family 2. Myxidiide, Thelohan, 1892. The trophozoites are typically free-living parasites in the fluids of the internal organs of their hosts; the spore has two polar capsules. Genus 3. Spherospora, Thélohan, 1892. With spherical spores. Four or five species, mostly from fish kidneys. Genus 4. Myaidium. Biitschli, 1882. Spores navicular, with polar capsules at each end. Seven or more species from kidney and gall-bladder of fishes and tortoises. Genus 5. Spheromyxa, Thélohan, 1892. Spores navicular with truncated ends and a polar capsule at each extremity. Polar filaments are short and thick, an somewhat conical in form. Three species from the gall-bladder of shes. Genus 6. Cystodiscus, Lutz, 1889. Trophozoites without ameboid movement or changes of form; spores symmetrical with the sutural plane running obliquely from one extremity to the other and with a polar capsule at the extremities of the oblique suture. One species, C. immersus, Lutz, from the gall-bladder of toads and Cystignathus in Brazil. Genus 7. Myxosoma, Thélohan, 1892. Spores flattened and ovoid in form and with the polar capsules crowded together at the narrow extremity. One species, M. dujardini, Thél., from the gills of Scardinius sp. Genus 8. Myzoproteus, Doflein, 1898 (Myxosoma ambiguum of Thélohan and Labbé). Spores somewhat pyramidal with spinous processes from the base of the pyramid. One species, M. ambiguus, from the bladder of Lophius piscatorius. Family 3. Chloromyxide, Thélohan, 1892. Spores with four polar capsules. Genus 9. Chloromyxum, Mingazzini, 1890. With the characters of the family. Several species (six or seven) known and distinguished by presence of appen- dages and distribution of polar capsules. Family 4. Myxobolide, Thélohan, 1895. Typical histozoic parasites rarely found in the ameboid form but usually as cysts filled with spores. Usually poly- sporous, the spores with one or two polar capsules. The sporoplasm contains vacuoles which are stained a reddish brown by iodine. Genus 10. Myxobolus, Biitschli, 1882. Spores ovoid or flattened into an ellipse. Polar capsules single or double. A great many species (about forty) known, and found in some organ or other of various fishes, and usually in the connec- tive tissue of such organs. The genus is usually split up into three divisions, the first of which contains the aberrant forms M. piriformis and M. unicap- sulatus from the tench, with a single polar capsule and with pear-shaped spores. In the second are species with spores having polar capsules of dissimilar size. In the third are the great majority of the species referred to this genus, all with polar capsules of similar form and size (Fig. 20, K). Genus 11. Henneguya, Théelohan, 1892. Ovoid spores with two polar capsules, the sporocyst prolonged into two long caudal processes which are not pene- trated by the sporoplasm. Four species from stickleback, pike, and perch. Genus 12. Hoferella, Berg, 1898. Spores broad and compressed with two tail- like processes at the posterior end. One species, H. cyprini, Dofl., from the carp. Order 2. Microsporidia, Balbiani, 1883. The trophozoites are more or less ame- boid; the spores are very minute, piriform, and with only one polar capsule which is invisible in the fresh state. They are typically parasites of inverte- 68 GENERAL ORGANIZATION OF THE PROTOZOA brates and usually of crustacea and other arthropods, where they are typically cytozoic. Family 5. Glugeide, Thélohan, 1892. With the characters of the order. Group A. Polysporogenea, Doflein, 1898. The trophozoite produces many pansporoblasts, each of which gives rise to many spores. Genus 13. Glugea (Nosema), Thélohan, 1891. With the characters of the group. Many species which are not satisfactorily worked out. The most famous species is G. bombycis, which caused the destructive epidemic among silk- worms from 1850 to 1865. Group B. Oligosporogenea, Doflein, 1898. The trophozoite produces but one single pansporoblast. Genus 14. Gurleya, Doflein, 1898. The pansporoblast produces four spores. One species, G. tetraspora, Dofl., from Daphnia maxima. Genus 15. Thélohania, Henneguy, 1892. The pansporoblast produces eight spores contained in small spherical or fusiform vesicles. Five species recorded, all from the muscles of crustacea. Genus 16. Pleistophora, Gurley, 1893. The pansporoblasts produce more than eight spores. Many species, some of fish, but mostly of invertebrates. Order 3. Actinomyxidia, Stolé, 1890. Sporozoa consisting of a double cellular envelope, three polar capsules, and eight spores arranged in ternary sym- metry. Genus 1. Hexactinomyzxon, Stolé, 1899. Spores in anchor form, with six branches. H. psammoryctis, Stolé, 1899, in the intestinal epithelium of Psammoryctes barbatus. Genus 2. Triactinomyxon, Stolé, 1899. Spore in anchor form, with three branches. T. ignotum, Stolé, 1899, in the intestinal epithelium of Tubifex tubifea, Miiller. Genus 3. Synactinomyxon, Stolé, 1899. Spores associated in a common envelope. S. tubificis, Stolé, 1899, in the intestinal epithelium of Tubifex rivulorum, Lam. Genus 4. Spheractinomyxon, Caull. and Mesnil, 1904. Spores spherical and without wing-like prolongations. S. stole’, C. and M., 1904, in the body cavity of marine oligochetes (Clitellis arenarius, O. F. M.), ete. Order 4. Haplosporidia Caull. and Mesnil, 1899. A group of little-known para- sites with obscure affinities and undetermined life histories. Caullery and Mesnil, 1905, group them in three somewhat ill-defined subdivisions, as follows: Family 1. Haplosporidiida, C. and M., 1905. Parasites of ameboid form, which reproduce by encapsuled merozoites, which may or may not be ornamented by spines or processes. Genera Haplosporidium and Urosporidium, with six species enumerated by C. and M., all parasites of annelids. Family 2. Bertramiide, C. and M., 1905. With two genera, Bertramia and Ichthyosporidium, and with four species parasitic in annelids, rotifers, and fish. Family 3. Celosporidiidw, C. and M., 1905. With three genera, Celosporidium, Mesnil and Marchoux, 1898; Polycaryum, Stempell, 1901; and (?) Blastuli- dium, Ch. Perez, 1903, mainly parasites of copepods. Doubtful forms, includ- ing the genera Schewiakowella, C. and M., 1905, parasite of Cyclops, etc.; Chytridiopsis, A. Schneider, 1884, parasite of Tenebrio mollitor and of Blaps; Celosporidium, Crawley, of Blattella germanica; Lymphosporidium, Calkins, 1898; and Rhinosporidium, Minchin and Fantham, the cause of nasal tumors in man. Order 5. Sarcosporidia. Sporozoa in which the initial stage is passed in muscle cells of vertebrates. Great sac-like spore cases are formed (Miescher’s tubules) with double membranes. Genus, Sarcocystis, Lankester, 1882 (Fig. 79, p. 186). CHAPTER II. PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA. EHRENBERG, in 1838, entitled his monumental work on the protozoa Die Infusionsthierchen als vollkommene Organismen (The Infusoria as Complete Organisms). Despite the great improvements that had been made in the microscope, and the vast collection of facts that had accumulated in connection with the structures of the protozoa, Ehrenberg’s point of view was but slightly advanced beyond that of Leeuwenhoek one hundred and fifty years before. ‘‘ Animalcula,” said Leeuwenhoek, “which swim in stagnant waters, and which are no longer than the tails of the spermatic animalcula, are provided with organs similar to those of the highest animals. How marvellous must be the visceral apparatus shut up in such animalcula!’”’ Ehren- berg sought to make out the various organs in this ‘‘visceral complex,” and with great ingenuity managed to find digestive tract, kidney, brain, heart, ovary, and other organs characteristic of metazoa. The red, so-called ‘“‘éye spots” were regarded by him as eyes, and the colorless lens upon which they frequently lie was interpreted as.a cerebral ganglion, or brain. The contractile vacuole became, for him, a beat- ing heart, and the collecting canals formed the vessels. The macro- nucleus was an ovary, the gastric vacuoles stomachs, while various chance inclusions were regarded as organs of one kind or another. While Leeuwenhoek’s and Ehrenberg’s interpretation made out these primitive animals as marvels of creation in miniature, how much more marvellous are the facts as we know them today and summed up in the statement that the functions of all of these organs of the highest animals are performed within the single cell! The protozoén has no digestive tract, but it seizes food, digests and assimilates it, and grows in size through the addition of such food. It has no heart or circulatory system, and yet it distributes the digested food throughout the body, takes in oxygen, and throws off carbon dioxide as does every many celled animal. It has no kidney, but disposes of the waste matters of oxidation none the less, and so every function of the highest metazoa finds its counterpart in the vital activities of the primitive forms. Nor is the importance of these simpler processes of the proto- zoa any the less, in that they come very close to the ordinary physical and chemical processes that we are familiar with in non-living matter. As complete organisms, therefore, in a sense quite different from that 70 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA meant by Ehrenberg, the protozoa today offer a field of research in physiology that is quite unique, for while they epitomize the vital activities of the higher animals, these activities are of such simple types that they may be more easily observed and correlated with the ordinary reactions in physics and chemistry, reactions which we do not associate with the vital processes of the higher animals. The warning may not be out of place here that despite the simplicity of function in the protozoén, and the analogy with reactions in the inorganic world, there is, nevertheless, a power of acting as a whole, a power of coérdination combined with factors of adaptation and Fic, 21 —-e----- -9 Food-taking. A, after Penard; B and C, after Biitschli. A, Raphidiophrys elegans, H. and L.; B, Oikomonas termo, Ehr.; C, Didinum nasutum, O. F. M.; f, food particles. evolution, which permit of development into more and more com- plicated structural units, which arises, per se, in all protoplasm, and raises it immeasurably above the most complex of non-living sub- stances; this power of adaptation is an inherent characteristic of living matter, transcending physical or chemical analysis, and justify- ing, perhaps, the often abused term vitalism. It must not be forgotten that, notwithstanding the simplicity of the single functions, the proto- zoa are units exhibiting a complex of these activities and an harmonious working of them all, no less surely than fish, bird, or mammal. In studying these simple functions it is well not to forget that each belongs in the same category of activities as the functions of much more highly PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 71 evolved organs. Consciousness, for example, an attribute of the brain and central nervous system in general, is not seen as such in the protozoa, but its prototype irritability, with the codrdinated responses to stimuli, is common to every protozoén, and such stimuli sometimes lead to reactions on the part of the protozoén which are often appar- ently directed toward a given end until we are tempted to interpret them as conscious acts. While most of the actions of protozoa are reactions to external stimuli, many are combinations of reactions that do not lend themselves to analysis. Such, for example, is the apparent choice of food or of building material for shells and tests, or the com- plex reactions that are frequently involved in the avoidance of some obstruction. Not infrequently such reactions have been interpreted as evidence that the protozoén acts wilfully, or with a certain amount of intelligence of the end to be accomplished, and they are frequently cited as examples of conscious activity on the part of these primitive forms. Many of these so-called conscious acts can be explained by the ordinary physical laws of fluids, and while one cannot deny that the protozoén’s actions may be conscious, it seems much more prob- able that these activities are the fundamental, often physical or chemi- cal, reactions which serve in evolution as the starting point for the infinitely more complex activities which we call our consciousness. In all animals there is a certain amount of work done in the daily life, and the energy put into such work comes from the oxidation, or physiological burning, of the body protoplasm. ‘There is, therefore, a constant waste of protoplasmic material which goes off as work done, as heat, or as residual waste matters comparable with the smoke and ashes of physical combustion. Such waste is made good by the addition of new raw materials in the form of food, which is made over into new protoplasm. The phenomena of waste and renewal are usually spoken of together under the name of metabolism—waste as destructive, repair as constructive, metabolism. Food getting, there- fore, becomes the first necessity of the living thing, and the chief end toward which the fundamental structures of the body are directed, and this, whether in the highest mammal or the lowest protozoén, becomes the chief economic problem to be solved (Fig. 21). The methods employed by different kinds of living things are widely varied, and the great problem is apparently well solved in many dif- ferent ways. Green plants are the starting point for all living things, for they manufacture not only their own food, but indirectly the food for all other living things. This they are able to do because of the chlorophyl or green colored matter which they possess and which has the power to utilize the energy of sunlight in reducing CO, and manu- facturing starch out of water and carbon. ‘The further changes of the starch into more complex substances, and these into protoplasm of the plant, are buried in the obscurity of unknown chemical processes 72 ‘PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA which take place in the plant’s protoplasm. Animals solve the problem of nutrition by living on plants, or by eating other animals which, either directly or indirectly, live on plants. Still other types live as parasites upon other animals, some, like the intestinal worms, using freely the foods that are prepared by, and for the use of, the host, while others, like some insects, suck the blood, or, like trichina, invade the cells and tissues, and live at the expense of the living protoplasm. In the group of protozoa all of these methods of food getting are found. Many forms possess chlorophyl, and like the green plants, Fie. 22 Synura uvella, a colony of phytoflagellates, often a source of disagreeable odors and tastes in drinking waters. (After Calkins.) manufacture their food directly from simple elements. These protozoa. are of considerable theoretical interest, for they stand upon the border- line between the animal and the plant kingdoms, and are sometimes classed as one, sometimes as the other. They are thus involved in what has been one of the most contested of biological problems, the limits of the animal and plant kingdoms, and the problem is the more difficult because some types of this intermediate group may on occa- sions make their food, while at other times they eat like undoubted animals and take in solid food (Chromulina flavicans, and some forms of dinoflagellata). The problem has but little significance in the present PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 73 day, for biologists recognize that it is only an academic matter after all, and merely affords further evidence of the artificiality of classi- fication. It is to these intermediate forms that we must turn for the causes of odors and tastes, which occasionally make potable waters unfit to drink. As shown in the previous chapter, the metaplastic products of vital activity are sometimes stored up in the cell as oils or fats, which, when liberated in a water supply, give rise to offensive odors and tastes (Fig. 2). Like all organisms which make their food, these sus- pended ieee require salts of different kinds. Many such salts are normal to drinking waters, the nitrites and_ nitrates being almost invariably present, and these are the very salts most needed for the maintenance of these forms of life. Hence, it follows that if an infected water supply can be freed from an excess of such nitrogen- holding salts, the protozoa will disappear. If inlet and outlet of a given water supply are closed, the organisms soon exhaust the avail- able food elements and die. While some forms of protozoa are thus holophytic, like the green plants, others combine the holophytie with the animal, or holozoic method, while still other protozoa, and, indeed, the great majority of them, are entirely holozoic. They seize their food in the form of other minute living things and digest it in much the same way that higher animals do, all of the organs of the cell playing some part in the pro- cess. Food-getting, therefore, more than any other function of the body, has been the most influential in leading to morphological development. Seizure of food is one of the most interesting of the protozoén pro- cesses, and is frequently accompanied by such complicated reactions on the part of the minute animal as to suggest wilful activity. In other cases it is quite mechanical, as, for example, in choanoflagellates, or in many ciliates. In these the motile organs, flagella, or cilia, create a current in the surrounding water toward the mouth, and this carries with it bacteria or minute pieces of disintegrated plant or animal matter. In Vorticella campanula and its allies the apparatus is most highly developed for this method of food taking. A powerful adoral zone of membranelles creates a vortex current toward the oral or vestibular opening, while within the vestibule a long, undulating membrane carries the current to the mouth opening. ‘The proto- plasmic area around the mouth is furnished with contractile muscle threads or myonemes, so that when any irritating object comes with the food current, the entire vestibular area, adoral zone and all, con- tracts into the cell body, while the myonemes of the ereaaid: stalk contract at the same time and draw the body away from the offending region. In other ciliates, like paramecium, colpidium, oxytricha, etc., the process is essentially the same except that the animal is not 74 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA attached nor provided with contractile fibrils. In all of these ciliated forms there is a definite and frequently very complicated mouth opening, but in the flagellated forms, as a rule, there is no permanent mouth, the entire anterior end of the cell forming a receptive area for food products swept toward it in the current created by the flagel- lum. This is a vortex current caused by the undulations of the long flagellum, which, at the same time, moves in such a way as to describe a cone whose apex is at the base of the flagellum and base at the tip. In some cases, as in the collared flagellates or choanoflagellata, the flagellum moves inside a protoplasmic, collar-like membrane, which, like a pseudopodium, can be thrown out or retracted by the animal. The surfaces of this collar are sticky, and small particles move down it to the floor of the collar pit, where they are taken into the body. As the flagella and cilia are in constant action, and as the mouth is always open for more, these protozoa become, as Maupas pointed out, the gluttons, par excellence, of the animal kingdom, while the oral apparatus becomes strikingly modified and diversified. Not all protozoa, however, are so persistent in food taking, and many of them, while provided with a mouth opening, keep the mouth shut until a food particle is to be eaten. Such forms live upon larger things than bacteria, and with them eating involves a regular swallow- ing process. In some cases this is combined with the food-getting activity of the flagella or cilia, and large particles of solid proteid matter, either in the form of small organisms or of disintegrated fragments of plant or animal brought with the current, are seized by protoplasmic processes, as in Oikomonas termo, or the mouth opens to swallow them, as in Didiniwm nasutum. There seems to be a remarkable power of distention in these mouth openings, for a didinium can take in an organism quite as large as itself (Fig. 21). In those forms of protozoa belonging to the group suctoria there is no mouth opening, nor flagella or cilia to create food currents, but the animals are provided with tentacles, often twice as long as the diameter of the body, with which they seize passing organisms. Once seized, the victim struggles for a short time and then becomes quiet, as though paralyzed. Its ‘protoplasmic contents are then sucked into the body of the captor, or, in some forms, the protoplasm of the captor passes into the body of the victim and there digests its meal. Many protozoa set a trap for their victims, so that they become entangled as in a spider’s web. This is the case with the majority of the great group of rhizopods, especially the foraminifera and radio- lane where the pseudopodia form a network of branching protoplasm, or a forest of protoplasmic spines, in which the streaming of granules is constant, passing from the inner protoplasm of the shell outward to the farthest tip of the pseudopodia. The sticky character of the pseudopodia makes it difficult for any small animal to break away, PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 75 while its struggles furnish the stimulus for an accumulation of more protoplasm about it, and this, armed with digestive fluids, soon kills Fic. 23 Allogromia, sp., with pseudopodial net and two diatoms. (After Calkins.) 76 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA the prey, which is then digested without even the formality of carriage into the shell of the captor (Fig. 23). Other rhizopods, as an ameba, throw out pseudopodia under the stimulus of the touch of some other living animal or plant. These Actinobolus radians with tentacles partially retracted and with five ingested halterias; swimming. (After Calkins.) surround the victim, which frequently does not begin to struggle until ensheathed in a wall of protoplasm, from which it rarely escapes. Large animals like rotifers, and relatively large plants like the des- mids are thus captured and digested. While most of the protozoa thus far described wait until the prey PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 77 comes to them, and take what they can get, others are predatory and go in search of food. These are the most interesting of all protozoa, for they are occasionally too fastidious, apparently, to take the ordinary run of the microscopic wilds, but seem to select their food with all the care of a gourmand. They are usually armed with offensive weapons in the form of trichocysts, which may be shot out from the surface of the body, or carried javelin-like, at the extremities of projectile tentacles. One of the most interesting of these types is Actinobolus radians, one of the most primitive and one of the surest of hunters (Fig. 24). “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 (Entz, 1883). When at rest, the mouth is directed downward, and the tentacles are stretched out in all directions, forming a minute forest of plasmic processes, among which smaller ciliates, such as urocentrum, gastrostyla, etc., or flagellates of all kinds may become entangled without injury to themselves and without disturbing the actinobolus or drawing out the fatal darts. When, however, an Halteria grandinella, with its quick and jerky movements, approaches the spot, the carnivore is not so peaceful. The trichocysts are dis- charged 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 contracted until the victim is brought to the body, where by action of the cilia it is gradually worked around to the mouth and swallowed with one gulp. Within the short time of twenty minutes I have seen an actinobolus thus capture and swallow no less than ten halterias.” (Calkins, The Protozoa, p. 50.) The complicated processes involved in this act of food-getting would certainly justify an Ehrenberg in the belief that actinobolus is capable of wilful actions to a certain end, and that in the apparent choice of food, and skill in bringing it down, it shows a high order of intelligence. It would be a natural tendency to interpret such activities in terms of our own consciousness, but it is much more probable that simple physical or chemical laws of attraction are at the bottom of it all, halteria possessing an attraction for the darts of actinobolus analo- gous to that between an iron filing and a magnet, or between various chemical elements. In all of the above cases solid food is taken into the body of the protozoon and there disintegrated and digested. Many other protozoa have no mouth opening nor chromatophores to manufacture their food, but absorb it through the general surface of the body, as does a tapeworm. Such protozoa, like some of the lower plants, are sapro- phytes and get their nutrition in the proteid matter from disintegrating 78 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA plant and animal tissues, dissolved in the water. Other saprophytes live upon the juices in blood or other fluids of the animal body which are similarly taken in by osmosis; these, however, belong to the group of parasites or commensals, the difference between the two being largely one of degree only, a parasite exerting some deleterious effect upon the host, while a saprophyte and a commensal are harmless. In all such cases the protozoa multiply in the region, such as a water supply, or the fluids of the body, where food is most abundant and Fie. 25 Digestion in a foraminiferon. (After Verworn.) A-E, successive stages in the disintegration of a ciliate (Colpoda) in a pseudopodium of Lieberkiihnia. where they are least disturbed by environmental factors. Thus, we would account for the immeasurable swarms of chilomonas in a meat infusion, or quantities of opalina in the frog’s rectum, or the myriads of cytoryctes and neuroryctes in skin and brain of victims of smallpox and rabies. In the higher animals solid food materials are taken into the food receptacles of the body, where a secretion from the lining epithelial cells is poured upon them, the food matter not coming in close contact PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 79 with the secreting cells. In the protozoa the solid food is taken directly into the living cell, and the processes of digestion are all within the living matter. Such a method is known as intracellular digestion, as Pemireisted with intercellular digestion of the higher pies (Fig. 25). When a rotifer or other cial animal is enwrapped by the pseudo- podia of an ameba, or swallowed by an actinobolus or other preda- tory form, a certain amount of water is taken in with it so that the victim moves freely within the body of its captor and in its normal water environment. ‘The water, with victim, forms a gastric vacuole or an “improvised stomach,” and is surrounded on all sides by a wall of living protoplasm, and this soon begins to pour a secretion into the vacuole. With the first changes in chemical nature of the surrounding water the prey begins to struggle, and ceases its efforts to escape only when killed by the secretion. This, according to the researches of Fabre-Domergue, Meissner, le Dantec, and others, is acid in nature, but, beyond the fact that it is some mineral acid probably hydrochloric as in other animals, nothing is known as to the exact chemical nature of this digestive fluid. Whatever it is, its manufacture is intimately Bearecied: with the chromatin material of the nucleus, for Hofer and Verworn have shown that digestion does not take place when the nucleus is absent. This was "determined by cutting an ameba into two parts, one of which contained the nucleus, the “other, a recently ingested animal. The enucleated protoplasm retained its vitality for from nine to fourteen days without any change in the gastric vacuole; the nucleated fragment, on the other hand, soon recovered from the operation and began to digest as usual. It is probable that the minute particles of nucleoproteids that are constantly arising in the neighbor- hood of the nucleus contain digestive ferments which stimulate the formation of the mineral acid in the vicinity of the gastric vacuole. In those protozoa in which the mouth is continually open, as in paramecium, vorticella, dileptus, bursaria, etc., the food is usually minute forms of Gercellnlar algze, or, most nee bacteria. ‘These are collected in water in the protoplasm at the base of the vestibular opening until a great number are massed together, or until the vacuole has assumed a certain size. It is then caught up in the flow of proto- plasm on the interior of the organism, and dragged away from the mouth, while a new vacuole begins to form. The process of digestion in one of the bacteria-eating goricellids, carchesium, has been eeudied by Greenwood, who found ‘that the ageregate of bacteria passes into a region of protoplasm i in the immediate vicinity of the horseshoe-shaped melee: where the water disappears, leaving ‘the bacteria in close con- tact with the protoplasm. This state of ‘ storage” lasts for from one to twenty hours, and during the time the many separate or individual bacteria are massed together into a compact ball of food. This mass is then again surrounded by fluid, this time having a decidedly acid 80 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA reaction. Through the action of this acid the compact mass of bac- teria is broken into minute fragments, which ultimately mix with the protoplasm as digested food. Although nothing further is definitely known about it, it is quite probable that the product of this digestive action is the formation of soluble peptones similar to the products of proteid digestion in the higher animals. This is rendered the more probable because of the extraction of a pepsin-like ferment from the myxomycete Fuligo varians by Krukenberg, and from the huge ameboid rhizopod Pelomyxa palustris by Dixon and Hartog. The problem of the nature of the digestive processes in protozoa has an interest in connection with other questions of more vital impor- tance. The nature of the digestive reaction in phagocytes in response to the food matters supplied are involved in the general subject of intracellular digestion. While the initial experiments of Engelmann, Metchnikoff, Le Dantec, Greenwood, and others showed that there is an acid reaction in the gastric vacuoles of certain forms of protozoa, their conclusion that digestion here is entirely due to the action of some ferment-like pepsin acting in an acid medium were apparently pre- mature. The extraction by Krukenberg from fuligo, and by Dixon and Hartog from pelomyxa, of a digestive ferment which dissolves proteid in an acid medium, undoubtedly lends support to their view. But, on the other hand, Mouton (’02) extracted a digestive ferment from ameba which disealy es gelatin and fibrin in an alkaline medium, while Mesnil and Mouton (’03) extracted a similar ferment from para- mecium. ‘These observers, therefore, insist that the digestive fluid is more like trypsin than like pepsin. An intermediate position was taken by Metalnikoff (03), who, on the basis of repeated observations, claimed that the reactions in the paramecium vacuole are first acid and then alkaline. Feeding paramecium with powdered alizarin, which is colored reddish violet in an alkaline medium in which paramecium lives, he found that the vacuoles are at first of this same color. In from five to fifteen minutes the color changes from red to yellow, showing an acid reaction, and this, after from ten to fifteen minutes more, is changed again to the red, showing an alkaline reaction. Not all vacuoles are thus colored, a few giving the alkaline reaction throughout. Metalnikoff concluded, therefore, that proteid digestion in these protozoa follows the same course as in higher animals, a ferment acting in an alkaline medium following one which acts In an acid medium. Nierenstein, repeating these experiments, confirmed Metalnikoft’s observations, but came to the conclusion that the acid medium plays no part in the actual digestion of the food, serving merely to kill the living organisms taken in. Metalnikoff, however, ina later publication maintains that the bacteria swell in the acid medium and thus undergo the first steps in the process of digestion. These results differ to some PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 81 extent from those obtained by Greenwood in the case of carchesium, where the acid reaction is not forthcoming until after the “state of storage,” a state varying in length of time from one to twenty hours. The chemical reactions in the later periods were not observed. The protozoo6n, therefore, like phagocytes, evidently has the power of secreting different kinds of ferments in response to the stimulus of different kinds of living food particles. Not only proteolytic, but other kinds of ferments as well are formed in the various types of protozoa, although not by all kinds. Thus, some types of protozoa are able to create starch dissolving ferments similar to the diastatic fer- ments of higher animals, or fat emulsifying ferments similar to steap- sin. In many forms, however, the starch grains, like other indigestible parts, are thrown out of the body untouched (Greenwood, Fabre- Domergue, Meissner). The granules that are formed by the breaking down of food par- ticles through the digestive process are ultimately distributed by means of the protoplasm streaming to all parts of the protozodn. Some are probably converted directly into protoplasm by an assimilative pro- cess that is as little understood in these forms as in the metazoa, a process involving synthetic changes whereby the relatively complex food elements are built up into still more complex protoplasmic molecules, thus leading to the repair of waste and to growth. Other granules are not immediately assimilated, but are stored up in the protoplasm as a reserve of nutriment. In these cases it is impossible to say whether such granules are utilized directly as fuel for functional activity through oxidation, or whether they are first built up into pro- toplasm and the protoplasm itself, or its products, oxidized. In all protozoa these reserve matters are present, giving the characteristic granular appearance to the protoplasm of these forms, and their dis- appearance may be easily followed by starving the individual. A paramecium, for example, when normal and active, has a character- istic granular appearance, while numerous gastric vacuoles are dis- tributed throughout the inner protoplasm. When it is starved these granules disappear first of all, and then, with continued starvation, the protoplasmic network is used as a source of energy for the active animal, and great vacuoles appear which increase in size with starva- tion, while the size of the cell decreases to an eighth or a sixteenth of the normal volume, the macronucleus alone, although frequently fragmented, retaining its normal volume. It often happens that some one of the many functions of metabolism fails to act, and the organism suffers from the failure to assimilate or from lack of oxidative ferments. I have frequently seen Paramecium aurelia so filled with these reserve food granules that its protoplasm appeared dense and black under the microscope (Fig. 26). In such cases there are no gastric vacuoles, food taking and movement stop, 6 82 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA a division stops, and the animal, unless treated, invariably dies. The trouble seems to be due to the lack of oxidative processes, possibly because the nucleus fails to provide the necessary ferments. The tension is relieved and activity again started up by treating such an organism with salts like potassium chloride or potassium phosphate, or with the more complicated salts contained in an extract of pancreas. It is possible that in the latter case the extracts from the pancreas have some direct effect upon the granules in question, but such an explana- tion cannot account for the successful results with the simple potassium salts, and it seems more probable that the explanation lies in the fact that the stimulants act directly upon the nucleus and cause it to resume a neglected function. This conclusion is borne out by the fact that the tension is first relieved in the immediate vicinity of the nucleus (Fig. 26), and then progressively toward the ends of the organism. Fic. 26 “ Paramecium aurelia in condition of protoplasmic ‘‘stability’’ (extreme left) and resumption of normal “‘labile’’ condition as a result of treatment with salts. The inner processes of digestion are entirely unknown in the sapro- phytic forms of protozoa and in the parasitic forms, but there is reason to believe that it is taken up at the point of granule formation in other, holozoic, forms. In parasites like trypanosoma living in blood lymph the nourishment is probably derived from the digested food materials carried by the blood and upon which the organisms, pre- sumably, live as saprophytes. Such forms are quite different, physio- logically, from intracellular .or intracorpuscular parasites, such as coccidia, malaria organisms, etc., which live upon the substance of the cells or blood corpuscles. PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 83 The free-living forms of protozoa are almost constantly at work; they are usually in motion, either in progressive movement, or, by action of their flagella or cilia, are creating currents toward the mouth. The energy for such work comes from the breaking down of complex molecules of protoplasm or possibly of digested food, which is accomplished by oxidation or physiological burning. The products of such combustion, as in physical combustion, are kinetic energy, heat, and residual matter, and the latter, like ashes, must be disposed of, or by accumulation they hinder and ultimately prevent the normal processes. The ordinary products of such physiological activity are solid or fluid matters consisting mainly of water, some mineral sub- stances, urea, and a gas, carbon dioxide. In higher animals the former are disposed of through the medium of the skin in part, but mainly through the activity of the kidney, while the latter are thrown out through the skin and lungs, or gills. In protozoa, while there is the same need of elimination of the waste materials, there is in many forms no especial organ for the purpose, elimination of urea and of carbon dioxide taking place, as in some intestinal parasitic worms, by osmosis through the general surface of the body. Such is the case in all of the foraminifera and radiolaria, and in individual cases among the other types of protozoa. In other forms of protozoa, however, there may be special organs for the disposal of such waste matters. These are the contractile vacuoles which fill with fluids from the interior of the cell and then contract, emptying their contents to the outside through a minute pore, as in the majority of infusoria, or breaking through the outer wall of protoplasm at any point where the vacuole may be at the time of contraction, as in amebea. The fluids of these contractile vacuoles are supposed to hold urea in solution as well as carbon dioxide, the experiments of Griffiths (’89) indicating the presence of urea, while biologists generally agree that carbon dioxide must also be present in the fluids discharged, although in no case has this been proved. Another function of the contractile vacuole may be, as Hartog early pointed out, the regulation of the tension in protoplasm and surrounding water and the prevention of large dis- ruptive vacuoles through the constant addition of water taken in by the crystalloids of the cell. Whatever may be the function of the vacuole, it becomes a very important element of the cell in the more complicated forms of protozoa, and is frequently associated with long, branching feeding canals, which to Ehrenberg were evidences of a vas- cular system, since they ramify through the protoplasm, collecting fluid which is emptied into the contractile vacuole. While the function of such contractile vacuoles is elimination of waste or regulation of density, they cannot be absolutely necessary to protozoa, nor the sole means of disposing of waste materials, since great numbers of protozoa are without them. 84 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA Oxygen, necessary for the various processes of oxidation, is taken in through the general surface of the body and from the surrounding water. Little or nothing is known regarding its action in the protozoan cell. Trritability.—‘‘This liberation of energy is the ‘response’ to an action of itself inadequate to produce it, and has been compared not inaptly to the discharge of a cannon, where foot-tons of energy are liberated in consequence of the pull of a few inch-grains on the trigger, or to an indefinitely small push which makes electric contact; the energy set free is that which was stored up in the charge. This capa- city for liberating energy stored up within, in response to a relatively small impulse from without, is termed ‘irritability; the external impulse is termed the ‘stimulus.’” (Hartog, 1906, p. 8.) The sensi- tiveness or irritability of protozoan protoplasm has been a favorite branch of protozoén research, and is especially interesting in the light of comparative psychology, for here is the prototype of higher animal consciousness. It is manifested in a great variety of ways, and the manifestations have been grouped into categories called taxes or tropisms. Nearly all of these reactions take the form of motion in some form or other, and are usually called out in response to stimuli, which may be of various kinds. Mechanical stimuli, light and heat rays, electricity, diffusing chemical substances, all exert some effect on the movements of protozoa, sometimes toward the source of stimu- lation (positive taxis), sometimes away from it (negative taxis). It is this irritability of protoplasm that frequently saves the life of the small organism, or provides it with food. Positive thigmotaxis is the name given to that reaction of a paramecium, for example, when it approaches and adheres to some larger object where its bacterial food may be concentrated; positive chemiotaxis is the reaction shown in the sudden extension of the tentacles of actinobolus; positive or nega- tive aérotaxis is that reaction whereby the organism so places itself in a medium that irritability is reduced to a minimum, and so on, all movement probably being a response to stimuli which owe their origin either to external or internal causes, the latter due, perhaps, to the varying conditions of hunger, fatigue, and the like. The most extensive and illuminating observations on this aspect of protozoan physiological activity have been made by Jennings, and the results of his long studies on the behavior of lower organisms are well stated in his own words in the following theses (Jennings, 1906, p- 261): 1. “First, we find that in organisms consisting of but a single cell, and having no nervous system, the behavior is regulated by all the different classes of conditions which regulate the behavior of higher animals. In other words, unicellular organisms react to all classes of stimuli to which higher animals react. All classes of stimuli which PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 85 may affect the nervous system or sense organs may likewise affect pro- toplasm without these organs. Even the naked protoplasm of ameba responds to all classes of stimuli to which any animal responds. The nervous system and sense organs are, therefore, not necessary for the Saas of any particular classes of stimulations. . “The reactions produced in unicellular organisms by stimuli are noe ‘the direct physical or chemical effects of the agents acting upon them, but are indirect reactions, produced through the release of certain forces already present in ‘the organism. Tn this respect the reactions are comparable with those of higher animals. It is true for ameba as well as for more differentiated protozoa. “Tn the protozoa, as in the metazoa, the structure of the organism plays a large part in determining the nature of the behavior. There are only certain acts which the organism can perform, and these are conditioned by its organization; by one of these acts it must respond to any stimulus. If the behavior of the metazoa is comparable i in this respect to the action of a machine, the same comparison can be made for the behavior of the protozoa. 4. “Spontaneous action—that is, activity and changes in activity induced without external stimulation—takes place in the protozoa as well as in the metazoa. Both vorticella and hydra, as we have seen, spontaneously contract at rather regular intervals, even when the external conditions remain uniform. Continued activity is the normal state of affairs in paramecium and most other infusoria. The idea that spontaneous activity is found only in higher animals is a totally ea one; action is as spontaneous in the j protozoa as in man. “Tn unicellular organisms, without a nervous system, certain a of the body may be more sensitive than the remainder, forming thus a region comparable to a sense organ ina higher animal. Whether such a part may become more sensitive to one form of stimulation while insensitive to others, as in higher organisms, seems not to have been determined. 6. “Conduction occurs in organisms without a nervous system. This is, of course, seen in the fact that a stimulus limited to one part of the body may cause a contraction of the entire body, or a reversal of cilia over the entire body surface. A strongly marked case is the con- traction of the stalk in vorticella, when only the margin of the bell is stimulated. 7. “Summation of stimuli occurs in protozoa, as in metazoa. ‘This is shown most clearly in Statkewitsch’s experiments with induction shocks. Weak induction shocks have no effect until frequently repeated. “Tn the unicellular animal, as in that composed of many cells, the reaction may change or become reversed as the intensity of the stimulus increases, though the quality of the stimulus remains the 86 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA same. Such a change in reaction has sometimes been claimed as a specific property of ‘the nervous system. ‘The protozoa ameba and stentor, as well as the metazoan planaria, move toward sources of weak mechanical stimulation, away from sources of strong stimulation. “In the protozoa, as in the metazoa, the reaction may change while the stimulus remains the same; that is, the animal may respond at first by a certain reaction; later, while the stimulus remains the same, by other reactions. This has been shown in detail in the account of stentor. The change may consist in either a cessation of the reaction or in a complete alteration of its character. ‘These changes are, as a rule, by no means due to fatigue, but are regulatory in character. The behavior thus depends on the past history of the organism. For such modifications of behavior a nervous system is then unnecessary. 10. “In the protozoa, as in the metazoa, the reactions are not invariably reflexes, depending only on the external stimulus and the anatomical structure of the organism. ‘The reaction to a given stimulus depends upon the physiological condition of the organism. In stentor we could distinguish at least five different conditions: each with its ae reaction to the given stimulus. “Tn unicellular, as ell as in multicellular, animals we find two chiet general classes of reactions, which may be designated as positive and negative. The positive reaction tends to retain the organism in contact with the stimulus, the negative to remove it from the stimulus. In many classes of stimuli we can distinguish an optimum condition. A change leading from the optimum produces a negative reaction, while a change leading toward the optimum produces no reaction, or a positive one. The optimum from this standpoint usually corresponds, in a broad way, to the optimum for the general interests of the organ- ism. ‘These relations hold equally for protozoa and metazoa. 12. “In both the protozoa and the metazoa that we have studied, the behavior is based to a considerable degree on a selection of certain conditions through the production under stimulation of varied move- ments. When the organism is subjected to an irritating condition, it tries many different conditions or many different ways of ridding itself of this condition, until one is found which is successful. “Altogether, there is no evidence of the existence of differences of fundamental character between the behavior of the protozoa and that of the lower metazoa. The study of behavior lends no support to the view that the life activities are of essentially different character in the protozoa and metazoa. The behavior of the protozoa appears to be no more and no less machine-like than that of the metazoa; similar principles govern both.” Growth and Reproduction. —In all of the constructive pro- cesses of the cell there is no doubt that the nucleus plays the most important part, and that it is, in a sense, the directive centre of activi- PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 87 ties. This is shown by the behavior and history of enucleated frag- ments, which, as we have seen, cannot digest food; other functions are similarly crippled by removal of the nucleus, and movement itself is greatly impaired. A contractile vacuole will reform and will contract to a certain extent in enucleated protozoa, but it will not act normally and soon ceases to contract, swelling then, with the continued addition of fluids, until the cell bursts, as in the characteristic phenomenon of diffluence. When the constructive activities of the protozoan body exceed the destructive, and when the addition of new raw material exceeds the waste, new protoplasm is added to the old and growth results. The dimensions of the cell are increased in all directions, the increase taking place in the fluid protoplasm apparently throughout all parts of the cell at the same time, a process of growth by intussusception. The mere accumulation of reserve food granules plays no part in growth, all growth ceasing when the cell becomes packed with them, but must take place only after the necessary constructive changes have con- verted such reserve stores into protoplasm. Growth continues until the cell has attained to a more or less definite, optimum size, and then it divides into two or more small cells according to the species. The explanation of growth is one of the unsolved problems of biology, and we get but little nearer the solution in the case of pro- tozoan organisms than in the higher forms of life. We know, indeed, that growth ceases with the elimination of the nucleus, hence, we conclude that the nucleus is a necessary factor in the process. Growth in the protozoa can be controlled in a variety of ways, and we know that certain conditions of temperature, of density, and the like, are necessary. While the explanation of the finer processes of growth is far away, the solution of the problem of cell division is almost equally remote, and no theory yet propounded satisfies the conditions as we see them in the various forms of life. Spencer’s theory of volume and surface is very seductive; indeed, it may be a step toward the final solution. Briefly stated it predicates that a normal relation exists between the protoplasm and the nucleus of the cell, and, if the form remains the same, this relation is disturbed by growth, for the surface of the organism increases as the square of the diameter, while the volume increases as the cube. Hence it results that the mass increases faster than the surface which provides the means of interchange with the environment (absorption and the like).. The changed ratio of surface to mass of protoplasm, according to Spencer and his followers, brings about internal changes which result in cell division. But after this theory is stated, we know nothing more about the ultimate causes of cell division than we did before. When the nature of the changes is understood, the reason for cell division will naturally follow. Leaving aside the causes of cell division, and looking at the phenomena 88 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA alone, we find a far more satisfactory state of affairs, for the details of the process are known in many different cases. Whatever the causes of cell division may be, whether limits of growth or sun spots, the fact is established that the first indications of the process in the majority of cases are found in the nucleus. Here we are dealing with a universal biological phenomenon, the division of a cell, and the protozoa are interesting in this connection because of the variations in the process which they present, and also because the structures involved are less complicated than those of higher animal and plant cells, and, therefore, more easily analyzed. In all tissue cells of normal character, division is brought about through the medium of a peculiar structure of the nucleus known as the mitotic or karyo- kinetic figure. Under ordinary vegetative conditions of the cell, the A micronucleus of Paramecium aurelia in division. nucleus contains chromatin substance in the form of granules arranged in a more or less definite network or reticulum. Prior to cell division these granules become rearranged in a much wound thread or spireme, and later the spireme thread is divided across into a number of short chromatin elements known as the chromosomes, the number of such chromosomes being constant for all of the cells of the same species of animal or plant. The number of these chromosomes in no way indicates the degree of differentiation of the organism, nor its position in the animal or plant scale, some protozoa, for example, having a larger number of chromosomes than does man. In the ordinary process of mitosis these chromosomes are arranged in the centre of a spindle-formed nuclear figure consisting of fibers of kinetic substance focussed at two poles, these poles characterized by the presence of PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 89 small granules of deeply staining substance, the centrosomes. The centrosomes, spindle fibers, and chromosomes, to which the spindle fibers are attached, are collectively known as the mitotic figure, and few cells that are known divide without the formation of this mitotic figure, or some modification of it (Fig. 27). It represents, therefore, the mechanism of cell division, and further, since the hereditary char- acteristics are now known to be connected in some way with the chromosomes, the mitotic figure becomes the mechanism of heredity. The chromosomes, while in the equator of this mitotic figure, or in some cases even before the mitotic figure is formed, are divided by a cleft which passes from end to end through the centre, and the two halves, as the daughter chromosomes, are apparently drawn apart by the mechanism of the mitotic figure; the cell body is then divided into two daughter cells by a constriction or cleft passing through the middle; the nuclei reform their characteristic reticular condition, and the two cells are then ready for further processes of digestion, assimi- lation, and growth. Ever since 1883, when Roux first called attention of biologists to the extreme care with which the chromosomes were halved and dis- tributed to the daughter cells, and especially since the publication of Weismann’s classical essays on the nature and constitution of the germ plasm, these elements of the cell have been recognized as the physical basis of inheritance, and their mode of origin and complete history have been the chief subject for study by cytologists. Not only the chromosomes, but the entire spindle figure as the mechanism by which they are divided, has also demanded the attention of biologists. In this branch of biological research the protozoa have played an important part, for in these cells we find the simplest types of the division figure and the simplest forms of the chromosomes, while cell division is found in every conceivable form, sometimes strikingly similar to the division of a metazoan cell, as in some heliozoa, some- times so highly modified as to be regarded as a type by itself, as in the budding forms. Cell division, therefore, which Spencer interpreted as marking the limit of growth of a cell, is inaugurated through some change in the relations of nucleus and cytoplasm, and some change which is entirely unknown. In many protozoa the process is so different from tissue- cell division that other names are given toit. We recognize: (1) Simple binary division of the cell into equal parts, or simply cell division. (2) Unequal division of the cell, the smaller part being pinched off from the larger as a bud. This is known as budding or gemmation, and is only a slight modification of cell division. (3) Spontaneous division of the cell into four or more, frequently a great number of daughter elements, each with a portion of the original cell nucleus, the process being known as spore formation or sporulation. 90 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA These various modifications of the process of division or reproduc- tion in its broadest sense may be conveniently summarized as follows: Cells dissociated (Protista). (iaeyes 7 Aivisioe [ Undifferentiated (Protozoa | ; : f Cells associated 4 colonies). Simple + | Differentiated (Metazoa, Meta- | phyta). Reproduction 4 [ Budding division (Buglypha, etc.). Gemmation JPExogenons, i | Endogenous. Multi é . pene | Multiple ls Soulation J Schizogony (without fertilization). : Sporogony (after fertilization). Fic, 28 4 ¢ Se Trypanosoma gambiense; stages in longitudinal division. Original from a preparation by I. W. Baeslack. In a number of protozoa, the cell before division draws in or throws off its motile organs, rounds out into a sphere, and then divides into two equal parts. ‘This is the case in some of the heliozoa, a nuclearia, for example, which is very plastic with freely moving and often branching pseudopodia, becomes spherical and then dintdes through the middle, the entire operation, as seen under the microscope, taking not more than a minute. The process becomes more complicated in those forms with com- plex motile organs. In some cases, as in some forms of trypanosoma, the flagellum is divided throughout the entire length, but in other Cases ihe basal body alone iailee, a second flagellum being formed PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 91 from the free half, while in still other cases it is discarded before divi- sion, and, as in copromonas, each daughter cell creates a new one (Fig. 28). Similarly with the infusoria, some forms like paramecium, colpidium, etc., have a cover of uniform cilia which are retained during the act of division; indeed, the organisms swim vigorously throughout the entire process, but in other forms, as Euplotes patella, oxytricha, stylonychia, ete., the more complex motile organs are discarded and formed anew by the daughter cells (Wallengren) (Fig. 29). In the flagellate Noctiluca miliaris (Fig. 30), the division is accom- panied by very complicated nuclear changes, and a division figure is formed which recalls the mitotic figure of the metazoan cells. The chromatin in the ordinary conditions of the cell is contained in a few Fic. 29 Euplotes patella in division. The macronucleus is not quite divided, the daughter nuclei being connected by a delicate strand. large chromatin reservoirs or karyosomes; these disintegrate prior to division, and the granules thus formed collect in lines, the chromo- somes, which are oriented toward one pole of the nucleus. At this pole, but on the outside of the nuclear membrane, lies a large centro- some or division centre, which divides during the time of disintegra- tion of the karyosomes and forms a central spindle between the two halves. The nuclear membrane next disappears in the region between the chromosomes and the spindle, but is retained elsewhere, and special spindle fibres grow out from each of the division centres and become attached to the ends of the chromosomes. The division centres then move apart and the chromosomes are drawn asunder, each having divided through the middle. 92 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA An entirely different mode of division is found in some of the more simple flagellates. Euglena, for example, divides without any rupture of the nuclear riecateane and without any definite mitotic figure (see Fig. 10, p. 30). The chromatin is in the form of granules distributed throughout the nucleus, and surrounding a oad deeply staining, larger granule, the division centre. When the cell divides this granule first divides into two equal parts, about which the chromatin granules are equally massed, and it corresponds to the entire mitotic spindle of metazoan cells. ‘This type of nucleus (the centronucleus) is quite common among the protozoa, and from it we can trace the evolution of the mitotic figure of higher animal cells through forms like noctiluca and the ieliozoa, In some forms among the flagellates, and in some infusoria, there is no definite nucleus, but the chromatin granules are distributed through- out the cell unconfined by a nuclear membrane. This is the case with some forms of tetramitus and with some ciliates like dileptus. In the Fic. 30 Nucleus of Noctiluca miliaris in division. The light streak through the middle is the groove in which the central spindle lies. former, the chromatin granules collect about the division centre at the time of cell division, and the nucleus then divides like one of the centro- nucleus type. In the latter each of the separate granules divides, although this does not mean that each granule is represented in both daughter cells; on the contrary, only those granules pass into a daughter cell that lie in the half of the parent organism represented by that daughter cell. Division here is a means of keeping the quantity of chromatin material and the active surface up to a standard (Fig. 31). Budding differs widely from simple division, in its external appear- ance, at least, for, in the majority of cases, the nucleus does not divide until the daughter individual is nearly formed. In many rhizopods, for example, the protoplasm swells out as a large protuberance from the surface of the cell until it is quite as large as the parent cell, and then the nucleus divides and the organisms move apart, each with a nucleus and an equal portion of the. protoplasm. This is the case in PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 93 forms like arcella, difflugia, or euglypha, where the cell is enclosed in a test or shell. Here the protoplasm wells out of the mouth opening of the shell until it forms a counterpart of the parent organism, then the nucleus divides, as stated, and the two individuals separate. Such a method is complicated, and to a certain extent anticipated, by the organism, for long before the cell divides the shell plates of a euglypha are formed and stored up in the protoplasm about the nucleus of the parent organism, to be used only when the bud has reached a certain size. ‘They then flow into the bud with the protoplasmic streaming, and arrange themselves on the outside of the bud protoplasm, where they form a tightly fitting shell (Fig. 5, see A, p. 23). In other cases the buds are much smaller than the cell which forms them, and Fie. 31 Dileptus, sp., with distributed nucleus in process of division. Each of the chromatin granules is drawn out in the form of a rod and divides (see Fig. 2, p. 19). they first appear as mere protuberances on the surface of the parent (Fig. 32, Z). This is the case in forms like spherastrum, for example, and several buds may form at one time. ‘These are frequently dif- ferent from the parent and are often provided with motile organs of a different type. ‘Thus, in the heliozoa the buds may have pseudopodia of the lobose type and move around like small amebe, or they may have flagella and move around like flagellates. The former are called pseudopodiospores by Lang, and the latter flagellispores. In all cases, however, the bud soon loses its larval motile organs and develops into an organism similar to the parent (see Fig. 11, p. 31). In the case of acanthocystis, the buds require five days for their com- plete development, the characteristic centralkorn and the ray-like pseudopodia appearing on the sixth day (Schaudinn). 94 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA Budding, in cases like the last, is very similar to spore formation, and can scarcely be distinguished from it. Many instances of budding are presented by different groups of the protozoa, and in all of them the process is characterized by the fact that the parent organism continues to live as an individual after giving rise to these motile offspring. In spore formation, on the other hand, the substance of the parent in the majority of cases is used in the formation of the offspring, and it loses its life as an individual. In noctiluca the buds are formed after the nuclei divide, and appear as minute swellings on the surface. The nuclei in these swellings divide repeatedly until about five hundred buds are formed; these Fie. 32 Entameba histolytica. (After Craig.) A, organism showing rods and granules of chro- matin in the nucleus, vacuole with some stained substance, and dense ectoplasm; B, the chromatin of the nucleus passing into the cell plasm, where it is distributed as chromidia, shown n C; D, aggregation of chromidia to form secondary nuclei (see Fig. 51, of Ameba limax); EZ, ‘spore formation”? by budding; F, spores of Entameba histolytica as seen in feces. develop two flagella similar to those of the dinoflagellata, and swim off. After a time one of the flagella turns into a tentacle, and the characteristic structures of the adult are then formed (Ishikawa). Budding is the characteristic method of reproduction of the suctoria, and is interesting from the fact that it may be either on the surface, as in ephelota, or inside the body, as in acineta (Fig. 33). The latter condition is derived from the former by the bud-forming area sinking below the surface and being covered over by a membrane so that a small brood pouch is created within which the buds swim about by means of their embryonic cilia before making their escape (Fig. 34). PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 95 This so-called endogenous budding is perhaps the forerunner of the curious method of spore formation, or, better, budding, which occurs in one group of the sporozoa, the neosporidia. Here the individual continues to live while forming buds, as in acineta, within its proto- plasm. Such buds, known as pansporoblasts, then form peculiar thread-bearing spores, the entire substance of the bud being used in Fie. 33 Ephelota biitschliana, a budding individual with five daughter buds. N, macronucleus, which forms a branching organ connected throughout. (After Calkins.) the formation of the spores, and these small bundles of spores are carried about by the grandmother organism until its protoplasm is loaded with them, and until it appears like a huge cyst filled with spores (see Fig. 61, p. 145). ‘These organisms are frequent parasites on fish, where they may be the cause of costly epidemics. Budding, furthermore, is frequently associated with the process of conjugation; the mother cell, loaded with chromatin granules in the 96 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA chromidia form, gives rise to numerous buds, each of which is pro- vided with chromidia, but with no part of the vegetative nucleus. Such buds ultimately form the conjugating gametes in forms like arcella, difflugia, centropyxis, etc. In parasitic forms like Entameba histolytica, the cause of tropical dysentery, or Neuroryctes hydrophobie, the cause of rabies, there is a similar bud formation, the buds having the characteristic chromidia; their further fate, however, is unknown, the sexual processes of these organisms not having been made out (see p. 803). A much more highly evolved method of division is found in some of the colony forms of protozoa, where, as in Gonium pectorale (Fig. 35), for example, each of the sixteen cells of the parent colony forms simultaneously a daughter colony of sixteen cells. Here Fic. 34 Endogenous budding in Suctoria. (After Biitschli.) A, B, two stages in the formation of the bud in Tokophrya quadripartita, Cl. and Lach.; c, the bud liberated as a ‘‘swarmer;’ C, buds (e) in Acineta tuberosa, Ehr.; d, a bud liberated. simple division is followed by association of the daughter cells, and individuals result which have passed through an actual, although primitive, ontogeny. In spore formation, finally, we find one of the most prolific methods of reproduction known. Here the organism breaks down simultane- ously into great numbers of daughter elements, each dissimilar to the parent in size if not in other characters. This process, involving as it does the cessation of normal vegetative life with its ordinary processes of digestion, assimilation, etc., usually takes place under the protection of an outer covering or cyst, such encystment being a common phe- nomenon among the protozoa, an outer covering of gelatinous material being thrown out on the surface of the organism whenever the condi- tions of the environment become unsuitable. This investment becomes PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 97 firm and membrane-like upon continued contact with the water, and, finally, if conditions continue unsuitable, it turns into chitin, which withstands drought or heat, and within it the reduced sphere of pro- toplasm is protected until conditions are again favorable. The chitin is then reduced or dissolved by enzymes from within the cell, or by external agents acting on it, and the organism creeps out and Gonium pectorale in reproduction. Each of the sixteen cells of the colony is dividing to form a daughter colony of sixteen cells. (After Calkins.) resumes active life. Within such protecting cysts many different types of protozoa go through the often complicated processes of spore formation. In some cases the protection seems to be hardly neces- sary, and spores are formed and liberated before the membrane has had an opportunity to harden. This is the case in colpidium and in Tillina magna, for example; in colpidium, four or eight daughter cells 7 98 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA may be formed within the cyst, in tillina, only four, and these are all alike, and, except for the smaller size, similar to the parent organism. In many cases only two daughter individuals are formed within the cyst, a fact showing that it is not a long step from the process of simple division to that of such so-called spore formation, and tillina and colpidium are examples illustrating the transition from the one mode of reproduction into the other. ‘Tillina rarely varies from the formation of four spores, and then only to revert to the apparently ancestral mode of simple division. Colpidium, on. the other hand, has progressed farther toward obligatory spore formation, and not infre- quently forms eight spores within the temporary cyst. Other forms of ciliate infusoria form a varying number of spores; in some, as in Holophrya multifilius, a great number of swarm spores are developed in the cyst, each similar to the parent. It is a question whether such reproductive elements are entitled to the name spore, for they are not formed by the simultaneous fragmentation of the mother organism, but by repeated division, the cleavages following one another in rapid succession; in some cases, indeed, as in tillina, the divisions follow so closely upon one another that the two planes of division are sometimes seen at the same time, and this activity is followed by a period of rest lasting for from twelve to twenty-four hours or longer, according to the vitality of the individual. If this is not simultaneous, it is very close to it, and the process in these ciliates must be due to the same, or at least to similar, physiological causes that bring about spore formation in other cases. Spore formation, apart from the spores that are formed in prepa- ration for fertilization, is uncommon among the protozoa and is found chiefly in the one group—sporozoa—which gets its name from this method of reproduction. In many of the flagellates, however, it seems to be a method of reproduction which follows conjugation. Thus, in Tetramitus rostratus and Cercomonas longicauda a cyst is formed immediately after conjugation of two similar cells, and within the cyst the protoplasm fragments into hundreds of minute flagellated organisms. In these cases the ordinary method of reproduction is by cell division, the spore formation appearing to be a special method that follows upon fertilization (Fig. 67, p. 155). It is in the group of the sporozoa that we find the highest develop- ment of the spore-forming power, and here it has been found necessary to distinguish between the spores that are formed sexually, 7. e., after fertilization, and those that are formed asexually, for they differ both in structure and in function. The spores that are formed after fer- tilization are protected by firm and resisting coverings, and are able to live outside of the body of the animal in which they are definitive parasites; the other type of spores, formed asexually, have no such coverings and cannot live apart from the host. With these various PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 99 differences to take into account, the use of the term spore has been very ambiguous and misleading, and protozodlogists have given it up for two other terms, sporozoite and merozoite, now generally adopted. The term sporozoite is used to designate those spores or germs that are Fic. 36 Life cycle of Coccidium schubergi. (After Schaudinn.) Sporozoites penetrate epithelial cells, and grow into adult intracellular parasites (a). When mature, the nucleus divides re- peatedly (b), and each of its subdivisions becomes the nucleus of a merozoite (c). These enter new epithelial cells, and the cycle is repeated many times. After five or six days of incuba- tion, the merozoites develop into sexually differentiated gametes; some are large and well stored with yolk material (d, e, 7); others have nuclei which fragment into many smaller par- ticles (‘‘Chromidien’’), each granule becoming the nucleus of a microgamete or male cell (d), h,i,j). The macrogamete is fertilized by one microgamete (g), and the copula immediately secretes a fertilization membrane which hardens into a cyst. The cleavage nucleus divides twice, and each of the four daughter nuclei forms a sporoblast (k) in which two sporozoites are produced (J). produced after fertilization, while merozoite is used for the asexually produced germs. The protected sporozoites have the power to carry the disease from one host to another, while the merozoites, as a rule, carry the infection only from one part of the host to another part (Fig. 36). Sporozoites, therefore, have the full potential of vitality 100 PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA of a new individual, while merozoites have a shorter life to run and a lessened vitality (see Chapter II). Merozoite formation is best illustrated by the coccidia, a group of cell-infesting sporozoa, and the genus adelea is an interesting type, because it combines asexual reproduction with sexually differentiated organisms. A word here as to the significance of this fact. In the sporozoa, both in the gregarinida and the coccidiidia, the cycle ends with the formation of sexually differentiated reproductive bodies, one of which is larger, corresponding to an egg cell, the other very minute and similar to a spermatozoén; the former is called a macrogamete, the latter a microgamete. The mother cells of these gametes are not visibly different in many cases, and it is impossible to tell whether a given cell will produce one or the other. Insome cases there is a slight difference either in size, or in possession or absence of granules, or in the make-up of the nucleus. These differences do not go far back, as a rule, and in the ordinary run, male and female cannot be distin- guished. In adelea and a number of other forms, however, the sexual differences do go back almost to the fertilized cell, and it is possible to distinguish any given cell as female or male. The formation of asexual reproductive elements, or merozoites, in these different parents is the same, and begins with the division of the nucleus into as many parts as there will be merozoites, in adelea usually twelve to sixteen. After their formation they occupy a peculiar and character- istic position, being rolled together like staves of a barrel, or like the segments of an orange, a peculiar arrangement which has given rise to the name corps en barillet, while the term eimerian cyst is also used designate the parent membrane cyst where they are formed (Fig. 20, A). The sporozoites differ but little from the merozoites when they are deprived of their protecting cases. After fertilization of the macro- gamete, which will be described in a later chapter, the nucleus of an ordinary coccidian, such as Coccidium schubergi, for example, divides twice and the protoplasm surrounds them in equal masses; these are the sporoblasts. ‘The nucleus of each sporoblast then divides again, while the protoplasm secretes a sporoblast membrane, one of the pro- tecting coats of the sporozoites. The second division of the nucleus in each sporoblast provides the nuclei of the sporozoites, two develop- ing in each sporoblast. The germs are then protected by the sporo- blast membrane, and by a membrane which is secreted by the original cell, and with this double safeguard the germs of the organism are thrown to the outside, where no further dev. elopment takes ‘place until the sporocysts are swallowed by some new host (Fig. 36, /). The variations in these processes of merozoite and sporozoite forma- tion are legion, and they are of great importance economically, as well as interesting biologically, but their description belongs rather to the special chapters dealing with protozoan diseases. PHYSIOLOGICAL ACTIVITIES OF THE PROTOZOA 101 The protozoa are, then, complete living organisms, in which no function found in the higher animals is lacking, and we have seen enough of their structures and functions to show how the scope of protozodlogy leads us into all fields of biological pursuits, from tax- onomy, the description and classification of living things, through morphology, physiology, cytology, psychology, and theoretical biology. In the following chapters I wish to show how this scope widens out and leads us into some of the most difficult, but at the same time fascinating, problems of biology. CHAPTER III. PROTOPLASMIC AGE OF PROTOZOA. Upon watching one of these simple organisms through the micro- scope there is a certain fascination in the idea that this minute bit of naked protoplasm has been continuously living since life appeared upon the earth. As a matter of fact, the same sensations might be experienced upon gazing at any of our fellow-beings, or, indeed, at any other living thing; but somehow we do not think of the latter in this way; we associate with them the ideas of age, of senile degeneration and natural death, concepts which do not seem to be associated with the free-living cell. It would appear, furthermore, that the ameba protoplasm wich we see under the microscope, and which has lived continuously for all of these ages, might continue to live for an indefi- nite time in the future. It would seem that this perfectly balanced cell, with its powers of growth and reproduction, would be self-suffi- cient, containing within itself the potential of an endless existence. Such, however, is not the case, protozoa, like metazoa, may die of old age. “In every higher animal we recognize certain more or less definite periods of physiological activity, and according to these we roughly divide the span of life into three stages, which are in no way sharply outlined. These we call the stages of youth, adolescence, and old age. Youth, characterized by a high degree of vitality, is the period of rapid cell multiplication and growth; organs are formed and perfected, functions are unimpaired and active and the body is a perfect living thing. The second period is characterized by functional and sexual maturity; the multiplication of tissue cells is less rapid; the organs strengthen and their functions are more perfectly correlated; growth comes to an end. In‘the perfected animal it is the period for per- petuation of the race, and in conformity with this great function sexual differentiation is fully established. The third period, old age, brings a marked change, the potential of vitality wanes, cells atrophy, and functions weaken; degenerations of all kinds appear; and cumu- lative weakness ends in natural death. ‘These three periods are characteristic of all of the higher many- celled animals, the last period being rarely seen in nature, because in the wild animals a violent death follows the early functional weakening and inability to fight off enemies. Do we find the same sequence of PROTOPLASMIC AGE OF PROTOZOA 103 ° physiological changes in the unicellular animals, and can we distin- guish periods of youth, maturity, and old age? Since the fundamental biological laws are much the same, on a priori grounds alone we should expect to find the same series of changes in protozoa as in the metazoa. But while we do find them in protozoa, they are manifested in a way that we would not at first suspect. We have been accustomed to look upon the single-celled ameba, or paramecium, or other protozo6n, as a complete individual in itself; but when we come to compare such an individual with a metazoén we do not find the analogous periods of vitality which in metazoa we recognize as youth, adolescence, and age. A protozoén is a free-living cell, a complete organism indeed, but as such it has no period of youth nor of sexual maturity, nor, by itself, old age. It is formed by division or some modification of division; it regenerates the normal form in a few hours, and then again divides; with division its individuality is lost, to be merged into that of two new individuals, these two into four and so on. Obviously such an individual cell presents nothing comparable with the sequence of stages so char- acteristic of the “individual” in higher forms of life. Students of the protozoa and biologists generally (e. g., Biitschli, Weismann, etc.) early called attention to the fact that not the single cell of a protozoén, but the entire succession of cells that may be formed from the period of one conjugation to that of the next, should be compared with the metazoén. In the latter, the fertilized egg cell gives rise to a multitude of body cells by repeated divisions; the cells are bound together to form a uniform and differentiated whole. In the former, the fertilized protozoén divides, but the cells do not remain bound together; they separate and live as independent units. If we could take such an entire succession of cells thus formed from the repeated divisions of a fertilized protozoén, and if at any given period could combine them in one mass of cells, we would have the analogue of a metazoén and would find that the protoplasm represented by the aggregate of cells would manifest the same suc- cessive periods of vitality as those of youth, adolescence, and old age in metazoa. We would find that the young cells divide more rapidly than they do later in the cycle; we would find that after a certain period they become sexually mature and able to conjugate and so to perpetuate the race; and we would find that, ultimately, evidences of weakened vitality and degeneration appear in the aggregate of cells, and that they would finally die of old age. Not only would such an aggregate show the characteristic periods of vitality, but with the changes from one period to another there would be, in a great number of cases, accompanying changes in the form of the cell body; changes of so great a nature that a casual observer would never regard such cells as belonging to the same 104 PROTOPLASMIC AGE OF PROTOZOA species as those of the younger generations. It is for this reason, mainly, that in recent years a number of biologists have strongly advocated the use of the entire life cycle of a protozoén rather than the cell, or many cells in the same stage of vitality, for the basis of species. While Biitschli (’76) was the first to note the differences in vitality in a race of protozoa, and Hertwig, Maupas, and a score of others added many observations on different periods, it was Schaudinn (1900) who first clearly perceived the importance of studying the com- plete life history of every species. It is because of this importance that the life cycle forms such a conspicuous part of the definition of protozoa as given at the beginning of Chapter I. Before outlining a typical protozoén’s life history, it will be necessary to understand clearly what is meant by age in protoplasm. It is quite evident, broadly speaking, that there is some protoplasm that does not die, the living things on the earth today testify to that, for they repre- sent protoplasm that has been continuously living since the advent of life on the earth, and which, through posterity, will continue for an indefinite time in the future. Such protoplasm forms the substance of the germ cells, and they alone of all cells have the potential of an indefinite existence. But this capacity to live without finite end is bound up with a biological phenomenon as little understood as life itself, namely, fertilization. Without the union of two germ cells even this endowed protoplasm would die no less surely than do tissue cells. The protozoa are like both tissue cells and germ cells, and consist of protoplasm which is differentiated into somatic and germinal parts, and this protoplasm, like that in higher cells, will die of old age if fertilization or its equivalent is prevented. The problem of age in protozoa, then, has to do with vitality as apart from the union of germ cells and as manifested in the ordinary processes of vegetative activity. J. A TYPICAL LIFE CYCLE. The manifestations of protoplasmic activity which occur in all cells from monads to man, involving processes of digestion, growth, irri- tability, etc., are easily studied in Paramecium aurelia, a very common infusorian that may be found in any stagnant ditch or pool (Fig. 37). To a trained eye it may be seen without the aid of a lens as a minute white spot of protoplasm which moves from place to place in an irreg- ular line of motion. When magnified it appears as an asymmetrical, cigar-shaped organism, with a somewhat spirally wound depression or “‘peristome” leading from one end toward the mouth near the centre of the body. Within the protoplasm is a large nucleus, macronucleus, usually ellipsoidal in form but subject to wide variations in size; and a smaller nucleus, known as the micronucleus, which is embedded in A TYPICAL LIFE CYCLE 105 the substance of the macronucleus. At each end of the infusorian is a bright spot which appears and disappears with considerable regular- ity; these are the contractile vacuoles, their function being to throw to the outside of the body the waste matters that are formed during the physiological activities of the cell. Each vacuole is supplied by a series of canals from various parts of the body, the waste matters in fluid form collecting in them to be emptied into the contractile vacuole and thence disposed of. The peripheral protoplasm of paramecium is filled with minute thread-like structures, the trichocysts, which are thrown out when the cell is irritated. On the outside of the body, finally, is a dense covering of minute lash-like whips which are con- stantly in action during life, and by means of which the organism moves about freely in the water, turning the while on its long axis. These are the cilia which are arranged in spirally wound lines around the body, while a somewhat more powerful set are located in the asymmetrical peristome and are used to direct a food current toward the mouth. Fic. 37 Paramecium aurelia. Macronucleus normal; micronucleus abnormally large. The food consists of any proteid matter small enough to pass through the mouth opening. The organism will take in bits of flesh, or parts of vegetable matter, or bacteria or lifeless matter, such as carmine or indigo granules, all with equal voracity. ‘The process of ingestion is hastened by the activity of an undulating membrane situated in the small gullet, and the bacteria or other food matters are collected in a vacuole which forms at the base of the gullet. Con- siderable water is taken in with the food, and when the vacuole is large enough it is caught up in the protoplasmic flow and carried away from the mouth opening. Numerous gastric vacuoles are thus formed and the food is digested in them. When the organism is fully grown it reproduces by dividing into two cells, each cell having the characters of the former one cell, which has disappeared, indeed, although it has not died. Its protoplasm is still living in the two daughter cells; these repeat the processes of digesting and growing, and finally, each of them reproduces by trans- verse division. ‘The metabolic processes leading to reproduction by division are thus repeated generation after generation, and, having all that is necessary in the form of cellular organs for an indefinitely 106 PROTOPLASMIC AGE OF PROTOZOA continued existence, they apparently offer some justification for the older view that protozoa are practically deathless, so far as old age is concerned. The matter of physical immortality can be easily tested, however. After a little practice, a single cell of paramecium can be isolated and fed on the bacteria which develop in a previously sterilized hay infu- sion made by boiling small pieces of hay in water. The organism is placed in a small chamber filled with the hay infusion and made by supporting a coverglass on pieces of glass. When it divides, which it will do within twenty-four hours, the daughter cells can be similarly isolated and fed on freshly made hay infusion, and in this way the vitality of that originally minute bit of protoplasm can be watched day after day and generation after generation of cell divisions, until natural death from old age ensues. ‘The writer successfully followed the life history of such a culture of paramecium from an initial cell to protoplasmic death from old age, giving fresh food medium and isolating the single cells day after day and generation after generation for a period of twenty-three months and 742 generations. ‘The obser- vations made during such a study deal with living protoplasm that is growing old more rapidly than in nature, and with the ageing process in an organism endowed with an initial potential of vitality. A paramecium which is thus followed from generation to generation shows surprisingly regular variations in vitality. Some of the more minute variations are Sige to temperature changes, a warm day, for example, increasing, a cold day diminishing, hele vigor. In the laboratory, however, such variations may be overlooked, for the changes in temperature from day to day are of minor importance. After much experimenting, a measure of vitality was finally found which made it possible to compare the activity of the physiological processes from time to time. ‘This measure was represented in the form of a curve, the points upon it being obtained by averaging the number of divisions made by all of the organisms under observation in periods of ten days, each average giving the ordinate for one period; the abscissas represent the arbitrary ten-day periods (see Fig. 38). Such a curve, representing the vitality of the paramecium ‘proto- plasm, shows that in a period of six months under cultivation, if the organisms are fed upon the same diet of hay infusion, there is a gradual exhaustion of vitality, the curve falling from an average of about twelve divisions in ten days in February. to an average of one division in ten days in July. As the curve shows, the average number of cell divisions sinks more or less regularly during the six months, but undergoes periodic rises and falls, until at he end of that time the organisms are unable to digest and assimilate the bacterial food and the cells begin to die, the minute cellular corpses being abundant at such a period. 107 A TYPICAL LIFE CYCLE *|[G ¥e azVInfuoos you plnoa stustues10 ay} apoAo ysvy oy. UL apIYAd ‘auNG [UN Udy Woy pus AleNUBL 0} IEqWOAON WOT} ata APTATIOR [ENS jo spotsed ay} ‘aja£o psi} pus pUooas ay} UL ‘UOTyeIaUaT puodesS-AJIOJ PUT poIpUNY UsAEs oY} UT SoUO ySUT OY} ‘ZOGL ‘Loquaseq UL yno perp ][B Inq ‘urrAq jo puv svadouvd yo syourzxo Aq oUTT} SITY ‘AT[VIOYTIG poyE[NUT}s UleSe oO JTBE “ZOGT ‘PUN UL papua Yor ‘apoAD pI} oY} UO poyTEys puv popE[NUIset A19M ‘87[VS BAUS 10 JOVIPXO Jood UBAIT 219M YOTYA ‘ySor OY} ofA ‘parp ]]e ‘JorzUOS sv UOISsNyUT AvY oy} UO 4yday a1aM YOIYA SUISTULSIO ay} JO FEY JY} Soyo -Iput aseq oY} 0} poltavo WesFeIp oY} UI aUT] OY, “ZOGI ‘Arvnuee ur pepue ‘pezivys A][eIoyTWE snyy ‘efoAo puooas ay “YRI}XE Jood YA peze[nUys duro SUISTURBIO OT} ‘TOG ‘A[NE UT papua afoAo 4siy OY], ‘SeuT} JUIaYTP ye ABPEITA YY Burieduiod Jo suveuwl JUS!UeAUOD B SULAIT snyy ‘spotsad Avp-Us} UI pesvIdAv a1IM UOTPAIASGO OpUN avi t[} JO S[ENPIArpul [Jv 1oJ Avp Jed suo[starp Jo sxequu ey], ‘“WNJepnvs tunoowvv_ Jo sapoAo oft] oY} Sulquesoidar wvsserd ‘i Th sia as as) = [trronofaat Aan oo tne et anor TAVA | ‘adv | ‘MV | ‘O34 pS stele pS paar SNAP A \ } 1 1 Nipo 183s ad Beck 7 aa SEW faa NVe | BOT \ cI on | i LOGE : ' EE ae ' ' 108 PROTOPLASMIC AGE OF PROTOZOA This exhaustion of the power to digest and assimilate is an unmis- takable phenomenon in the life history of a protozoén, and marks a somewhat indefinite phase of the life history, which was designated the “period of depression.” Many other observers have noted it in connection with protozoa of different kinds; the first, Biitschli, in 1876, in relation to paramecium, without noting the sequence of stages lead- ing to this depression period, observed that the organisms become reduced in size and sluggish in movement, and that while in such condition they conjugate, an observation which led him to his famous suggestion that conjugation is not an act of reproduction, but a means of renewing the vitality of the organisms, including the power to repro- duce; in other words, a Verjungung of the protoplasm. Later observers, including Maupas and Hertwig, likewise studying the organisms en masse, noted a similar stage of lowered vitality, the former concluding that it indicates a senile degeneration of the nuclei, the latter, that it indicates a changed relation between the volume of the nucleus and that of the cell. Woodruff and Gregory, as graduate students in the Columbia laboratory, have followed out, generation by generation, the life history of different protozoa, the former in connection with Oxytricha fallax, one of the hypotrichous infusoria, which he followed for 860 generations of cell divisions, requiring twenty-one months, the latter in connection with Tillina magna, one of the holotrichous infusoria, which was followed for thirteen months, dying out in the 548th generation. Periods of depression were observed in these organisms as in paramecium, and the same physiological derange- ments were noted by both observers, the first period of depression carrying off all the cells of tillina. What is the explanation of the depression period? The organisms have abundant food; they are able to take in food up to a certain time, but they appear abnormal in structure, and if left to themselves they would die. ‘The protoplasm at this period is markedly different from that at other times; in paramecium the endoplasm lacks the characteristic vacuoles of the ordinary organism and appears dense and homogeneous (Fig. 39), an appearance due to the aggregation of granules. The lack of vacuoles signifies a concentration of the cell protoplasm and, therefore, a reduction in size of the organism; the macronucleus, in the meantime, retains its full size, and it Pug appears that the volume of the latter is relatively greater than it is under normal conditions. ‘This is perhaps one reason why Hertwig, Popoff, and others have concluded that the cause of depression is the change in relative volume of nucleus and cytoplasm, but such a change in relative volumes may be equally well an effect of the depression and not its cause. Woodruff noted the same reduction in size of the cell in oxytricha (his figures 1 and 9) during the period of depression and a corresponding change i in nature of the cytoplasm, which, in oxytricha, A TYPICAL LIFE CYCLE 109 became vacuolated instead of granulated. There is no doubt, from these daily observations on the same organisms, that there is a change in physiological activity, which cannot be interpreted as due to the difference in the relative sizes of nucleus and cytoplasm, but must be traced to some more deeply lying cause. After two similar periods of depression had been successfully offset by artificial means, a fourth and final period, in which the protoplasmic structures were quite different from previous conditions, carried off the last generation of the race, 742d generation (see p. 129). Fie. 39 Paramecium aurelia at period of depression, showing (at left) the dense granular condition of the protoplasm, which, if not relieved artificially, invariably ends in death. The central and right hand figures show the effects of such artificial relief in the vicinity of the nucleus, while the extremities are still dense. While these initial experiments would seem to indicate a certain normal length of life (approximately 200 to 800 generations), it does not follow that all paramecia have the same endowment. Different races of paramecium, like different human individuals, vary in the initial potential of vitality, and are capable of living for different lengths of time upon the same medium. ‘Thus, other cultures of para- mecium, carried on at the same time as those described, yielded 376 and 379 generations before evidences of depression set in. A con- stantly changing medium, furthermore, may tend to offset the cumula- tive physiological weakness and so to prolong the life of the race. Such an experiment on paramecium has recently been carried out by Woodruff (’08), who, instead of constant hay infusion, used infusions of leaves, grass, etc., from natural pond water, frequently changing 110 PROTOPLASMIC AGE OF PROTOZOA the source of such food material. Upon such a continually changed diet he carried on a race of Paramecium aurelia through several hun- dred generations without the advent of a period of depression. It appears, therefore, that in the constantly changing conditions of nature a race of protozoa may live much longer than under the conditions of laboratory experiments on a single diet. It is probable that the salt contents of the medium rather than the food are of importance in this connection, since the bacteria of the laboratory air, with which all food media were inoculated, were presumably the same. II. MORE COMPLICATED LIFE CYCLES AND THE PERIODS OF “YOUTH,” “MATURITY,” AND “AGE.” With different types of protozoa the three periods of vitality may be recognized with quite the same facility as in any of the lower forms of metazoa. There is no sharply defined difference between them, but, as Maupas first, pointed out, there is a fairly definite period of proto- plasmic or “individual” maturity, which is preceded by a period that may be designated “youth,” and is followed by a period that may be called ‘‘old age.” The period of maturity is so frequently accom- panied by well-marked cellular changes, which distinguish the organ- isms at that period from the ancestral cells which gave rise to them, that we are justified in the attempt to generalize, if only for descriptive purposes, and to speak of periods of youth, maturity, and age in protozoa. In the life history of Paramectwm aurelia the three periods, youth, maturity, and age, of the life cycle are not so clearly marked by struc- tural and functional manifestations as in some other forms of protozoa. Nevertheless, there is a physiological difference which becomes appar- ent when one follows out the complete history. The period of youth is marked by a high rate of division energy and by the fact that con- jugation does not occur if many of them are put together in a limited space. After some time in culture, however, usually when the rate of division has begun to decline, the protoplasm of the cell body changes slightly in physical and chemical make-up, so that two or more cells upon meeting fuse and conjugate. The entire race of para- mecium in such a “culture may become sexually mature at the same time, and “‘epidemics” of conjugations may be thus obtained. At the last period of depression, however, in the experiments cited, there were no conjugations, a fact indicating, ‘possibly, the exhaustion of the germ plasm. Such a final period of old age may be easily identified, involv- ing, as it does, the curious vacuolization and degener ation of the protoplasm and exhaustion of the physiological energies. MORE COMPLICATED LIFE CYCLES Sa A. The Period of Youth.—As with the fertilized egg of a metazoén, this first period of vitality of the copula or fertilized cell of a protozoén is characterized by the distinct excess of constructive over destructive metabolism, which indicates a high potential of vitality and great powers of cell reproduction, which may take the form of division, budding, or spore formation according to the difficulties successfully overcome by the type in the struggle for existence. These young forms show a well-marked conformity to type, and this feature, occur- ring when the greatest numbers of representatives of the species are in evidence, undoubtedly has given a false impression of the stability of form of the protozoan species. The protoplasm, as a rule, is trans- parent and without reserve matters, metaplasm products, and the like, and the nucleus is often without the characteristic structures of the later forms. It is along physiological lines that the young forms are most promi- nently marked. ‘This is the period, for example, of the greatest resist- ance to adverse conditions in the surrounding medium, and in patho- genic forms it is the period of greatest malignancy. It is a well- known fact that in many parasitic forms of “protozoa attempts to inoculate from animal to animal are either failures altogether or result in a weakened infection, the failures being due, presumably, to the inability of the organisms in a more or less weakened condition to withstand the natural immunity of a new host. The matter of malig- nancy is so intimately connected with restored vitality that in yellow fever, for example, it is almost sufficient to indicate that fertilization processes and renewal of vitality must have taken place in the body of the intermediate mosquito host. At this period, also, is the greatest power of self-preservation in other ways than by resistance of a chemical nature; thus, the firm protective cysts are formed at this period within which the fertilized cell may resist heat, cold, and drought, as in many of the free forms of protozoa when the organisms live thus through the winter, or in parasitic forms like the sporozoa, when the organisms are protected in the interval of changing hosts. The difficulties in determining which are young and which older cells of a life cycle are great, and much must be left for inference. It may be accomplished, however, in one of several ways: (1) By culture experiments for which cells are isolated immediately after conjugation, a method that may be easily employed for the larger free protozoa. (2) By inoculation of uninfected hosts with the spores of the form to be studied, a method which may be employed with sporozoa or with encysted amebe. (3) By natural inoculation through the opera- tion of intermediate hosts, such as insects, ticks, or leeches. Few observations, however, have been made upon the young forms, prob- ably because the morphological characteristics of the mature cells are much more apparent than those of the young. 112 PROTOPLASMIC AGE OF PROTOZOA “ The high grade of vitality of the young protozoan is undoubtedly due to the perfection of the cellular structures and to their harmonious working. ‘This was very well illustrated in some observations on so- called Paramecium caudatum (Calkins, 1906). This species has been generally regarded as distinct from another very similar form, Para- mecium aurelia, which is regarded as much more rare than the former. The main difference between the supposed two species is the presence Fic. 40 r 1 ro AUGUST Hwwrogn-t | | MARCH APRIL | JUNE | JULY, Diagram to show the relative vitality of the caudatum and aurelia forms of paramecium. The dotted line represents the division rate (average for ten-day periods) of an ex-conjugant from the same culture which reorganized normally, 7. e., as a Paramecium ‘‘caudatum.’”? The solid line represents an ex-conjugant that reorganized abnormally, 7. e., as Paramecium “aurelia,’’ but which changed into a normal form during the month of June. Note the rise in division rate with the assumption of the normal condition. (After Calkins.) in the latter of two micronuclei as against one in the former, while certain physiological differences, as indicated by the rate of division and the rate of movement, were noted by Maupas (’89) and Simpson (01). The observations mentioned were made upon some ex-con- jugants from a culture of the more common “‘caudatum” form. The two cells derived from such a union were isolated, and one of them was maintained for months in culture, the other dying shortly after MORE COMPLICATED LIFE CYCLES 113 isolation. In the reorganization of the cell following separation two micronuclei instead of one were left in the cell. This abnormality for the “‘caudatum” form was the “normal” condition for the ‘“aurelia”’ form, and was maintained for more than three months, the animals showing every characteristic of form and function that have been ascribed to Paramecium aurelia. The movement was sluggish and the rate of division much lower than in the case of “caudatum”’ forms which had been isolated at the same time and carried along as a con- trol (see Fig. 40). At the expiration of three months in culture the cells here and there showed the loss of one of the micronuclei, and ulti- mately all of the so-called ‘“aurelia’”’ forms had become “‘caudatum”’ forms and with the typical characteristics which mark this species. The rate of division rose to a much higher average than before, and the cells became much more animated and larger in size. The average number of divisions in ten-day periods rose from 11.3 from March 1 to June 10, to 19.3 in the time from June 10 to September 1, that is, during the time when the nuclear relations were normal. It is evident, therefore, that Paramecium caudatum and Paramecium aurelia are not distinct species but merely variants of the same species, and that the abnormal condition of the cell organs resulted in strongly marked physiological derangement. B. The Period of Maturity.—There is no definite limit to the period of youth in protozoa, the changes which characterize the period of maturity coming on slowly and imperceptibly as they do in higher forms. The morphological characteristics of this period, when arrived, are clearly marked, however, and unmistakable. Such changes affect both the cell body and the nucleus, and may accom- pany either vegetative or germinal activities, or both. 1. Protoplasmic Changes at Maturity—While the most important characteristic of the period of maturity is a general decrease in func- tional activity, with decline in the rate of multiplication, these physio- logical activities are accompanied by well-marked morphological changes which may be of a sexual character. In a single cell or specimen of a protozoan species there may be no clue to its position in the life cycle unless it is in some phase of sexual activity, and where sexual dimorphism does not exist it is quite impossible to tell from morphology alone. ‘Thus, in the mature paramecium the sexual differences are so minute that unless one is following out the life his- tory in culture the period of maturity passes unobserved. Nevertheless, the cells of paramecium do undergo a physical change at this period; the peripheral protoplasm becomes sticky and highly miscible, so that, in some cultures, two organisms upon meeting will adhere at any point, and groups of from six to nine cells may be seen whirling about in aimless movement among the normally conjugating pairs. This miscible state indicates a well-marked difference in the physical 8 114 PROTOPLASMIC AGE OF PROTOZOA make-up of the protoplasm, for in the early periods of activity the body wall, while plastic, always retains its firm contour and cortical density. Pearl (’07), furthermore, has shown by biometric analysis that con- jugating paramecia are markedly smaller and less variable than non- conjugating forms. Similar changes in density mark this period in other kinds of pro- tozoa. Thus, among the flagellated forms like tetramitus or cerco- monas the ordinarily firm contour of the cell becomes plastic and highly changeable in form, and two of them upon meeting fuse in conjugation. Here again a physical change is well illustrated. Fic, 41 Polystomella crispa. Liberation of pseudopodiospores from the microspheric individual. (Photo by J. J. Lister.) Still more remarkable is the change in form which some types of sarcodina undergo at this time. The thizopods are especially note- worthy in this connection, Schlumberger (’83) noting for the first time a peculiar dimorphism in the shells of foraminifera (Fig. 42), a differ- ence which Schaudinn (’03) and Lister (’05) were the first to explain. These observers found that the young forms, immediately after fer- tilization, give rise to what Schlumberger termed the “microspheric” type of shell. Upon reproduction, sue tha cell ultimately gives rise to: pseudopodiospores which leave the old shell and secrete new ones of a different type, termed the “me galospheric” type (Fig. 41). The latter generation, when fully grown, gives rise to flagellispores which conjugate and thus complete the cycle (see Fig. 52, p. 123). MORE COMPLICATED LIFE CYCLES 115 Even more marked is the change in trichospherium where the chemical composition of the skeleton parts changes with advancing age. The young forms resulting from conjugation grow into an adult characterized by a gelatinous membrane and radial spicules of magnesium carbonate. This adult reproduces by the formation of pseudopodiospores, which grow into organisms similar to the parent, or after advanced age (presumably) to a second adult type characterized by a firm membrane and entire absence of radial spicules. This second type, as in the foraminifera, finally gives rise to flagellispores, the progeny from different parents uniting and thus Megalospheric (A) and microspheric (B) shells of Biloculina depressa, Lam. (After Schlumberger.) Dimorphism is shown by the central chamber c. completing the cycle. Such secondary types are morphological evidences of changed metabolic conditions characteristic of the second period of vitality. The possibilities of similar alternations in the life history of parasitic and pathogenic forms have hardly yet been realized. 2. Nuclear Changes at Maturity.—‘‘ Chromidia.’’— While changes in the body form are often characteristic of the second period of vitality, there are great numbers of protozoa in which the external structure gives no clue to the state of affairs within. The nucleus, however, undergoes changes at this period which are not only more widespread throughout the phylum, but are of far more theoretical and practical importance. These changes have to do with the formation of so-called “ chromidia”’ and with the maturation phenomena of the cell (Fig. 43). 116 PROTOPLASMIC AGE OF PROTOZOA The first definite observations upon chromidia formation were made by Hertwig (99) in connection with the minute structure of the shelled rhizopod Arcella vulgaris. Previous observers had noted that chromatin-like granules are distributed throughout the cell body in many of these types, but Hertwig was the first to describe the origin of this material from the nucleus in arcella and to show that it forms a dense zone of granules in the protoplasm (Fig. 44). At that time Hertwig described this material under the name of “chromatin net,” but later, in 1902, he called it the “‘chromidialnetz,” because of the reticulate structure assumed by the granules en masse. The function of this extranuclear chromatin was not made out, however, until the following year, when Schaudinn (’03) worked out the origin and fate of similar masses of granules in several different kinds of sarcodina Fic. 43 “‘Chromidia” in rhizopods, Arcella vulgaris (on left) and Ameba proteus (on right). The dark granules are the idiochromidia distributed throughout the cytoplasm. (Polystomella crispa, Centropyxis aculeata, Chlamydophrys stercorea, and Entameba colt) and found that the nuclei of the conjugating gametes were developed solely from this extranuclear chromatin. He thus interpreted the material of the chromatin net of arcella and its allies as sexual or racial chromatin and correctly compared it with the micronuclei of the infusoria. In the meantime the subject became more complicated by Hertwig’s further observations upon extranuclear chromatin in the heliozoén Actinospherium eichhornii. 'These observations, first noted in 1897, were confirmed and extended in 1904, when it was shown that in starving forms and as well in forms that had been overfed, the nuclei all disintegrate and the chromatin contents becomes distributed throughout the cell body (Fig. 45). The distributed chromatin thus MORE COMPLICATED LIFE CYCLES 117 formed was named by Hertwig, in 1902, “‘chromidien,” from which the term chromidia is derived, a term now universally employed by protozodlogists. According to Hertwig this latter material in actino- spherium cytoplasm is prophetic of the death of the animal, for when it is thus formed the renovation of the cell is impossible (1904). Arcella vulgaris. (After Calkins.) mic union. sspherium ei A, primary nucla ; B, complete tre It thus appears that we have to do with two kinds of chromatin masses in the cell body and no little confusion has arisen in conse- quence of the mixed terminology applied to this material, which is alike in origin from the nucleus but very different in function. Chromidia, in Hertwig’s sense, is functionless extranuclear chromatin, but Schau- dinn and others have used the term to designate the sexual chromatin which is equivalent to the chromidialnetz in Hertwig’s terminology. 118 PROTOPLASMIC AGE OF PROTOZOA Subsequent observers have tried to straighten the tangle by giving new terms for the different kinds of extranuclear material. Calkins (’04) proposed the term protogonoplasm for the gamete-forming substance; Goldschmidt (’04) proposed the terms ‘‘chromidia” and “sporetia” for chromidia and “‘chromidial net” respectively, and Mesnil (’05), the terms “‘trophochomidia” and “‘idiochromidia.” Goldschmidt’s suggestion is a good one, but the term sporetia is not indicative of the function, while Mesnil’s term idiochromidia expresses the fate exactly and will undoubtedly supplant the other names. In the present instance the terms ‘‘chromidia” and “‘idiochromidia” will be used, the former on grounds of priority, the latter on expediency. Arcella vulgaris. Secondary (gametic) nuclei (n) forming from the idiochromidia ch; o, mouth opening of shell. (After Hertwig.) (a) Ip1ocHRomip1a Formatrion.—As might be expected, the method of formation of the idiochromidia differs widely in the different types of protozoa, and frequently in the same type. Although all methods, in their final analysis, may be traced back to the same physiological causes arising during this period of maturity, the different types may be separated “for purposes of description into three groups, as follows: (a) Idiochromidia formation by nuclear transfusion; (6) by dissolu- tion of nuclear parts; and (c) by nuclear fragmentation. Nuclear Transfusion —This method of idiochromidia formation is most characteristic of the rhizopods, and has been worked out mainly in connection with arcella, centropyxis, difflugia, and other mono- MORE COMPLICATED LIFE CYCLES 119 thalamous forms. In arcellait has been described by Hertwig (’99) and Elpetiewsky (’08), and the process here may serve as a type for all. The normal vegetative cell of arcella contains two nuclei which at an early period begin to secrete chromatin materials, which collect in masses about the nuclear periphery (Fig. 44). With continued Gametes and copulation of Arcella vulgaris. C, copula. (After Elpetiewsky). Fic. 48 Stages in development of Mastigella vitrea and Mastigina setosa. (After Goldschmidt. xX 1270. A, flagellate stage of M. vitrea; B, same, somewhat older and before chromidia formation; C, same during chromidia formation; a, entire cell; 6, nucleus only, showing transfusion of chromatin to form chromidia; D, young Hagella stage of M. setosa, with heap of chromidia; EL, same, older form with pseudopodia, compact chromidia, and food vacuole; F, same, young form with peripheral ‘‘bristles;’’ G, same, formation of gametic nuclei a, from idiochromidia, b. growth, and at maturity of the cycle, these masses become distributed throughout the cell body in the form of deeply staining chromatin granules (Fig. 43). When fully mature the protoplasm breaks down into a number of pseudopodiospores, each with distributed chromatin, and these form new arcella shells in which the protoplasm ultimately 120 PROTOPLASMIC AGE OF PROTOZOA breaks up into ameboid gametes, in which the nuclei are formed, as in centropyxis, by fusion of the idiochromidia granules (Figs. 46 and 47). Not only in rhizopods, but in flagellated protozoa as well, the idio- chromidia arise in this manner. ‘Thus, in the case of Mastigina setosa, Goldschmidt (’07) has shown that the idiochromidia accumu- late in heaps about the nuclear membrane, as in arcella or centropyxis, before being scattered throughout the cytoplasm, where they ultimately form the nuclei of gametes (Fig. 48). Nuclear Dissolution There is probably no great difference between the above-described method of idiochromidia formation by transfusion, whereby the chromatin materials percolate through the nuclear membrane in fluid form, and that by nuclear dissolution, whereby the peripheral portion of the nucleus becomes scattered in granular form throughout the cell body. Nor is this second method Fic. 49 Ameba limax (group of five on left) and Chilomonas paramecium to show alveolar structure of protoplasm prior to idiochromidia formation. Two of the amebz are in process of division. different, save in degree, from the third, which I have called nuclear fragmentation. The distinctions have, at best, only a descriptive value. Nuclear dissolution, in substance, was described more than thirty years ago by Hertwig (’76) in connection with the radiolarian acan- thometra. In this form there is a great increase in the thickness of the chromatin at the periphery of the nucleus and at the expense of the karyosome, and this cortex ultimately breaks down to form quan- tities of minute secondary nuclei of the macro- and microgametes (see Hertwig, 1907). Here, then, the peripheral rind of chromatin is little more than a condensed zone of idiochromidia, and is closely associated with the karyosome. In Ameba limax (Fig. 49) there is no MORE COMPLICATED LIFE CYCLES 121 such condensation, but the idiochromidia granules collect in a loose shell or rind about the karyosome, and from it granules of chromatin are discharged into the surrounding protoplasm prior to encystment (Fig. 50). During encystment these distributed granules are abun- dant in the cell while the karyosome becomes indistinct and ultimately degenerates. Under proper environmental conditions (which may be brought about artificially by changes in temperature) the idiochromidia fuse into sixteen groups of secondary nuclei (Fig. 51). A similar method of idiochromatin formation was described by Schaudinn (03), and more recently by Craig (’08), in the case of Entameba histolytica. Not only idiochromidia, but chromidia as well, may be formed by this method of nuclear dissolution. Thus, in some coccidia and grega- rines according to the observations of Siedlecki (’07) and Léger (’07), on caryotropha and ophryocystis, respectively, a similar disposal of the peripheral rind of chromatin gives rise to degenerating granules which, possibly, according to both observers, may have some vegeta- Fic. 50 Ameba limax. Chromidia forming from nucleus and collecting in the cytoplasm prior to encystment. tive function in cell metabolism. The latter, therefore, apparently agree with Hertwig’s chromidia in actinospherium. Nuclear Fragmentation.—Idiochromidia formation by fragmenta- tion is widely scattered among protozoa, and has been described by numerous observers, first by Schaudinn (’94), and by many others since, in connection with various forms of foraminifera, rhizopods, flagellates, and sporozoa. The most widely recognized example of this mode of idiochromidia formation is the case of Polystomella crispa, one of the foraminifera. Here, according to the independent obser- vations of Schaudinn (’03) and Lister (’05), the nuclei of the micro- spherical generation increase by division until a large number are formed. The older ones then disintegrate, or fragment, into minute chromatin granules, which are ultimately distributed throughout the protoplasm. Later aggregations of these idiochromidial granules give rise to the nuclei of the conjugating gametes (Fig. 52). Similarly in the coccidian Klossia octopiana, according to the researches of Siedlecki the nuclei of the microgametes, and in Gregarina cuneata, according 122 PROTOPLASMIC AGE OF PROTOZOA to Kuschakewitsch (’07), the gametic nuclei, are formed by nuclear fragmentation. A slight modification of this method of idiochromidia formation is found in Ameba proteus, where, according to Calkins (’07), the primary nucleus divides repeatedly until about seventy nuclei are present in the cell. These primary nuclei then give rise to secondary nuclei, which form from the chromatin granules inside of the primary nuclei. The chromatin substance of the primary nuclei is thus metamorphosed into secondary gametic nuclei, and these conjugate two by two. Here the process may be interpreted as a precocious development of the gametic nuclei, a development taking place before the primary ones are com- pletely fragmented (Fig. 53). Fic. 51 Ameba limax. Aggregations of idiochromidia to form sixteen secondary nuclei, which then unite to form eight. The vegetative distributed chromatin granules or true chromidia, as seen in Actinospherium eichhornit are formed by similar nuclear fragmentation; it is quite obvious, therefore, that the method of formation of these distributed chromatin granules has little or nothing to do with the subsequent function. (b) Tue SIGNIFICANCE oF IpiocHRomipia.—It is quite apparent from even the few cases cited above that we cannot generalize as to the function of the deeply staining granules of nuclear origin in the cyto- plasm of protozoa. In some cases (e. g., actinospherium, ophryo- cystis, caryotropha, etc.), whatever may be their significance in the MORE COMPLICATED LIFE CYCLES 123 cell, they certainly are not connected with the formation of the gametic nuclei. On the other hand, there can be no doubt of the propagative nature of such distributed granules in the great majority of protozoa, and in such cases we may, with reason, speak of a definitive germ Life cycle of Polystomella crispa S. (Lang and Schaudinn). A young form derived from the union of two flagellated gametes (4) develops into an organism with microspheric type of shell. The nucleus increases by mitosis until many nuclei are present when they break up into granules of chromatin (B). The protoplasm fragments into reproductive bodies, equivalent to merozoites (C), each having several granules of the distributed chromatin (‘‘Chromidien’”’). Each reproductive body (D) develops into an adult with a macrospheric type of shell, and with nuclei in the form of small chromatin granules (Z). When mature these forms fragment into hundreds of flagellate gametes (F) which conjugate, and so com- plete the cycle. (See, also, Fig. 41, p. 114.) plasm as contrasted with the somatic plasm. With such an assump- tion we are brought in touch with a problem of high theoretical interest in general cytology, and with the protozoa, as with the metazoa, we have this question to consider: Are there two kinds of substances in 124 PROTOPLASMIC AGE OF PROTOZOA the nucleus, the one superintending exclusively the processes having to do with germinal life, heredity, and the race, the other having to do only with the metabolic processes of the individual? In connection with higher animals and plants we meet with con- flicting answers to such a question. Weismann, Roux, and their followers maintain—and their contention is strengthened by the con- stantly increasing evidence as to individuality of the chromosomes and their connection with specific characteristics of the adult organism— that a specific inheritable substance—idioplasm—is always present in the cell from the start, and is gradually sifted out with growth as the various organs are formed. Others, notably O. Hertwig, take the view that nuclear materials are fundamentally the same, and that as growth advances, environmental changes affect and alter the original homo- geneous stuff. It is in connection with the latter point of view that R. Hertwig approaches the problem of dualism in the protozoan nucleus (1907). He believes that ‘‘functional degeneration” becomes localized in certain substances of the cell nucleus, so that a dualism is gradually brought about through such degenerative changes, and indicated, morphologically, by the different chromatin elements scattered throughout the cell. Chromidia, therefore, according to this point of view, would be the same as idiochromidia save for a difference in potential, the latter having the possibilities of continued existence, the former not. Neglecting, for the present, the question of original dualism in nuclear substances in protozoa, we must accept the fact that there are, at times, specific germ substances within the cell and localized in the chromatin of the cell. In the higher animals the analogous germ plasm becomes segregated and separated from the somatic plasm in the form of germ cells or germinal epithelia. Differentiated from somatic plasm during ontogeny, this racial protoplasm becomes functional only after the period of maturity is reached. Similarly with protozoa, there is, at periods of maturity, a definite germ plasm distinct and separate from the somatic plasm. In some cases, like the germ cells of higher animals, this specific racial substance is early differentiated from the vegetative, functional, or somatic plasm. Such is the case in infusoria, where, in Paramecium aurelia, for example, germ nuclei and functional vegetative nuclei are differ- entiated as micronuclei and macronuclei, respectively, after the third division following conjugation; and such is the case in arcella and allied forms where the germ plasm is not aggregated in a compact micronucleus, but as idiochromidia is scattered throughout the cell. In other cases the germinal and somatic parts are not separated until later in the life history, or in some cases not until full maturity, when for the first time chromatin of conjugation and of vegetative function can be distinguished. Such is the case in Ameba proteus, in MORE COMPLICATED LIFE Fie. 53 Idiochromidia formation in Ameba proteus. CYCLES (After Calkins.) 5 126 PROTOPLASMIC AGE OF PROTOZOA Polystomella crispa, in gregarines, and coccidia, where the residual primary nucleus, or the Restkérperchen, may be interpreted as the now functionless somatic chromatin. Idiochromidia, or germ plasm, therefore, must be interpreted, in some cases at least (infusoria), as a definite and distinct substance of the cell. In other cases its segregation and separation from somatic chromatin occurs only during the second period of the life cycle, and its formation is the index of advancing age (sarcodina). It is, in point of fact, the chief morphological feature characteristic of the period of maturity in protozoa. 3. Sex Differentiation—At the present time the hypothesis first advanced by Montgomery (01) is widely accepted, that during maturation of the germ cells the reduced number of chromosomes is brought about by union, two by two, of chromosomes representing the same characteristics of the adult in maternal and paternal ancestors. Of such characteristics, none are more marked than those primary and secondary characters which distinguish the sexes. Wilson’s obser- vations, following and enlarging upon those of McClung, Stevens, and others, on the structure of the germ nuclei in insects, have prac- tically demonstrated that sex here, like other adult characteristics, is a matter of inheritance. In protozoa, sex differentiation, when present, is, apparently, the final expression of the period of maturity. We have seen that, with advancing age, the structure of the protozoan cell may become materi- ally altered, and that these alterations may give rise to similar con- jugating gametes, or, directed possibly by inheritance, may give rise to male or female germ cells. In the former case (isogamy), conju- gating elements may be similar in size to normal cells or only slightly reduced, as in paramecium, didinium, and the majority of infusoria; or both may be reduced to small-sized equal cells (isomicrogametes), as in many gregarines and rhizopods. In the latter case (anisogamy) one cell, macrogamete, may be similar to the ordinary vegetative cells (as in vorticella, coccidium, ete.), or only slightly changed, while the other cell (microgamete) may be relatively minute ” (vorticellidee, coccidiidia, etc.); or both cells may be reduced and of dissimilar size (as in polytoma, centropyxis, schaudinnella, stylorhynchus, and other gregarines). In sexually dimorphic gametes there is no difference between the early cells in the majority of cases, differentiation coming only as a last step in maturity (hemosporidia, coccidium, and cooeldiidia gener- ally); in some cases, however, notably in adelea (Siedlecki, 1899) and cyclospora (Schaudinn, 1902) among coccidia, and in trypanosoma (Schaudinn, 1904) among flagellates, the sex differences are said to extend as far back as the schizont stage immediately after fertiliza- tion; hence, if this is true, it is possible to speak in some cases of male MORE COMPLICATED LIFE CYCLES 127 and female protozoan individuals. The evidence for this conclusion is in every case somewhat inconclusive; the differences seemingly are not beyond the range of individual variation. In the majority of free forms, gamete formation, with their libera- tion, is accomplished in the ordinary medium in which the organisms live, although these processes may be hastened or influenced by artificial changes in the environment. Thus, Hertwig (’98) noted that the quantity of food had much to do with these phenomena in the case of actinospherium, and Klebs, Dangeard, Greeley, and others have found that changes in temperature or in density of the medium may induce gamete formation in different kinds of flagellates. Similar changes in environment seem to be a sine qua non for sex differentia- tion in many parasitic forms, the most notable and best-established case being the malaria organisms where microgametes are formed only in room temperature, in the mosquito’s gut, or, in general, in a colder (denser?) medium than the blood. ' In the great majority of cases where gametic differentiation obtains, if the gametes do not conjugate they die. This is invariably true of ‘the microgametes, and their fate is probably due to the extreme specialization which they have undergone. In the female forms this is not the invariable fate, for in some cases the cells undergo partheno- genesis, a process of renewal which is accompanied by nuclear activi- ties of a special kind. (See Chapter IV.) C. The Period of Old Age.—Protozoa quickly die after the period of maturity is passed, and old age, the final period of a life cycle, is rarely seen or recognized. Maupas (’89), however, using the culture method, gave a very graphic description of oldage in certain forms of infusoria. Thus, in Onychodromus grandis the body of the cell becomes much reduced in size, loses cilia and cirri, while other organs both external and internal, atrophy, and the organisms die of senile exhaustion. In Paramecium aurelia the circumstances accompanying old age have been described above, but in this case the metabolic processes had been restimulated, and apparently the cell organs were suitable for a continued activity, but something was wrong and the race died. ‘This “something” had to do with the germ plasm, for, as stated, the micronucleus was hypertrophied and divisions were abnormal. “The first clearly marked period of depression came in July, about six months after the cultures were started. It was characterized by a well-defined reduction in size (down to 109 microns) and by yacuolization of the endoplasm, while the ectoplasm did not appear to be much involved. Many of the individuals were characterized by great vacuoles similar to those in starved forms, which dis- torted the body almost out of recognition; in others the nuclei were fragmented into two or three parts, and in all there was a marked 128 PROTOPLASMIC AGE OF PROTOZOA absence of the larger food granules and gastric vacuoles which characterize the normal animals, and this, notwithstanding the fact that bacterial food was present in abundance (see Studies I). As stated in these Studies (III), the organisms under these conditions still take food, and in some cases the endoplasm appears opaque with the undigested food balls, but the decrease in size continues and the endoplasmic vacuolization is not prevented by the presence of the food. It is the digestive function, apparently, which becomes ineffec- tive at such periods, and if this is a correct assumption, this function can be stimulated, as I have shown by the experiments. “Identical results were obtained in the period of depression in December, 1901, a depression which was again overcome by the use of beef extract, while the individuals of the series which had been con- tinued on the hay diet all died. These became smaller and smaller, and again gave morphological indications of starvation, notwith- standing the fact that the individuals which had been stimulated with the beef extract were living and reproducing normally in the same food medium. They became much reduced in size, the endoplasm became distorted with vacuoles, and they died with absolutely no indication of disease through parasites. “These observations show, therefore, that starvation effects may be produced, even though the animals are living in a medium rich in food. It is trite to say that to prevent starvation we must have not only food, but the ability to digest and assimilate it, yet common as this observation is, it is important in the present connection, and involves a factor which cannot be overlooked in any discussion on old age. “Tn the June period, as stated previously, the same conditions were not observed, for the organisms, in part at least, had been treated with the beef extract every week during the first three months, since the previous period of depression. The division rate began to run down in the case of the B series in April, in the A series in May, and in all of the material that had been continued on the beef the characteristic structure was a densely granular endoplasm (Fig. 26, p. 82). In the specimens that had not been treated with the beef since the preceding December this character of the endoplasm was not noted. These unstimulated individuals died out in about the 508th generation (B series) after becoming much emaciated and reduced in size, and with reduced nuclei. . . . The unstimulated A series did not die out until about two weeks later. At the time when the B individual described above died (May 12) the unstimulated A series was char- acterized by somewhat reduced size, a declining division rate, and absence of the dense protoplasmic granules. In the stimulated A series, on the other hand, (Al and AQ) of about the 560th genera- tion, the structures were normal, gastric vacuoles were numerous, and MORE COMPLICATED LIFE CYCLES 129 divisions were frequent. Toward the end of June, however, when the A series nearly died out in the 620th generation, the conditions were very different. Fig. 26, left, is froma specimen in the 615th generation; its size is below the normal; its endoplasm is choked up with granules, and there is no trace of vacuoles save the contractile vacuole near one end. ‘he macronucleus is definitely granular, and its contour is irregular, as though devoid of nuclear membrane. The micronucleus is elongate and spindle- formed. The ectoplasm is not deformed, and save for the absence of trichocysts it appears to be normal. This was the condition of the protoplasm when the usual large number of culture individuals was reduced to 6 A’s and no B’s, and a condition from which the A series was rescued only with the greatest difficulty by the use of pancreas extract. “From this time until the race died out the division rate was slug- gish. The conditions of the protoplasm in the latter individuals was decidedly characteristic. Throughout the fall individuals would appear with densely granular protoplasm, which is invariably the Fic. 54 Paramecium aurelia from culture in 741st generation. The macronucleus and endoplasm are normal, the micronucleus is abnormal, and the cortical plasm is filled with vacuoles. (After Calkins.) sign of death, unless the animals are stimulated in some way. In such fae the macronucleus may or may not be normal, whereas the micronucleus, as a rule, becomes hypertrophied and the ectoplasm full of great vacuoles. Fig. 54 is a good representation of the condi- tions ae this time. The endoplasm. is apparently normal; there are food vacuoles and endoplasmic granules and vesicular structure, but the micronucleus is spherical end vesicular, has lost its usual place in a niche in the macronucleus, and shows evidence of granular modification of the previously homogeneous chromatin. “One of the two oldest of the A series (742 generations) showed the following points while alive: ‘A12 was alive this morning and was picked out ae examination. It had two contractile maciele: situated dorsally and close together. The astral canals were absent; in their place was a row of dorsal feeding canals, such as those Uhencrnisne of the more generalized holotrichida ( e.g., Chlamydodontide). The rest of the body contained eight or ve large vacuoles not contractile. The macronucleus was slightly hy pertrophied and visible, indicating 9 130 PROTOPLASMIC AGE OF PROTOZOA the approach of disintegration. The papillze of the cuticle were plainly visible, and what I have taken to be apertures of the trichocysts were more or less numerous. (This is shown in the preserved cell, Fig. 54.) A few trichocysts remained in the cortical plasm, but there were many vacuoles in this layer, indicating that when the trichocysts were discharged they were not reformed. The peristome was normal and the mouth had a vigorous oral membrane. The size was large, fully as great as any of the preparations that had been made at any time during the 749 generations. Movements vigorous us slow, with a tendency ¢ on the part of the animal to remain stationary.”* “Tt was while the organisms were in this structural condition that the many attempts to rejuvenate the race were made as described in the previous pages, and it was in this condition of the protoplasm that the race finally died out from exhaustion. Before dying, however, the individuals, as indicated in the above paragraph from my notes, were of full size and were filled with gastric vacuoles and partly digested food, while the body form was normal. “Tt must be admitted that these forms were capable of individual growth at this period, and since the macronucleus was normal in the last individuals, while the micronucleus was considerably changed, it must be further admitted that the vegetative metabolic processes were presum- ably re-invigorated; on the other hand, the functions of reproduction, that 1s, of division, were degenerated possibly, if not probably, because of the apparent degeneration of the micronucleus and of the cortical plasm, whose functions were not reinvigorated by the artificial means which were tried.” We are not in a position yet to demonstrate the nature of the cause of the depression periods. It is probably to be sought in the chemical make-up of the constituents of the cell, the chemical changes necessary for the functions of digestion, such as the formation of proteolytic ferments, oxidizing farments. and the like, being no longer possible with the same food. We may compare a paramecium or oxytricha with a storage battery, the one having, at the outset, a certain potential of physiological activity, comparable with the initial electric charge of the battery. With the same food for a period of six months the initial charge of vitality is drawn upon, as work done draws upon the initial potential of the battery, until in a period of depression the resources of the cell are exhausted and the organism dies by what Hertwig calls “physiological’’ death. The ‘battery, however, to continue our analogy, can be recharged and is good for another period of work. So can \ the paramecium pro- toplasm. The six months of culture does not exhaust the germinal possibilities of that protoplasm; in the cultures referred to, the organ- 1 From my notebook. MORE COMPLICATED LIFE CYCLES 131 isms, or rather the race, were in the 200th generation at the time of the first depression, but the vitality of the protoplasm was not exhausted until the 742d. Woodruff’s race of oxytricha protoplasm was in the 235th generation at the first depression period, but lived through 860 generations. ‘There is no doubt whatsoever that all of the cells of paramecium would have died in the first period of depression had nothing been done to revive them. Joukowsky, in 1898, followed paramecium through 170 generations, when they all died during a period of depression; Simpson, in 1901, noted the gradual loss of vitality and death in his three to four months’ cultures of paramecium. My cultures would have disappeared in a similar manner had it not been for a change of diet, by which it was found that beef extract, if given to paramecium for several days during this depression period, would restore the vitality and start the organisms off on another cycle of cell generations. In this way the few surviving organisms of the original culture were stimulated to new activity, or, to carry out the analogy with the battery, were given a new potential of vitality and a potential which again lasted through a period of six months, and through approximately the same number of generations (actually, 198) (see Fig. 38, period, August, 1901). How can the renewal be interpreted? Obviously, the change in diet gave the cells an entirely different assortment of chemical substances, and it is to this fact that we may attribute the artificial rejuvenescence. Woodruff found that the same expedient renewed the vitality of his race of oxytricha, the effect being slower than in the case of para- mecium. It was also found by Calkins that a change in the salt con- tent of the usual food media would produce a similar stimulating effect, and dilute solutions of potassium phosphate were used, the organisms experimented with being allowed to swim in the solutions for half an hour (a longer period being followed by death in a few days). This simple salt, like the beef extract, was enough to renew the vitality, and the stimulus thus given was sufficient to enable the organisms to live again in the same medium for another cycle of 193 generations. The effect of the change on the organism’s structure is of interest, and is represented by Fig. 39. The cell in a depressed condition is shown on the left; a cell twenty-four hours after treatment is shown in the centre, where a lighter area in the vicinity of the nucleus will be noted, the ends meanwhile showing the same densely granular struc- ture as that of the depressed condition, thus indicating that the organ- ism is recovering from the disease, if we may so designate its trouble. It is important, in this connection, to note that the reéstablishing of the normal structure occurs first in the neighborhood of the nucleus, a fact that indicates that here is the region of greatest chemical activity in the cell. A cell forty-eight hours after successful stimulation is shown on the right. These show that the “labile” condition of the 132 PROTOPLASMIC AGE OF PROTOZOA protoplasm is now extended nearly throughout the cell, the extremities alone retaining the granular structure of the depressed condition. After such successful stimulation the digestive processes recom- mence, the organisms divide, and the division rate, as indicated by the curve, rises to an average of more than one division per day (see Fig. 38). Three times in the history of this paramecium culture were the cells stimulated to new activity by this artificial means. The first time, as stated, was after the 200th generation, the stimulant being beet extract; the second time was after 198 generations more (398th of the race), the stimulant being beef extract and potassium phosphate; the third was after about 193 generations more (about the 600th of the race). ‘This third period of depression was most interesting, for it was found that the same stimulants that had been previously used with success were now without effect; beef and potassium salts of various kinds were tried in vain, and the final extinction of the race was threat- ened; indeed, one race, which was called the B series, died out entirely in the 540th generation. Only six cells were left, finally, for experi- mentation, but some of these were successfully stranlated: by treatment with an extract of pancreas, which contains many different salts in solution. The effect of this last stimulation was a renewal of the vitality, but the potential given to the protoplasm was not so great nor so clearly defined as in the previous periods of depression, and after another six months, in which the organisms showed great sluggishness, the race died in the 742d generation. This fourth cycle is the most important for our present purpose, since it represents the period of old age in the protoplasm under observation. The cells divided only 123 times, and toward the end manifested curious and hitherto unobserved degenerative phenomena, which deserve special attention. The protoplasm of the cells in this final period of depression had at first the same appearance as the protoplasm of the organisms at previous periods of exhaustion; the cell body became granular, the size decreased, and the general appearance was similar to that w ash had been successfully met at previous periods. The same stimulants were used; the diet was changed for short periods as before; and, singularly enough, the same effect on the structures of the cell was produced. The granules disappeared, the nucleus and cy toplasm appeared perfectly normal, and the organisms were able to take in food, digest, and assimilate it. The normal size was restored, and it seemed, “from morphological grounds, that the depression period had been successfully overcome for a fourth time. Still, the cell divisions were very infrequent and irregular, while the few that did take place were mostly of a pathological nature, complete fission not taking place, the result being monsters of different size and form (Fig. 55). The macronucleus was perfectly normal in the last cells of the race, but the MORE COMPLICATED LIFE CYCLES 133 micronucleus, which has but little part to play apparently in the ordinary functions of vegetative life, now appeared enlarged and vesicular, and entirely different in structure and size from the micro- nucleus of the ordinary paramecium. The protoplasm was not granu- lar nor chemically stable, and was apparently as active as ever. Still the organisms died, and death was not due to infection or disease. Something in the cells that had been operative before had given out, and the only part of the cell which had not responded to treatment was the micronucleus. Here, then, was a pathological condition which could not be met, and the organisms died. @ Fic. 55 A ‘‘monster”’ formed by incomplete division of Paramecium aurelia as an indication of the exhaustion of division energy. (After Calkins.) Was it death from old age that carried off the race under obser- vation? ‘There seems to be no other alternative to consider, and by old age we mean the wearing out of an organ and the cessation of a function. If old age may be thus defined in a simple organism like paramecium, it follows that three times previously had the race been weakened by old age, since the organisms were unable to digest and assimilate food. As soonas this power was restored by artificial means, old age was overcome and cell division was resumed. The cells would have died without any doubt had they not been stimulated, so that we are justified, as I believe, in speaking of this condition of paramecium as “physiological” old age, which leads to physiological death through the cessation of one or more of the vegetative functions. It is obviously death from a different cause that carried off the last cells of the race, and since the ordinary vegetative functions were apparently in perfect working condition at this final period, it follows that the cause of death must be looked for in the cessation of some other than the ordinary vegetative activities. The history of the micronucleus in conjugation (see next chapter) shows that this is the organ of the paramecium cell endowed with the characteristics of the race; in other words, that it alone of all the structures of the cell, must contain the 134 PROTOPLASMIC AGE OF PROTOZOA germinal elements. It is to be compared with the germ plasm con- tained in the germ glands of the many-celled animals, while the macro- nucleus and the cytoplasm are to be compared with the relatively much more voluminous somatic tissue of the higher animals. Its degeneration, therefore, indicates an exhaustion of the potential of activity of the germinal functions, including the power to divide, and with this exhaustion comes the death of the race, but death due to “germinal” rather than physiological exhaustion. While physiological death may be averted by stimulants of different kinds, germinal death, at least in the experience of all investigators up to the present time, cannot be offset, and with this comes the inevitable death of the race of protoplasm or death from germinal old age. Still paramecium are plentiful in ditches and ponds, a fact indicating that there is some natural way in which germinal death can be averted. Here is where the process of fertilization comes into play, and with fertilization the protoplasm of an exhausted paramecium is made over into a new “individual,” in the same way that the protoplasm of a germ cell of a bird, mammal, or man is made over into a new individual. These various experiments indicate, therefore, that natural death from old age under the conditions of the laboratory is actually inherent in protoplasm as little differentiated as in these single-celled animals, and they fail to confirm Weismann’s claim that natural death is a penalty which higher animals must pay for the privileges of differenti- ation. They likewise fail to show that natural death by old age is due to any malevolent action on the part of certain structures of the body, as Metchnikoff would have us believe is one cause of old age in man. It is a natural condition of all protoplasm to grow old, and if we find the phenomenon in the generalized cells of the infusoria, how much more probable is it in the highly specialized somatic cells of the body. Each paramecium has a certain allotment of natural life and division. I have called it the potential of vitality. When this is exhausted under given conditions the protoplasm dies. It also has a certain allotment of germ plasm, so that by exhaustion of the physiological potential it still may retain a certain capacity for cell division, the germinal potential not being exhausted. It may, therefore, be stimulated by artificial means. In different kinds of animals and in different indi- viduals of the same species it is probable that the initial potential varies, in some representing a longer, in others a shorter, life. In paramecium and the protozoa generally we find the greatest relative germinal potential, but as we go higher in the animal scale the ten- dency is for the germinal plasm to concentrate in a definite tissue of cells, the ger minal epithelium, while the somatic cells have a corre- spondingly low degree of germinal plasm. ‘To illustrate, while in all probability every cell of the paramecium race is capable of becoming or of giving rise to a germ cell, the same is not true of the animals next MORE COMPLICATED LIFE CYCLES 135 higher in the scale of animal forms, such as the hydroids and jelly fish. A very small fragment, indeed, of a hydra will reproduce the entire animal, but one cell of the hydra will not do so; each of the two germ layers must be represented in the small piece. In worms and in still higher forms of the invertebrated animals this power to regenerate the entire animal decreases pari passu with the differentiation of the animal, and although not absolutely true, it may be stated in general that the higher the differentiation the less is the power to regenerate lost parts. In other words, something is lost from the highly differ- entiated somatic cells, something which is segregated in the germ cells and something which we find in each cell of the lower forms of inver- tebrates, but most widespread in the unicellular protozoa. It has to do with the racial characters of the organism, that is, with the germ plasm. In hydra and in some of the worms the cells retain enough of this germ plasm to reproduce the entire organism, but in the mammals the somatic cells have so nearly lost this germinal power that regen- eration of an organ or limb is no longer possible, and is limited to the mere repair of an injury. In this sense, therefore, Weismann’s claim that natural death is the penalty higher animals must pay for differ- entiation is justified. The so-called “noble” cells (Metchnikoff) of the body, that is, the cells of brain, liver, kidney, and other important centres of physio- logical activity, are somatic cells in which this regenerative power is reduced to a minimum; the potential of germinal activity in them is less than in connective-tissue cells, and after an injury their power of repair is less than that in connective-tissue cells. This is seen in the fact that a wounded epithelium is repaired less by the proliferation of the neighboring epithelial cells than by the adjacent connective tissue, and the “‘scar” tissue which results is composed of these “baser”’ cells. Like the physiological activities of paramecium, all somatic cells of the body are endowed with a certain potential of physiological activity, and like paramecium, when exhausted the particular function of those cells ceases; they have reached the limit of their activity, and when enough of them are so worn out a general impairment of the body functions results. This condition of the exhausted cells may be relieved by stimulants which, we imagine, may come from the general body itself, or from artificial treatment, as in the case of paramecium. But we have no reason to believe that in the human somatic cells this stimulation can be repeated indefinitely. If in the generalized proto- zoon there comes a time in which the potential of germinal activity of the cell gives out, how much more probable would it be that the somatic cells, with their low potential of germinal activity, likewise fail to respond to the stimulants. Unable to reproduce by division, with their potential of physiological activity reduced to a minimum, 136 PROTOPLASMIC AGE OF PROTOZOA these ‘‘noble” cells atrophy, their positions being taken by the con- nective-tissue cells. Here, then, is the condition of old age; the somatic cells lose what germinal power they possess through physiological usury; their potential of physiological activity is greatly reduced; the function of the organ is impaired and the entire organization correspondingly weakened; the useless cells are attacked by phagocytes (?) (Metch- nikoff), and they are replaced by the non-functional connective tissue. Old age, therefore, is a biological condition of protoplasm, char- acteristic alike of the lowest protozoén and the highest mammal. Its progress is inexorable, its advent inevitable, while the only per- manent plasm is that which has the highest power of germinal activity, and this is contained in the germ cells. Here, however, that other unfathomable mystery of life—fertilization, or its equivalent—is essential for the proper stimulation of the latent developmental activity and the distribution of the somatic and germinal cells in a new indi- vidual organism. How this occurs in paramecium and other protozoa will be shown in the following chapter. While the experiments on the lowest animals show that old age is a necessary condition of vitality and inherent in all protoplasm, it does not follow that man or any other animal has made the best possible use of the vital endowments. It may very well be, as Metchnikoff maintains, that the traditional three score and ten is not an adequate allowance for man, and it is conceivable that the normal length of life may be increased by careful living to four or five score of years or more. If there is a certain amount of vitality upon which one can draw, it is obvious that the faster it is drawn the shorter will it last, and conversely, the more saving one is by careful living, the longer will it endure. Only one thing are we sure of, and this is that somatic vitality, whether in protozo6n or man, is a peau de chagrin which con- stantly diminishes with use until finally nought is left. CHAPTER 1. CONJUGATION, MATURATION, AND FERTILIZATION. 1 the preceding chapter it was shown that the protoplasm of which a protozodn is composed, as demonstrated by continual observation, gives evidence of advancing age no less surely than does a many- celled organism. It was shown further that the advance from youth to age in such protoplasm is indicated by more or less well-marked physiological and structural changes, the former being characterized by the onset of a noticeable “period of depression,” the latter by morphological changes, of which the most important is the develop- ment of a well-defined germ plasm. Experimental work on free-living protozoa has shown that the cells die a natural death during such periods of depression, but also, in some cases, that these periods may be overcome by artificial stimulation. They show, also, that a final depression, distinguished from ordinary physiological or metabolic weakness, and characterized by loss of the germinal protoplasm, could not be thus overcome. Apart from death by violence, therefore, the free-living protozoén may lose its life by what Hertwig calls ‘“ physio- logical death” at some period of physiological depression, or by “‘verminal death” occurring with the exhaustion of the division energy and degeneration of the germ plasm. Notwithstanding the many natural enemies which a paramecium or other protozo6n has, and in spite of the fact that if it escapes such enemies it may die from physiological or germinal “old age,” it still exists in more or less abundance in natural waters, and will probably continue to exist in the future. In natural waters, salts, changes in the local environment, and other external causes undoubtedly tend to stimulate lagging physiological activities and to do on a large scale what we have Houed in the laboratory; but in nature, as in the labor- atory, such means of rejuvenation probably have their limits, and we must turn to other vital activities for an explanation of the continued existence of these living cells. There is little reason to doubt that the explanation lies in the secrets of the same mysterious and at present unfathomed phenomena which underlie the newborn infant; which are repeated in all living things with the creation of a new individual; and which are univer- sally regarded as among the subtlest of vital activities. These secrets are deeply hidden in the phenomena of fertilization, and philosophers today, like the ancients, have only speculations to offer 138 CONJUGATION, MATURATION, AND FERTILIZATION in explanation. The phenomena of conjugation and maturation of the germ plasm which accompany fertilization are more easily inter- preted, for they are largely matters of observation and deduction. In protozoa we have a particularly rich field for investigation of these problems, for the union of germ plasms is accompanied by phenomena of such relative simplicity that they are more easily observed, con- trolled, and interpreted than with metazoa. In interpreting the phenomena of fertilization of protozoa we are in accord with those naturalists who, since the time of Harvey, have advocated some ‘“‘dynamic” theory or other. (See Wilson, The Cell, p. 178.) In recent times this explanation is usually based upon the facts of decreasing vitality with advancing age, and, as expressed by Hertwig, fertilization is the means of restoring to a labile condition the protoplasm which, with continued physiological activity, has become stable in physical and chemical equilibrium. It is, therefore, essentially a process of rejuvenation. Opposed to this point of view are those who, with Weismann and his followers, maintain that protozoa do not die of old age, and that conjugation with fertilization is an incidental occurrence in the life ofarace. Fertilization, in higher forms, is a means of bringing about variation within the species, and at the same time a means of keeping the species true to its structural type. Weismann still maintained his contention in regard to the immor- tality of infusoria after Maupas’ classical experiments had demon- strated old age, and held that conjugation does not alter the indi- viduality of the cells, since that individuality is retained after con- jugation. Such a point of view would seem to be, however, merely an expedient to save the argument, for the essential part of the fer- tilized protozoén, like the metazo6n, results from the union of two germ plasms, the protoplasm resulting from this union being a new individual in both cases. Like the metazoén, the protozoén is physi- cally immortal only in the same sense of continuity of the germ plasm, for, with each fertilization there is a re-organization of the protoplasm, new chemical and physical combinations, and new individuality. There is no difference in kind in protozoa and metazoa, only a differ- ence in degree. The essential feature of fertilization appears to be the union of two masses of chromatin. We can only conjecture as to the significance of such union, but whatever hypotheses are framed to explain it, they must take into consideration a great variety of conditions under which the phenomenon is manifested. It is quite evident that complicated processes in metazoa are the highest and last steps, so to speak, in the elaboration of this universal biological phenomenon, and it is probable that they differ only in degree from the lowest and most primitive steps shown by the simple syngamic processes in protozoa. FERTILIZATION BY AUTOGAMY 139 In this lowest group of animal forms we find every grade in com- plexity in the sequence of syngamic processes, from those of undoubt- edly primitive character to processes quite as complicated as in many metazoa. We may pass from cases where only the one cell is involved, fertilization taking place by union of two chromatin masses derived from the same primary nucleus (autogamy); through cases where the chromatin has had the same ancestry but is derived from different cells (endogamy); to cases where sex differentiation and maturation processes are quite as complicated as in higher animals and plants (exogamy). With our present incomplete knowledge of the life his- tory of lower forms, no great value is to be attached to such a classi- fication, but its main purpose is served in providing a convenient frame for attaching the manifold variations presented .by the phe- nomena of syngamy in protozoa. A, FERTILIZATION BY AUTOGAMY (AUTOMYXIS, HARTMANN). In the primitive forms of protozoa, as in those of plants, this method of fertilization is widespread, and whatever may be the significance, its wide distribution among the most diverse of these lower forms and under the most varied conditions of life, indicates a natural and simple, if not primitive, fertilization phenomenon. Even in these more primi- tive cases, however, grades in complexity of the processes involved are to be observed, and the transition from autogamy to endogamy Fic. 56 Ameba limax budding, division, and idiochromidia forming stages. may occur in the same group. So far as the protozoa are concerned, the most primitive methods are to be found among the free and parasitic amebie, but even here there are indications of a more advanced process. The main element that enters into the complexity of these more primitive cases of autogamy is the formation of so-called secondary nuclei from idiochromidia and the differentiation of somatic and 140 CONJUGATION, MATURATION, AND FERTILIZATION germ nuclei. But in the simplest form such complication is not appar- ent, for the idiochromidia becomes segregated in masses without nuclear walls, and these masses fuse. This is the case in Ameba limax, a small free-living ameba common in ponds or decaying matter. It may be easily cultivated on artificial culture media, such as agar, in connection with various types of bacteria serving as food. Under normal conditions of temperature, salt contents, etc., the amebz reproduce by simple division and by budding (Figs. 56 and 57). Under certain conditions of the cultures, conditions which have not been thoroughly investigated, the organisms encyst and remain so until transplanted to new culture media. Occasionally, and again under conditions unknown, they form sexually mature cells, but this latter condition may also be brought about by suitable temperature changes. Fic. 57 he Ameba limax. Nucleus in upper cell in full mitosis; in lower cell (right) in anaphase of the mitosis. Syngamic nuclear union is always preceded by idiochromidia formation within the cyst, but the formation of this material does not necessarily imply sexual maturity. In all cultures, after a time, the nucleus, which consists of a central kar yosome and peripheral chro- matin, gives rise to idiochromidia by dissolution of the peripheral portion. The idiochromidia become scattered throughout the cell, and, under ordinary conditions of the culture, are evenly diffused. If the cultures be subjected to rapid changes of temperature, the idio- chromidia may be caused to accumulate in masses about the periphery (Fig. 51, p. 122). Sixteen of these masses are usually formed, and then by fusion two by two the number is reduced to eight. This fusion possibly represents a sexual union, or, more strictly speaking, takes the place of sexual union, being the fusion not of secondary FERTILIZATION BY AUTOGAMY 141 nuclei, but of masses of idiochromidia which in other protozoa become differentiated into such nuclei. The karyosome and some of the peripheral chromatin form a degenerating ‘‘somatic” nucleus which takes no part in the later processes. The further fate of the encysted form thus brought about has not been followed, but in Entameba histolytica, according to the observa- tions of Schaudinn and, later, of Craig (’08), such a stage is followed by spore formation. Schaudinn (’03) observed, and his observations have been confirmed in every detail by Craig (08) upon living and fixed material, that in this ameba the nucleus fragments into idio- Entameba histolytica. (After Craig.) A, organism showing rods and granules of chro- matin in the nucleus, vacuole with some stained substance, and dense ectoplasm; B, the chromatin of the nucleus passing into the cell plasm, where it is distributed as chromidia, shown in C; D, aggregation of chromidia to form secondary nuclei (see Fig. 51, of Ameba limax); EF, “spore formation’? by budding; F, spores of Entameba histolytica as seen in feces. chromidia (chromidia) which collect in masses at the periphery, and these masses, with some cytoplasm, are protruded from the surface as buds. The buds become covered with a hard and resistant membrane which is so deeply colored by the intestinal fluids that further internal processes could not be followed (Fig. 58). Neither Schaudinn nor Craig observed union of these idiochromidia masses, and the resem- blance to Ameba limax can only be inferred from the similarity of preliminary processes. In the closely allied forms, Entameba coli, Entameba muris, and Ameba proteus, the process of autogamy is somewhat more compli- 142 CONJUGATION, MATURATION, AND FERTILIZATION a cated because of the formation of definite nuclei from idiochromidia, and because of so-called maturation divisions of these nuclei before union (coli and muris). Here, again, the early observations of Schaudinn (’03) upon Enta- meba coli have been fully confirmed by Craig (708) and their conclu- sions have been fully supported by Wenyon C07) i in connection with E. muris, a closely allied intestinal parasite of the mouse, and by Hartmann (’07) upon Entameba tetragena in man. Schaudinn’s excellent description was not accompanied by illustrations, but the Autogamy in Entameba (ameba) muris. (After Wenyon.) A, ordinary ameboid form with nucleus in process of division; B, ordinary individual encysted and with one nucleus; C, nucleus divided; D, chromatin has passed into cytoplasm, leaving no definite nuclei in the cyst; E#, two small nuclei reformed from the scattered chromatin, other chromatin residue and food remains are being voided; F, two nuclei and so-called ‘‘reduction’’ bodies remaining in cyst; G, a cyst with two spindles, food remains, and some waste chromatin; the two spindles give rise to four nuclei which conjugate two and two; H, cyst with two recently conjugated nuclei which next divide to form four (J) and finally eight (J) spore nuclei. corresponding stages may be illustrated by Wenyon’s figures of E. mauris. Here and in E. coli the organisms encyst after a period i in the intestine; the nucleus of the ency sted cell divides (Fig. 59, A, B, C) and the cell body indicates a corresponding division into two parts, but the connections between these parts is never lost, and we are thus dealing at the beginning of fertilization with a binucleated cell. The nuclei next fragment, forming idiochromidia, from which two much smaller nuclei (D, Z) are formed by segregation of the scattered granules. Each nucleus then divides twice, one-half of each division forming nuclei which degenerate in the cell (reduction nuclei) and two FERTILIZATION BY AUTOGAMY 1438 fertilization nuclei finally result, each of which divides again, this time with the long axes of the spindle parallel with one another; the final daughter nuclei which are formed fuse two by two, the cleft in the cell disappears, and an encysted ameba results with two fertilized nuclei. Each of these nuclei divides twice, and eight spores are formed about the eight resulting nuclei. Hartmann (’07) mentions a similar process of autogamy in the case of an ameba from the frog and in one of the free-living imax forms, but describes a quite dissimilar process in Entameba tetragena. In these cases, therefore, there is a concentration of the idiochro- midia in secondary nuclei which then undergo so-called maturation processes. A still greater complexity is shown by Ameba proteus, where, according to the observations of Calkins (’07), there is no formation of diffused idiochromidia, but the secondary conjugating nuclei are formed directly from chromatin granules within the primary nuclei, which, prior to this stage, had divided repeatedly until about 70 are present. ‘These secondary nuclei next fuse two by two in the cytoplasm and give rise to spore-mother cells (sporoblasts), of which there may be as many as 250 within one parent organism (Fig. 60), while at least one of the primary nuclei remains unused and finally degenerates in the cell. In Ameba proteus, therefore, the organism forms not one spore-mother cell, as in the parasitic amebze, but many such spore-forming centres. In all of the above cases of autogamy, we have to do with the fusion of chromatin materials which at one time or another were parts of the same nucleus of the same cell. In all of them, with the exception of the free-living Ameba limax and the parasitic Entameba histolytica, where further observations are much to be desired, the union of the “oametic”’ nuclei does not take place until after two or more divisions of the primary or secondary nuclei; that this fact has some signifi- cance cannot be doubted, but there is no inkling as to what the significance is, unless, indeed, it is evidence of an earlier gamete- forming stage, autogamy thus being, as Hartmann (09) suggests, a degenerative rather than a primitive phenomenon. With the myxosporidia the process is much more complicated, involving the formation of vegetative and germinal nuclei. It is well described by Schréder (07) for the case ofa parasite of the seahorse, Spheromyaa labrazest, where the multinucleate ameboid body of the parasite appears to contain two kinds of nuclei distinguished by size and structure. Within this protoplasmic body small areas become differentiated from the surrounding matrix. These areas character- istic of the myxosporidia, termed pansporoblasts (Gurley), contain two nuclei, one of each kind (Fig. 61, K, Q). With development of the pansporoblast each nucleus divides in such order that seven daughter nuclei finally result from each, the fourteen nuclei being 144 CONJUGATION, MATURATION, AND FERTILIZATION characterized as follows: Two are destined to degenerate as ‘‘reduc- tion nuclei,” four become the centres of shell formation of the spores, four become centres of pole capsule formation, and four remain as Fic. 60 Autogamy in Ameba proteus. In upper figure secondary (gametic) nuclei are shown emerging from the primary nuclei. In central figure is pictured the union of gametic nuclei together with some undeveloped ones in a primary nucleus. In lower figure is shown the mass of sporoblasts which develop from the fertilized gametes. (After Calkins.) Conjugation in myxosporidia. A to 7, Myxobolus pfeifferi, Th. (after Keysselitz); A to Q, Spheromyxa labrazesi, Lay. and Mes. (after Schréder); A, B, formation of gametoblasts; C to G, union of sporocysts and multiplication of nuclei; H, young sporoblast with polar capsules forming and gametic nuclei not yet united; /, spore with capsules (not filled in) and gametic nuclei united; A, young pansporoblast of spheromyxa, with dimorphic nuclei; L, pan- sporoblast with fourteen nuclei; 2, pansporoblast divided into sporoblasts, each with two pole capsules (p), four globules present (1) and with two central reduction nuclei; N, sporo- blasts having two shell nuclei (s), two polar capsules, each with a nucleus and two germ nuclei; O, young spore, shell nuclei disappeared, capsule (p) and germ nuclei (gg) compact and lying in a row; P, same, with union of gametic nuclei in the sporoplasm; @, same, ripe spore with polar capsules and sporoplasm. 10 146 CONJUGATION, MATURATION, AND FERTILIZATION germinal nuclei. The sporoplasm of the pansporoblast divides into two parts (J/), the sporoblasts each containing six of the fourteen nuclei, while the reduction nuclei remain outside. The six nuclei in each sporoblast are thus differentiated into somatic and germinal nuclei, four in each case going into somatic modifications of the spores (shells, pole capsules, and threads), and two, presumably one of each of the original two kinds, remaining as pronuclei (.V, O, P). After the spores are mature and only traces of the somatic nuclei remain, these germinal nuclei fuse, so that the spores, when taken into a new host, are uninucleate (P, Q). If, as Schréder suspects, the multi- nucleate ameboid adult is formed by fusion of two or more cells, then such a process would be like that of the mycetozoa and exogamic rather than autogamic (p. 150). Observations, however, are wanting to confirm this supposition, the many obstacles in the way of observa- tions to this end making confirmation extremely difficult, but the other matters relating to number of nuclei formed, their fate, etc., are well corroborated (see actinomyxide, and Myxobolus pfeifferi, Fig. 61, A, J). B. FERTILIZATION BY ENDOGAMY (PEDOGAMY, PROWAZEE). The transition from autogamy into endogamy, whereby the sexual union is between descendants of the same original cell, is marked by numerous intermediate stages which are sometimes described as autog: amous. The difference is largely one of degree only, and among these intermediate forms, at least, to include them under one or the other heading is mainly a matter of expediency. The principle under- lying the distinction is , however, of considerable theoretical i importance, and the difference which exists between the partially divided cell in Entameba coli (see above) and the union of separated parts within the same parent cell (see myxobolus and other cases below) is a differ- ence which becomes magnified in higher types into all of the differential characteristics which distinguish exogamic processes. The transition from autogamy to endogamy i is well shown in myce- tozoa and myxosporidia, where, as may be seen, the difference is only one of degree. There are numerous examples of the phenomenon, from which we select a few showing different grades in complexity, and it should be noted that the same arguments as to the possible exo- gamic nature of the processes apply here among the mycetozoa and myxosporidia as well as in the cases cited above. Keysselitz (0S) has quite recently described the process of pan- sporoblast formation in a myxospore (Wyxobolus pfeiffer?) which differs in one important respect from the process in spheromyxa. Here the pansporoblasts which Keysselitz names the ‘‘ propagation. FERTILIZATION BY ENDOGAMY 147 cells” arise in the plasm of the adult organisms in the same way as in other myxosporidia, but the nuclei and with them the cell body of the germinal area divide (Fig. 61, 4, B, C). These propagative cells later unite two by two, and are separated only by a thin cell wall, which later disappears. Within this united mass the nuclei divide until there are fourteen, as in spheromyxa; their formation differs in some unessential details, but their fate is the same in both cases, two germinal nuclei finally resulting which conjugate in the mature spore (Fig. 61, D, I). Caullery and Mesnil (05) have carefully described the process of spore formation in spheractinomyxon, one of the actinomyxide, an aberrant group of myxosporidia named by Stolé (’90). Here the process is a little more complex than in the case cited above, but it agrees in essence with that described by Keysselitz. The youngest stages are found as intestinal parasites of the tubificid worm clitellio, and are either uninucleated or binucleated. ‘The observers are inclined to believe that the uninucleated stage comes first and that it repre- sents, possibly, the youngest form, or sporozoite, and that the binu- cleated stage represents the first division of this nucleus. If this pos- sibility is not well founded the fertilization process here must be taken out of the present category. Whatever may be the origin of these nuclei in the binucleated stage, they divide, and two of the first four nuclei formed become somatic nuclei and are connected with the formation of the cyst wall, within which the further processes take place. With the division of the nuclei the cell body also divides until there are sixteen independent, nucleated subdivisions. These unite two by two, the process of fertilization being thus affected, and eight spores ultimately result. The interpretation of this interesting case, as Caullery and Mesnil point out, depends entirely upon the mode of origin of the early binucleated stage. If these two nuclei represent a plastogamic union of gametes, as Léger (04) believed to be the case in an allied form triactinomyxon, then the process might be one of exogamy, but, as Caullery and Mesnil contend, this would involve two sexual processes in the life cycle, which seems improbable. The subject certainly needs further study. The endogamous process in the mycetozo6bn Plasmodiophora brassice is somewhat less complex than in the forms just described. Here, as Prowazek (’05) has shown, the protoplasm breaks down into many centres, each containing a sexual nucleus, and these centres —gametes—fuse two by two, a spore wall being formed about each copula (Fig. 62). In the majority of parasites the probability of endogamous fertiliza- tion is readily apparent, and the fusion of gregarines, for example, two by two, may be a union of cells from the same sporocyst or dif- ferent sporocysts. In such cases it is impossible to state definitely, 148 CONJUGATION, MATURATION, AND FERTILIZATION therefore, whether the process is endogamous or exogamous, and the same obscurity obtains in the union “of free flagellated or ciliated gametes. In some cases, on the other hand, there i is no doubt about the union of nearly related cells. Schaudinn (94) described the union of gametes of the same brood in Hyalopus dujardinii, and it is proved in the case of Basidiobolus lacerte by Loewenthal (03); in Actinospherium eichhornii by Hertwig ( 98); in yeasts by Guillier mond (’02), and in cultures of free-living infusoria ( Paramecium aurelia) by Calkins (02). Fic. 62 Endogamy in Plasmodiophora brassice. (After Prowazek.) A, portion of plasmodium showing ordinary vegetative nuclei; B, reconstruction of the gametie nuclei; C, division of same; D, union of gametes formed about gametic nuclei; E, F, stages in fusion of nuclei and formation of the spore. In basidiobolus, an intestinal fungoid parasite of the turtle, the organism forms straight or branched hyphz composed of sister cells lying end to end, and at maturity two adjacent sister cells conjugate, a process recalling conjugation among the lower plants (conjugate, diatoms, etc.). In actinospherium the phenomena of fertilization are ae more complex and have been made the subject of careful study y Hertwig (’98). The first evidence of the process is the encys stment if the adult organism and excretion of waste matters contained in the protoplasm. The many nuclei of the ordinary forms are here reduced to about 5 per cent. of the total by a process of fusion and absorption in the protoplasm, and after this has occurred the mother animal fragments into as many daughter cysts (cytospores No. 1) as there are FERTILIZATION BY ENDOGAMY 149 nuclei remaining (from one to twenty). Each of these daughter cysts secretes a gelatinous envelope about itself, and the nucleus of each divides by mitosis. This mitotic division is followed by division of the cytospore into two daughter cells (cytospores No. 2), and in these there are two successive nuclear divisions resulting in four nuclei. Three of these nuclei degenerate (‘polar bodies”’) and one remains as a pronucleus. The cytospores of the second order next unite again, reforming the cytospores No. 1, and the fertilization is completed by Fic. 63 Endogamy in Actinospherium eichhornii. (After Hertwig.) A, two gametes (cytospores No. 2), resulting from the division of cytospore No. 1; B, both polar bodies are formed in the right gamete, the second one forming in the left gamete; C, later fusion of the gametes, the nuclei now uniting and the polar bodies being absorbed at p; D, young actinospherium leaving cyst. fusion of the pronuclei. Thus, by a process of union of sister cells (endogamy) fertilization is brought about after complicated matura- tion processes (Fig. 63). Finally, in Paramecium aurelia, Calkins (02) found that cells removed by not more than eight or nine divisions from a common ancestral cell would conjugate normally, and that such fertilized cells were able to live through an entire cycle of cell generations (379 actually). Conjugation ‘between closely related forms, therefore, is quite as potent as between those of diverse ancestry. 150 CONJUGATION, MATURATION, AND FERTILIZATION C. FERTILIZATION BY EXOGAMY. It is not at all improbable that some of the cases that have been described as autogamous may be in reality exogamous. In the multi- nucleate forms, in order to decide such a matter it is necessary not only to observe the union two by two of such nuclei, but their mode of origin must also be known. Thus, in the mycetozoa the plasmodium from which the sexual nuclei are generated is formed by the fusion of two or more ameboid cells at an early period of development, hence the nuclei which later fuse may be derived from different ancestral cells, and such fusions would not be examples of autogamy, but of exogamy. In some cases of sexual reproduction among myxosporidia (notably in the actinomyxide and possibly in Spheromyzxa labrazest) a similar derivation of the conjugating nuclei has been suspected. Such cases of possible exogamy are well illustrated in almost any of the higher types of mycetozoa, and one such has been well described by Kriinzlin (07) for Arcyria cinerea and Trichia fallax, and by Olive (07) and Jahn (07) for Cerattomyxa hydnoides. Without going into the details the process may be summarized shortly as follows: The young ameboid or flagellated spores, after assumption of the ameboid state, fuse into plasmodia of considerable size. Cell boundaries are entirely absent and the nuclei have an opportunity to become thor- oughly mixed in the protoplasmic streaming. Fructification ensues after a longer or shorter vegetative life and in these fruiting bodies, or before their formation, the nuclei unite in pairs, the union being followed by synapsis and double divisions and formation of the ripe spores. A somewhat similar union has been described by Hartmann and Nagler in the case of Ameba diploidea, H. and N., where the organism is binucleated throughout the ordinary vegetative stages and until the period of maturity, when two cells place themselves side by side within a common cyst. The two nuclei of each cell then unite, forming a single synkaryon in each cell. The two adjacent cells finally unite by dissolution of the cell walls that separate them, and the recently fertilized nuclei, after some very questionable so-called maturation processes, assume the characteristic position of the vegetative forms. Here, then, if this observation is accurate, there is an exogamic fertili- zation, but the end stage does not occur until the next following period of maturity (Fig. 64). In the majority of protozoa the germ of the new individual, as in metazoa, is produced by the union of cells from different ancestors, and these cells, for the most part, show characteristic evidences of the period of maturity. In some cases there is but slight difference, if any, between the conjugating cells and the normal ones, the conditions FERTILIZATION BY EXOGAMY 151 of maturity manifesting themselves in other ways than by size changes. In other cases the conjugating cells are reduced in size, but without differences of a sexual character, and in still other cases there is a Fic. 64 Cc Ameba diploidea, Hartmann and Nigler. © @ So . h on, £38 a Tertian Estivo-autumnal Malarial Plasmodium. Oliver’s Modification of Wright’s Stain. 1, 3, 4, 5, 6, 7, 8, 9,10, and 15. Ring forms of tertian estivo-autumnal plasmodium. 2, Intracellular form, 11, 13, 14, 16, and 17. Pigmented ring forms. 12. Red corpuscle, showing infection with two “ring forms.’’ 18 and 19. Pigmented forms, just prior to seg- mentation. 20, 21, 23, and 24. Round and ovoid forms developed from crescents. (After Craig.) . Macrogamete. wo vy 9 5 to 36. Crescentic forms of estivo-aut plasmodium (tertian), nae 29. Ovoid form. 37. Segmenting form. 38. Sporozoites. a. Segmenting form of quotidian estivo-autumnal Plasmodium. THE PATHOGENIC HEMOSPORIDIA 285 ever, see p. 287). After some twenty-four hours the plasm of the ring form collects at one point, giving the effect of a signet ring, and the pigment granules first appear in the thickened portion. After about thirty hours the majority of the parasites have disappeared from the peripheral circulation, although a few may be found, especially in the Italian forms of the disease. In such cases the parasites, after thirty- six hours, appear round or oval and very sharply contoured, occupying from one-fifth to one-fourth of the whole volume of the corpuscle, which now begins to shrink. The chromatin divides (Plate I, 17 to 20, a) and the body of the parasite breaks up into from 12 to 16 merozoites, although the number of these may vary anywhere from 8 to 24 (Ziemann). By analogy with other parasitic protozoa this process of asexual multiplication may continue for a longer or shorter time, or until the vitality is exhausted. A period finally ensues, the conditions being unknown, in which the merozoites develop into the sexual phases of the organism. ‘These are the macrogametocytes and microgametocytes, the former female organisms, the latter mother cells of the male organisms. ‘The stages in this development in the case of Plasmodium vivax are shown in Plate III, Fig. 1. The female organism is a large cell with reserve granules and a well-developed nucleus. The male mother cell is less granular and its nucleus divides by a multiple divi- sion into a number of daughter nuclei which migrate to the periphery and there become the long-drawn-out nuclei of the flagelliform micro- gametes. The female nucleus, before fertilization, divides to form a small nucleus, which is extruded from the cell, this corresponding to the polar body equivalent of other protozoa and metazoa (Schaudinn). The processes thus briefly outlined do not all occur in the human blood. The early stages of gametocyte formation occur there while the remaining stages, viz., gamete formation and maturation processes, occur in the gut of a mosquito. Schaudinn had reason to believe that these sexual reproductive stages, especially of the microgametocytes, degenerate in the blood and come to nothing unless stimulated to development by the action of a cooler medium, such as room tempera- ture or the cool medium of an insect’s body. ‘The organisms ready for _ this further development are constantly in the blood after the first few paroxysms, and when sucked up by the mosquito, the further changes take place rapidly in the latter’s stomach and fertilization is brought about by the penetration of one of the slender microgametes into a macrogamete. The fertilized cell, called by Schaudinn the odkinet, now makes its way by a peculiar vermiform movement (giving rise to the name vermicule) to the epithelial cells lining the gut; it penetrates . the mucous membrane and comes to rest in the submucosa. Here it rapidly grows into an organism of the size of a coccidium, the nucleus divides, and the cell body, at about the third or fourth day, forms 286 THE PATHOGENIC HEMOSPORIDIA a permeable outer membrane and by the sixth day divides into as many portions as there are nuclei. These are special reproductive centres corresponding to the sporoblasts of the coccidia, and, as in the coccidia, each sporoblast forms by division a number of germs, the sporozoites. Unlike the sporoblasts of the coccidia, however, there is no protecting membrane or capsule about these plasmodium sporo- blasts; the sporozoites are naked and unfitted by this very fact for a free existence outside the body of some host. When mature, after a period of about fourteen days in the mosquito, they are liberated from the sporoblasts into the body cavity of the insect where, by the cir- culation of the body fluids, they are carried to all parts of the body, collecting, however, in the region of the head. Here they make their way into the salivary glands in the thorax and pass into the proboscis of the insect and thence into the human blood at the time of the first meal subsequent to their maturity. There is, perhaps, no better instance in the realm of biology of the delicate relationship existing between these intestinal parasites and the infected host. If the human blood of a malaria victim is taken by a mosquito belonging to the genus culex, the blood and its parasites are alike digested by this mosquito’s digestive fluids; no stage of the organism remains alive. But it is quite different with the species of mosquito belonging to the genus anopheles. Here the digestive fluids kill the ordinary asexual forms of the parasite, but the gametocytes have in some manner acquired immunity to the digestive ferments of these mosquitoes and continue to live in the gut and to reproduce in the tissues lining it. Ross, in India, showed that this very phenomenon occurs in the case of bird malaria, in which the organism Plasmodium precox is digested by the fluids.of anopheles, but immune to those of culex or stegomyia (Newmann), so that species of culex and stegomyia are the carriers of bird malaria, but harmless to man, for the organisms of bird malaria do not live in human blood. It is generally supposed, also, that mosquitoes may become immune to all kinds of blood para- sites, that is, capable of digesting all of the organisms, gametocytes and schizonts alike, and thus become quite harmless to man. This is the interpretation given to the fact that, although anopheles is common in England, there is no malaria. The phenomena of sporogony in connection with other forms of malaria are not essentially different from those of the tertian organism (Plate III, Figs. 2, 3,4). The macrogamete of pernicious malaria is, however, distinguishable from those of other forms of malaria by its sausage or crescent form (Plate III, Fig. 3). A number of observers (Grassi and Felletti, Mannaberg, Ziemann, et al.) have observed the binary division of such macrogametes, a method of reproduction which recalls the multiplication of the female organisms in trypanosomes. Schizogony and sporogony in the case ‘of Plasmodium precox, the EXPLANATIONS OF FIGURES IN PLATE III. Fig. 1.—Tertian Malarial Plasmodium. (After Craig.) 1. Hyaline form. 8. Flagellate form. (Microgametocyte.) 2. Pigmented ring form. 9. Non-flagellate form. (Macrogamete.) 3 to 6. Pigmented forms. 10. Segmenting form after destruction of red 7. Segmenting forms. corpuscle. Fig. 2.-Quartan Malarial Plasmodium. (After Craig.) 1. Hyaline forms. 8. Segmenting forms after the destruction of the red corpuscle. : 9. Flagellate form. (Microgametocyte.) 6 and 7. Segmenting forms. 10. Non-flagellate form. (Macrogamete.) 2 to 5. Pigmented forms. Fig. 3.—Tertian Estivo-autumnal Malarial Plasmodium. (After Craig.) 1 and 4. Hyaline ring form. 9. Segmenting forms. 3, and 7. Pigmented ring form. and 6. Pigmented forms. 8. Young intracorpuscular crescent. 10. Flagellate form. (Microgametocyte.) ob 11 to 14. Crescentie forms. Fig. 4.—Quotidian Estivo-autumnal Malarial Plasmodium. (After Craig.) 1 to 4. Hyaline ring forms. Some cells show 9. Plagellate form. (Microgametocyte.) infection with more than one organism. j s i ba ; 10, 11, 13, and 15. Crescentic forms. 5 to 7. Pigmented forms. In 6 one hyaline form. . 12. Ovoid form. S. Segmenting forms. Segmentation complete within infected red blood corpuscle. 14. Non-flagellate forms. (Macrogamete.) PLATE. Lit Fie. 1 a ie Fig. 2 oe « joe 8 THE PATHOGENIC HEMOSPORIDIA 287 cause of bird malaria, are not different in essentials from similar phenomena in human parasitic forms. Of great importance in the malaria problem is the fact of latent and recurrent malaria. In many cases, months after the first attack and apparent cure, the victim suffers anew from the parasites, and this without new infection. The matter has been studied carefully by many observers, among others by Craig and by Schaudinn, and it has been found that parasites, even after apparent cure, are stored up in the spleen and the bone marrow, where they live a comparatively pas- sive existence, getting into the peripheral blood when the conditions for their further development are favorable. What these conditions are is the one remaining obscure point in our knowledge of the malaria organisms. Schaudinn claims that certain of the forms of Plasmodium vivax, which under ordinary conditions would form the macrogameto- Fie. 113 Regression and merozoite formation (parthenogenesis) in Plasmodium vivax. (After Schaudinn.) A, macrogametocyte in blood with nucleus differentiating into a denser and a lighter part; B, the denser part of the nucleus now divides preparatory to schizogony, C, D, while the paler portion with a part of the original cell degenerates; D, numerous merozoites formed about the divided nucleus. cytes, undergo a process of parthenogenesis (Fig. 113), whereby the vitality is again renewed and with this the ability to withstand the natural or acquired immunity of the host. Craig, on the other hand, describes the conjugation of two schizonts within the human blood cell, an observation which Ewing (’01) and Wright (’01) had also made, although in the last two cases in connection with the normal infection and not with recurrence, while the occurrence was stated as rare and exceptional. Craig (’05 and ’07), however, claims that the union of schizonts is a normal process in every infection, and sees in this fact a means by which the organisms renew their vitality and thus bring about recurrence. Minchin doubts the interpretation of this fusion as given by Wright and by Ewing, and regards it as a process of plasto- gamy without sexual significance. Craig’s view is certainly enticing, but we must not forget that plastogamy is a very common phenom- enon throughout the group of protozoa and occurs frequently when 288 THE PATHOGENIC HEMOSPORIDIA there is no subsequent reproduction. It happens in most of the common rhizopods, for example, and has been described for cases of arcella, difflugia, centropyxis, ameba, etc., and it has been shown that these unions have nothing to do with the actual process of fertilization. It is impossible to state that no stimulation whatsoever results from such a plastogamic union, especially if it is followed by nuclear union or karyogamy, according to the account given by Craig; but it is diffi- cult to believe that two widely different processes of fertilization should exist in the same organism. My experiences with the free living para- mecium in cases of depression where the organisms were stimulated to new activity and new reproduction by purely artificial means opens the possibility, at least, that some analogous stimulation in the human system may start up the flagging energies of the malarial parasites. It is not inconceivable that minute changes in the con- stitution of the blood, especially of the salt contents, act upon the parasites in the same manner that potassium phosphate acts upon the weakened paramecium. Apart from the clinical effects of the different malaria parasites there is not much difference between them. ‘The cause of quartan fever, Plasmodium malaria, for example, agrees in all of its phases with Plasmodium vivax, the most important difference being the period elapsing between successive sporulating phases, requiring seventy-two hours as against forty-eight. The forms assumed by the gametocytes agree in all essential features, and fertilization in the mosquito follows the same history as in Plasmodium vivaz. There is evidence that at least two kinds of parasites causing pernicious malaria exist, one giving rise to a daily and the other to a forty-eight-hour recurrence. The difference in form of the macro- gametocyte was considered evidence of sufficient morphological value to justify a different generic name, and Grassi, therefore, gave it the name Laverania malarie. The grounds seem hardly sufficient for this, however, and the name Plasmodium falciparum, as given by Welch, is the one we adopt. (Pl.immaculatum, accepted by Schaudinn, was shown by Blanchard to be the name given by Grassi and Felletti to parasites occurring in birds.) In this parasite the macrogamete assumes the form of a crescent before maturity, but rounds out into a perfect sphere before fertilization. The action of quinine on the malaria organisms is particularly interesting, since it is one of the best-known specifics against any of the protozoan diseases. Introduced into Europe, in 1640, by del Cinchon, it was immediately recognized as a specific and was used as a diagnostic therapeutic test for malaria. Just how it acts upon the malaria organism was, of course, unknown until more or less of the life history of the parasites was known. Marchiafava and Celli, Schaudinn, and, in short, all who have studied the matter carefully THE PATHOGENIC HEMOSPORIDIA 289 have come to the same conclusion, that the drug acts directly upon the parasite, killing it with more or less distinct evidences of disintegra- tion of the organism. Marchiafava and Celli conclude that the treat- ment is most effective during the period of sporulation and upon the young stages of the organism, and practically without effect during the period of pigment formation and full growth of the schizonts. 19 CHAPTER X. THE PATHOGENIC RHIZOPODA. THE biological conditions which underlie parasitism are but little known, but, as with free-living protozoa, the dominant factor is the problem of food-getting. ‘The causes which lead an organism to invade a specific organ or tissue must, in the final analysis, be traced to this function, and reproduction leading to complete annihilation a cell or group of cells follows a parasite’s life in a suitable food medium. ‘There is a limit also to the kinds of parasites that can become cell-infesting forms, for the organism must have either the mechanical or cytolytical power of breaking down the barriers of a cell, and physical force enough and of a certain kind, to enable it to penetrate the cell membranes and cytoplasm. For such a function cilia are not useful, nor flagella, and we find that ciliates and ordinary flagellates rarely become intracellular parasites, and then only after losing their motile organs; unless, as in trichonympha, pyrsonympha, etc., they are provided with special anterior boring organs, by which they pene- trate the cell membranes, or unless, as in spirocheta, they possess the power of undulatory motion independent of flagella action (Fig. 114). Spirochetes may thus become cell-dwelling as well as fluid-dwelling forms, and some, like Sp. microgyrata or Treponema pallidum, work their way through the tissues of an infected host and not infrequently bore into the cells themselves. The ciliated and flagellated protozoa, however, are typically fluid-dwelling forms, and when they attack the epithelial cells of an organ it is usually only for purposes of attach- ment, as in trichonympha and pyrsonympha. There is considerable evidence, however, to indicate that one of the ciliates, balantidium, is occasionally found inside the mucosa of the intestine, and even within the muscular coating of the colon, while collections often appear in the epithelial cells and, apparently, cause the ulcers that are found there. ‘Two kinds of these ciliated parasites are common in man, Balantidiwm colt, frequent in the rectum, and Bal. minutum, and, according to Strong, Brooks, and Stengel, with others, the parasite becomes an important etiological factor in catarrhal inflammation of the intestine (Fig. 115). Other observers, including Malmsten, Opie, Doflein, and others, hold that these forms are quite harmless, increasing in number with dis- orders of the digestive tract, and for this reason are not uncommon in the intestinal tract of victims of cholera, typhoid, dysentery, or diar- THE PATHOGENIC RHIZOPODA 291 Fie. 114 A A, Multicilia lacustris, Lauterb. (After Lauterborn.) B, Pyrsonympha vertens, Leidy, with attaching organ. (After Porter.) 2, vibrating band in the inner protopiasm. Flagellated and ciliated intestinal parasites. A,B, Megastoma (Lamblia) entericum. Grassi; C, Balantidium entozoén, Ehr. 292 THE PATHOGENIC RHIZOPODA rhea. Brooks has given strong evidence to show that Bal. coli was the cause of a fatal disease resembling dysentery, in some valuable apes belonging to the New York Zodélogical Society, and from his observa- tions it is evident that these ciliates must be taken into account in searching for the causes of certain types of intestinal trouble, for, if not themselves the direct causative agent, they may be the bearers of some more pernicious organism. While ciliates and flagellates are not adapted morphologically for an intracellular parasitic life, the rhizopods have no such disadvan- tage, and by virtue of their ameboid movements, and of the cytolytic ferment which they are apparently able to secrete, they make their way into tissues and cells and then live upon the fluid elements of the living protoplasm. Thus, Plasmodiophora brassice, while in the young amebula stage, works its way into the root cells of a cabbage or turnip plant, absorbs and grows upon the fluid protoplasm of the plant cells, forms a plasmodium, and reproduces within these cells (see p. 209). Certain human diseases, notably dysentery, hydro- phobia, and smallpox, are characterized by the destruction of tissue cells, the agent being minute ameboid forms which we interpret as protozoa. In dysentery the organism causes the destruction of the epithelial cells of the digestive system; in hydrophobia, the nerve cells of the brain are destroyed, and in smallpox, the epithelial cells of the skin. In none of these cases is it generally agreed that the structures found within the diseased cells are the causes of the several diseases, and, indeed, in the last two, hydrophobia and smallpox, pathologists do not agree that the structures found within the diseased cells are organisms at all, much less the causes of the troubles. Unfortunately, cultivation of such organisms upon artificial media, and in pure cul- tures, has never succeeded. Indeed, up to the present time no one has succeeded in cultivating a cell-infesting rhizopod, and Liihe goes so far as to state that it will never be done, although success with forms like the Leishman-Donovan bodies makes such sweeping generaliza- tions unsafe. ‘The only means of determining whether such things are organisms rests upon morphological evidence, and lacking cultural possibilities the only proof that they are the cause of disease is to find them in every case of the disease. The morphological evidence, to most pathologists, is insufficient, and to most of them these organisms are more probably artefacts or degeneration products of the human cells caused by the disease, than etiological factors. To a proto- zoologist, however, the morphological evidence of organic structures of these protozoa is far more convincing, for he is familiar with the many variations in size and structure, in the different phases of the life history, of hundreds of different kinds of protozoa, and the struc- tures seen in these questionable inclusions become to him convincing THE PATHOGENIC RHIZOPODA 293 evidence of their protozoan nature. Such is the situation at the pres- ent time in regard to the inclusions found in trachoma, molluscum contagiosum, hydrophobia, and smallpox, while those in dysentery (although still in dispute as to etiology) are universally recognized as ameboid organisms. In the present chapter, I purpose to give some of the evidence upon which the protozodlogist bases his conclusions that the more questionable inclusions referred to are actually organ- isms of the rhizopod type, and if, thereby, I am able to impart some of my personal convictions in regard to them, the matter of etiology will take care of itself. In order to provide a basis for comparison of these disputed organ- isms it is necessary to consider first the variations in structure that occur during the life histories of widely different types of rhizopods, and then to show that, despite the minor differences, they all conform to a common type. The full life histories of many different kinds of rhizopods have been worked out on free living material, so that there is no ground for cavil as to whether such types are living organisms or artefacts. As fully shown in Chapter III, the life histories of free living rhizopods, involving many form changes, are characterized, at certain periods of maturity, by diffusion of the nuclear material throughout the cell and by the formation of exceedingly minute gametes. The curious diffuse idiochromidia are known to be no artefacts, nor abnormal features of the cell, but specific and highly important elements whose chief function is in sexual reproduction. It may be expected, therefore, and reasonably so, that similar structures should be characteristic of parasitic as well as of free living rhizopods, and the idiochromidia of chlamydophrys, of entameba, of neuroryctes, and cytoryctes, features of these organisms which many observers are reluctant to regard as evidences of organic structure, have the same importance as elsewhere. It is upon this feature of these organisms that we may reasonably depend for the assurance of the protozoa nature of the cell inclusions in trachoma, molluscum contagiosum, rabies, and smallpox. There is no reason to believe that the life cycle of a parasitic rhizopod should be essentially different from that of a free living form, unless, indeed, there may be an acquisition of some special means of overcoming the unfavorable condition of parasitic life, such as exposure to antibodies, acids, alkalies, etc., in the body fluids of the host, or to difficulties in transmission from one host to another. These are, in the main, provided for by the phenomenon of encystment, the organism within its cyst being amply protected against unfavorable conditions. Such a function, however, is shared with the free living rhizopods, encystment playing an important part in the life history of both shelled and shell-less forms. 294 THE PATHOGENIC RHIZOPODA A transition from the free living to the cell infesting rhizopods is afforded by one species of shelled forms—Chlamydophrys stercorea— and by different species of ameba—Entameba colt and Entameba histolytica—the life activities in all being singularly in conformity with the examples given above. Chlamydophrys stercorea, first described by Cienkowsky in 1876, is a rhizopod provided with a transparent glass shell of silica, found in animal feces. From its type of pseudopodia it would be classed with the reticulosa rather than with the lobosa or ameba type, and comes closer, therefore, to polystomella than to arcella or centropyxis. Schaudinn (loc. cit) found it in the feces of many different mammals, including cow, guinea-pig, turtles, and man, and was able to follow its life history by infecting his own digestive tract with encysted forms of the organism. The protoplasm of the cell contains one nucleus, many fine par- ticles, which are destined to form the shell of the daughter individual, contractile vacuoles (one or more), and idiochromidia in the form of a densely packed mass of granules about the cell nucleus. Like arcella, centropyxis, euglypha, and other shelled rhizopods, the organ- ism reproduces asexually by budding division, the plasm flowing out of the shell opening until a daughter mass is formed equal in size to the parent; the nucleus then divides by mitosis, one-half passing into the bud organism. The idiochromidia do not flow into the daughter protoplasm with the protoplasmic streaming, as in euglypha and centropyxis, but adhere to the nuclear membrane, so that when the nucleus divides, the germ plasm is likewise divided into two parts, the daughter organism thus getting its proportion of the important idio- chromidia. The sexual development is quite different from that of centropyxis. There is no dimorphism, and whereas in centropyxis the idiochromidia-bearing swarmers move out of the shell, leaving the disintegrating primary nucleus and residual protoplasm in control of the parental abode, here the residual parts are thrown out of the shell opening and the idiochromidia remain in the shell. The idio- chromidia next give rise to a small number of secondary nuclei, usually eight, by segregation of the chromatin granules, and the protoplasm then divides into as many parts as there are nuclei. Each part assumes an oval form, develops two flagella at the pole, and swims out of the shell and away. ‘Two swarmers (flagellispores) from different ances- tors fuse, form a hard, protecting cyst which becomes brown in color and irregular in contour, and within these the fertilized cells with a high potential of vitality, live until conditions are again suitable for development. With characteristic patience and ingenuity Schaudinn kept these cysts in damp chambers for a period of many months without observing any change, and finally inoculated himself: “I swallowed on November 17, 1899, for the first time, the contents of THE PATHOGENIC RHIZOPODA 295 eight moist chambers, in which were innumerable permanent cysts of chlamydophrys, which had lain unchanged for two or three months, and found on the 20th two typical chlamydophrys in an infusion made from solid feces of the 18th, while by the 24th they were so numerous that every preparation contained from one to two individuals.”’ (Schaudinn, loc. cit., p. 562). When he found that the organism would live in other digestive tracts, he gave up experimenting upon himself and used mice. One phase in the life history of this organism was earlier (1896) interpreted as a distinct species and named Leydenia gemmipara. (Schaudinn, 1903, p. 563). Chlamydophrys, therefore, behaves like centropyxis and arcella in its vegetative activities, but resembles polystomella more closely in its formation of isogamous gametes. ‘The chromidia are the same in all, being the substance of the nuclei of the conjugating cells. A transition from the lumen dwelling to the intracellular rhizopods is afforded by the intestinal amebze, which, since the time of Lésch, in 1875, have been closely associated with the problem of dysentery. These are minute amebe which penetrate the tissues by forcing the cells apart, and although they apparently do not enter the cells, they cause destruction of the cells by cutting off the food supply, exposing them to the materials of the intestine, or disturbing the ordinary pres- sure relations by infiltration with round cells and edema. Different observers have described many kinds of ameba in the human intestine, both during health and disease, and while some of these observations warrant careful consideration, the majority of them are not zodlogi- cally satisfactory. There are few points of structure in the parasitic amebee upon which to base species, and all attempts to create new species on account of size differences, nature of the pseudopodia, vacuoles, and the like, are insufficient; the only safe taxonomic basis is the life history, or the “individual” in the larger sense. At the present time very few of the many described amebze have been followed in their life history, and, although there are probably more, we recognize only two species of intestinal amebz, the one, Entameba coli, regarded by Casagrandi and Barbagallo, Schaudinn, Craig, and others as a harmless commensal in the human intestine, and Entameba histo- lytica (dysenterie, Councilman and Lafleur), regarded by pathologists generally as the cause of amebic dysentery. A third form, Entameba buccalis, is found in carious teeth (Prowazek). The life history in both of the intestinal species was worked out by Schaudinn, and the specific features were established by his demonstration of the char- acteristic differences in mode of reproduction. Lésch, in 1875, was the first to describe the simple structures of these amebz, which he also was the first to regard as an additional irritant, if not the cause, of dysentery. He named it Ameba colt. Later observers, finding the organism in so many cases of the normal 296 THE PATHOGENIC RHIZOPODA intestine, denied the pathogenic character of ‘‘ Ameba coli,” claiming that it is an organism of wide distribution and quite harmless. Casa- grandi and Barbagallo were the first to prove, although not the first to suggest, that the ordinary form of the ameba is harmless, a proof which was confirmed by Schaudinn, who inoculated himself with Entameba coli and without any disturbance, a result which he also repeatedly obtained with young cats. From the medical side Council- man and Lafleur, in 1891, first demonstrated that dysentery is not all one type of disease, and that amebic dysentery is both clinically and etiologically different from other kinds. ‘They suggested the name Ameba dysenterie for the organism causing the intestinal ulcerations, and Ameba colt, Lésch, for the harmless form; but their suggestion was not followed by enough morphological data to warrant the creation of a new species, and zodlogists did not accept the new terms. Casagrandi and Barbagallo, working on A. colz, came to the conclusion that the generic name ameba should not be stretched to include forms like Ameba proteus, on the one hand, and these small intestinal parasites on the other, and so called the latter entameba, while the specific name hominis was substituted, without justification, for Lésch’s term colt. Schaudinn, finally, overlooking Councilman and Lafleur’s observa- tions, adopted Casagrandi and Barbagallo’s name entameba for the genus, and named the harmless form Entameba coli, and the patho- genic form Entameba histolytica, a better name, but not prior to Councilman’s “dysenterie.” Entameba coli is widely distributed in the human intestine, this distribution varying with the locality and with the people. Schaudinn found it in about 20 per cent. of the feces investigated by him in Berlin, while in the region about Rovigno, in Istria, he found it in 256 cases out of 385, and other observers have noted a like variation in the per- centage of healthy persons infected. It is an organism to be obtained without much difficulty, and is more prevalent in persons suffering from intestinal disturbances. During the ordinary inactive phases there is little or no differentiation into cortical plasm (ectosarc, ecto- plasm) and endoplasm, but when it moves, a hyaline sheet of proto- plasm moves out from the body, and this is similar to the cortical plasm of fresh water amebee. This ectoplasm is only momentary, however, for the endoplasm quickly flows into the advanced part. The nucleus is vesicular, with a distinct membrane and with one or more karyo- somes of chromatin and plastin, while the numerous chromatin granules are distributed throughout the space of the nucleus, with a tendency—of frequent occurrence among the protozoa—to collect at the periphery. The abundance of chromatin makes the nucleus stand out prominently in stained preparations. Multiplication of the parasite is accomplished asexually by simple division and by multiple division or spore formation into eight daughter THE PATHOGENIC RHIZOPODA 297 organisms. The centronucleus, with its single division centre divides, according to Schaudinn, by amitosis, but, as in the flagellates it is a primitive mitosis. Spore formation is accomplished after a peculiar fragmentation of the nuclear chromatin into minute granules which collect in a rim around the inside of the nuclear membrane, the cell body, in the meantime, throwing out all foreign matter and ceasing its movements. The peripheral chromatin next collects in eight centres, the nuclear membrane is ruptured, and the eight small nuclei pass into the cell body. The protoplasm divides into eight parts around the nuclei, and eight small amebee finally creep out. As with all protozoa that have been carefully investigated, the reproduction by asexual means, in this case leading to auto-infection of the host, cannot be maintained indefinitely, and there comes a period when the organisms encyst, the conditions under which encystment takes place being somewhat indefinite in Schaudinn’s account. The cell throws out foreign matter and products of its own metabolism, and becomes more compact, smaller, and spherical, and then secretes a thick and slightly refractive gelatinous membrane. The nucleus then divides by primitive mitosis into two nuclei, which are separated from one another by the entire diameter of the spherical cell. The idiochro- midia characteristic of the rhizopods is then formed by disintegration of the two nuclei, the protoplasm of the cell in the meantime dividing into two incompletely separated parts around the two nuclei. In some cases the entire nucleus disappears in a mass of chromidial granules, in other cases there appears to be a secretion of chromidial substance as in arcella, but in all cases a part of the nuclear material is thrown out of the nucleus to degenerate, and this portion represents the eliminated and unused nuclear parts of the free living rhizopods. The fertilization process, following this preliminary division of the nucleus, is autogamous and similar to that in Ameba proteus and in the heliozoén actinospherium, as observed by Hertwig. The organism fertilizes itself in the following remarkable manner, ; the processes of maturation recalling those of the ciliate paramecium: From the disintegrated chromatin or idiochromida of the divided cell within its cyst membrane a new and a smaller nucleus is formed in each of the halves. This divides by a primitive mitotic process into two nuclei, one of which immediately degenerates, the shrunken nucleus remaining as a highly refractive irregular mass in the cell body; the other daughter nucleus then divides again, so that three nuclei lie in each half of the double organism, or six altogether, two of these undergoing degeneration. ‘Two of the remaining four nuclei then begin to shrink ‘and to degenerate like the first one, ‘until there are only two functional nuclei left. After this process, which Schaudinn inter- prets as equivalent to the reduction and polar body formation of metazoan cells, the final encystment takes place. The gelatinous 298 THE PATHOGENIC RHIZOPODA membrane disappears, and in its place is secreted a thin but much more refractive membrane, the definitive cyst membrane. The contents of the cyst become again closely united, and the two remaining nuclei are brought closely together. Then follows a third division by mitosis, characterized by long connecting strands which lie parallel with one another in the centre of the cell, so that the daughter nuclei of the two parent nuclei lie side by side in pairs. These nuclei then fuse, an eighth part of one of the original nuclei uniting with an eighth part of the other, while the outer membrane hardens and thickens. Each cyst thus contains two fertilized nuclei, the process recalling the phenomenon in paramecium where, from the same primary nucleus, a wandering and a stationary nucleus is formed. In the fertilized Entameba coli each of the two nuclei divides, forming four nuclei; then each of these divides again, making eight nuclei in the cyst, and in this condition the encysted parasite passes into the intestine of a new host, where the protoplasm of the cell divides into eight parts around the eight nuclei, the cyst membrane is dissolved off and eight small amebee start a new infection with a new potential of vitality. This complicated life history has been confirmed in part by other observers, Wenyon (’07) and Craig following out the sexual history in E. muris and E. coli respectively (see p. 142). The possibility of union of two amebze before encystment is not excluded, nor is the possibility of pseudoconjugation, as seen in the gregarines, beyond question. Autoconjugation, while recognized in many different kinds of animals, is too unusual to be granted without the surest proof, and further research on the life history of these parasites is urgently needed. The structure of Entameba histolytica, according to Schaudinn, is somewhat different from that of E. colt, and makes it better adapted for its cell destroying function. This is shown by its definite cortical plasm, a layer of firm protoplasm with distinctly higher refractive index than the internal protoplasm, which gives a more rigid character to the pseudopodia, by which the organism is able to force its way between the epithelial cells of the intestine and into the more deeply lying tissues. Schaudinn has watched the organism thus make its way into the epithelial tissue of a freshly extirpated, infected cat intestine, its active movements often lasting an hour, while its own body assumed the greatest variety of forms. The nucleus is difficult to see during life of the organism, a feature in marked contrast to the nucleus of Entameba coli, which Schaudinn recommends as a par- ticularly favorable object for the study of the changes of the living nucleus. The nucleus of E. histolytica has very little chromatin matter as compared with the nucleus of the other species, but there is a single central karyosome and a slight collection of chromatin around the periphery. While the nucleus of Entameba coli is only slightly vari- THE PATHOGENIC RHIZOPODA 299 able, usually spherical, and without much change in position during the activities of the body, that of E. histolytica is highly variable, bend- ing and turning with contact with objects in the cell, or flattening into a disk in the cortical plasm. The ordinary vegetative increase of Entameba histolytica takes place by simple division or by budding on the periphery, the formation of eight spores never being seen. Division takes place while the organisms are lying between the cells of the gut tissues, and may be either equal or unequal, the unequal division passing by imperceptible grades into bud formation. The buds are apparently similar in their mode of formation to those of acanthocystis (see p. 31), the nuclei arising, according to Schaudinn, by amitosis (Fig. 32, p. 94.) Permanent cysts are not formed during the height of the disease, but are first found during periods of healing, and after the organisms have reproduced again and again by division. The beginnings of the preparations for spore formation are first manifested in the nucleus. Here the peripheral zone of chromatin granules becomes thicker, the membrane of the nucleus disappears and the granules are ultimately disseminated throughout the protoplasm in a typical chromidium form similar to that of centropyxis (see p. 150), while the residual nuclear parts, with some protoplasm, degenerate. Spores are formed by the protrusion on the surface of the cell of small buds containing chromidia, and these buds are transformed into spores by secretion about themselves of a definite resisting membrane, while the central protoplasm, with the residual nucleus, degenerates. The further his- tory of these buds was not ascertained by Schaudinn beyond the fact that they were capable of infecting normal cats with amebic dysentery, so that the processes of conjugation are still unknown. It will be an interesting study for some student of the group to see if conjugation follows the pattern of Entameba coli or that of centropyxis, where the idiochromidia bearing spores are gametes which unite after leaving the parent cells. It is not the place here to discuss the question whether or not these parasites of the human intestine are the causes, or the sole causes, of acute enteritis in man.’ Pathologists, in the main, are in accord that one type, at least, of dysentery is traceable to these rhizopods, but there is a difference in opinion as to whether the rhizopods create an enzyme or poisonous product which acts as a direct agent on the tissues, or whether they are passive in this respect, but cause mischief by the mechanical irritation of their movements between the cells. Shiga and Flexner have shown that one type of dysentery is to be traced to a bacillus, and Prowazek suggests that these parasitic amebze may play an important part as carriers of bacteria into the deeply lying tissues 1Prowazek has recently given evidence to support the view that flagellates of the genus Lamblia megastoma (Fig. 115) are capable of causing acute intestinal trouble of like nature. 300 THE PATHOGENIC RHIZOPODA of the intestine which they are incapable of reaching by their own movement. On the other hand, the nearly pure cultures of the ameba which Strong, Musgrave and Clegg, and others have succeeded in raising and in causing the disease in normal animals, and Schaudinn’s experiments on kittens with dried spores of E. histolytica, speak for their specific pathogenic nature. Musgrave and Clegg (’04), indeed, are so positive of the pernicious effect that they maintain the patho- genic nature of all intestinal amebze, and claim that ordinary pond or soil dwelling amebee may become pathogenic on entering the intestine. Taking all into consideration, there is no doubt that the intestinal rhizopods are dangerous, and are either the causes of certain types of the disease, or pernicious accessories of the cause. ; If skepticism exists as to the pathogenic nature of entameba and the causes of dysentery in general, what can be said as to neuroryctes and cytoryctes and the causes of hydrophobia and smallpox? With entameba, skepticism never reaches the level of denial of the organism, but with these other organisms not only does doubt exist as to their connection with disease, but their claims to relationship with living forms are widely denied. The problems are certainly very difficult, and with the immense numbers of degenerations, secretions, and the like which may be imagined in tissues under diseased conditions, it is easily possible to be mistaken when morphology is the sole criterion. But it is not inconceivable that these difficulties are overestimated, and that the questionable structures in diseased tissues are actual organisms. Certainly no one doubts that rabies and smallpox are germ diseases, and it is equally certain that no other cause, apart from these cell inclusions, is known. There is a strong a priori reason, therefore, for believing that these intracellular structures in cells which are known to be the seat of the disease are the actual causes and not the product of the diseases. Thus, the Negri bodies (Neuroryctes hydrophobie) are constant inclusions in the brain cells of victims of rabies, and the Guarnieri bodies (Cytoryctes variole) are equally constant inclusions in the skin cells of man and apes infected with smallpox. So strong is the morphological evidence of the nature of these inclusions that there is no doubt whatsoever in my own mind as to their protozoan nature and to their affinities with entameba and other rhizopods. The transition from the intercellular to these intracellular para- sites of the rhizopod type is shown by such unquestionable ameboid forms as Plasmodiophora brassice, while recently a number of other forms of similar nature have been described. Among these the genus which Prandtl (’07) describes under the name of allogromia is very instructive. This is a parasite of free-living protozoa, such as Ameba proteus, arcella, nuclearia, or even paramecium, unicellular hosts which become infected with the sexual generation of the allo- THE PATHOGENIC RHIZOPODA 301 gromia. These grow to maturity and form gametes which escape and conjugate in the surrounding water, the resulting copula devel- oping into a biflagellated organism which subsequently becomes ameboid and grows into an adult allogromia (Fig. 116). While there is reason to doubt some of the developmental stages of this life history, the essential fact remains that here is a clearly defined rhizopod Fie. 116 “‘Allogromia,” sp. (After Prandtl.) A, an individual from Ameba proteus with nucleus undergoing fragmentation to form chromidia; B, aggregation of distributed chromatin into secondary nuclei; C, A, Vampyrella, sp., infected with Allogromia, sp.; D, allogromia from Ameba proteus shortly before ripening of the gametes. Fia. 117 Single and multiple infection of ameba nuclei by Nucleophaga amebe. (After Pénard.) one stage of whose life history is passed as an intracellular parasite. The history of its nucleus is important as furnishing a possible interpre- tation of the distributed condition of the chromatin in neuroryctes and cytoryctes. The cell plasm of this so-called allogromia becomes filled with idiochromidia which are derived from the nucleus (Fig. 116, A, B). It is probable, as Doflein points out, that this organism is not an 302 THE PATHOGENIC RHIZOPODA allogromia in the sense of Rhumbler’s organism of that name, but that it is a species of a still more striking intracellular rhizopod first described by Dangeard in 1895, under the name of Nucleophaga amebe and subsequently identified by Gruber, Pénard, and Doflein. It is a fairly common parasite of Ameba proteus and similar fresh- water forms, penetrating the nuclei and forming relatively large spherical reproductive bodies within the nuclear membrane (see Fig. 117). ‘The nucleus becomes more and more hypertrophied with growth of the parasite, until finally the membrane gives way and the mass of spores is left in the enucleated body of the host. Under the name of Karyoryctes cytoryctoides the author described a similar Fie. 118 Nucleophaga, sp., an intranuclear parasite in the macronucleus of Paramecium aurelia. (After Calkins.) intranuclear parasite of Paramecium aurelia in 1904 (Fig. 118). Being unfamiliar at the time with Dangeard’s work, I was under the impression that the parasite in question was a new organism, and described it as such, pointing out its close resemblance to the intra- nuclear forms of the smallpox organism. There is no doubt, however, that the parasite is a species of nucleophaga, and the name karyoryctes must go. The striking similarity between the smallpox organisms and these intranuclear parasites leaves little room to doubt the close rela- tions of the two, while the structures and life phases, also, of neuroryctes are almost identical with those of nucleophaga (Fig. 120). We are justified, therefore, at least until more convincing evidence to the con- trary is forthcoming, in regarding the Guarnieri bodies of vaccinia and THE PATHOGENIC RHIZOPODA 303 smallpox, and the Negri bodies of rabies, as protozoan organisms of the nucleophaga type. Neuroryctes hydrophobie, Williams, the ‘‘Negri body,” offers the best evidence of the rhizopod affinities of these intracellular inclu- sions, the mammalian brain cells, better than the skin cells, lending themselves to rapid fixation and study. When Pasteur and his immediate followers were working on the antirabic serum in connection with the cure of hydrophobia, they were obliged to wait from two to three weeks to tell whether the treat- ment they were giving a supposed victim was necessary or not. ‘This was due to the fact that many days were required for the disease to develop in laboratory animals inoculated with the virus of the sus- pected animal, and, as may be imagined, it was a period of great suspense for all concerned. In 1898 the inoculation period was shortened to about nine days by Wilson’s substitution of guinea-pigs for rabbits, these animals taking the disease more quickly than rabbits as used by Pasteur. Still, the time was far too long for diagnosis. Today it is possible to determine rabies in “mad” animals off the street in one-half hour. This wonderful practical advance in technical methods of the laboratory is due to the discovery by Negri, in 1903, of minute, characteristic inclusions in nerve cells of brain and spinal cord of animals with rabies, and by a special “smear” method of demonstrating them devised by A. W. Williams in 1904. The value of the Negri bodies in diagnosis was quickly recognized by pathologists throughout the world, and contributions confirming and extending Negri’s discovery poured into the press. At the present time it is recognized that these characteristic structures occur in 100 per cent. of definite cases of street rabies, and that they are found nowhere else in diseased tissues. What claims have these specific structures to be regarded as organisms, and if organisms, where do they belong? Negri regarded them as protozoa belonging to the class sporozoa, but was not particularly clear as to their classification. Previous observers, notably Di Vestea, in 1894, and Grigoriew, in 1897, had mentioned structures in the nervous system of rabic animals and had described them as protozoa, but the things observed were apparently quite unlike the Negri bodies. Others, notably Volpino, in 1904, followed Foa, Schaudinn, and Prowazek in their interpretation of the Guarnieri bodies in smallpox, in believing that the real organism of hydrophobia is the granule, more often multiple, found in the substance of the “body,” while the bulk of the “body” consists of material secreted by the cell (hence Prowazek’s term “chlamydozoa’’) about the parasite. Williams’ and Lowden’s work, in 1906, and Negri’s later papers leave no grounds for such an interpretation, the former believing that the granules represent distributed chromatin so characteristic of many forms of protozoa, and placing the Negri bodies as protozoa in, the 304 THE PATHOGENIC RHIZOPODA suborder microsporidia, while Williams later gave the name Neuro- ryctes hydrophobie to the Negri body. The life history of Neuroryctes hydrophobie, despite the admirable researches of Williams and Lowden, cannot yet be regarded as estab- lished, nor do I think the stages observed by Negri, Williams, and others justify us in assigning the organism to the sporozoa. ‘The variable form, the uninucleate condition leading to the condition of distributed chromatin, and the budding phenomena are not charac- teristic of sporozoa, but are common to parasitic rhizopods, and the distributed chromatin is, in all probability, the idiochromidia, which, we have seen, is a characteristic phenomenon of all rhizopods. Fie. 119 , : : ¥ ry J ‘ 6 * eo 1 £ . e Pind: *‘Negri bodies,’’ or Neuroryctes hydrophobie, in different stages of chromatin distribution. (After Negri.) The organism is most abundant in the region of Ammon’s horn, less abundant in the nerve cells of the cerebral cortex, cerebellum, medulla, and cord. In many cases, especially in street rabies, the organisms are large and ameboid in form, measuring up to 18 « (Wil- liams) (to 23 #4, Negri), while minute forms, one-half a micron and less in diameter, are characteristic of the organism after the virus has been THE PATHOGENIC RHIZOPODA 305 repeatedly inoculated in animals of the same kind, and, owing to their very minute size, such organisms are easily overlooked in this “fixed” virus. It has been found by Remlinger, Schiider, Bertarelli, and others that the virus is still effective after filtration through a Berkefeld filter, a fact used as an argument against the specific pathogenicity of these structures; but the well-known variations in size of ameboid protozoa and the small size of some stages of the organism, combined with plasticity, which suggests ameboid movements, explains the ability to pass a filter. Other protozoa, notably spirocheta and trypanosoma, likewise pass through the Berkefeld. It is probable, therefore, that an organism as variable as neuroryctes in size would have some stages minute enough to escape filtration. Negri was the first to make out the typical nucleus of the organism and to call attention to the distributed granules, although he did not Fic. 120 Form and size changes of the organism of rabies, with evidence of budding in some cases. (After Williams and Lowden.) interpret these correctly, Williams and Lowden, in 1906, being the first to interpret them as granules of distributed chromatin. Negri, in 1905, found that the nucleus has either a solid or reticular structure, according to the success in staining (Fig. 119), while the cell body contains a variable number of chromatin granules. Reproduction of Neuroryctes hydrophobie, according to Williams and Lowden, occurs by simple division and by budding. The division is either an equal binary fission, in which nucleus and chromatoid material are distributed to the two cells, although nothing like mitosis was observed. In budding, small buds are pinched off, these buds being single or multiple in number and containing granules of chro- matin. The possibility of conjugation was suggested by Williams and Lowden, and illustrated by figures, but it is equally possible, and more probable, that the cases cited and illustrated were cells in division. Finally, what appears to be a spore-containing cyst (Fig. 120) was also described. 20 306 THE PATHOGENIC RHIZOPODA With the exception of the rhizopods, the entire range of protozoa offers no analogies to these stages of neuroryctes. The series of forms, following more or less closely the clinical history, agrees with the his- tory of the parasitic amebze so far as the general outline goes, while further details and careful study are necessary before the life history can be stated. With our present knowledge it appears that the organ- ism, as seen in its smallest forms, is uninucleate; that as it develops into a larger ameboid form, the nucleus, either by fragmentation (as in polystomella) or by diffusion (as in centropyxis or Entameba histolytica), gives rise to the diffused chromatin or idiochromidia. In its mode of asexual reproduction it apparently follows Entameba histolytica in binary fission and in budding. Its sexual reproduction Fic. 121 “Negri bodies in nerve cells.’ (After Wolbach.) A X 2000; B x 1000. is as yet unknown, the union of two cells, as pictured by Williams and Lowden, being quite unlike any authentic account of conjugation in rhizopods or sporozoa. The nature of the “fixed” form, also, is enigmatical, but may be looked upon as a biological response on the part of a highly variable organism to long-continued conditions of the same nature. Further work is needed on Neuroryctes hydrophobie in respect to the mode of division and budding, and with especial reference to the nuclear phenomena; further, in respect to the nature of the permanent forms, encysted or otherwise, which might be expected to exist in animals shortly after recovery from rabies; and finally, work is needed in connection with the sexual phenomena whereby the potential of vitality of the parasite, and with it its capacity for further mischief, is restored. The early illustrations published by Negri, Luzzani, and others of the organism of rabies showed an irregular body with numerous THE PATHOGENIC RHIZOPODA 307 vacuoles (Fig. 121), and sections of infected tissue not properly fixed and stained give no satisfactory pictures of the organism, the place of chromatoid granules and nuclei being taken by the vacuoles. Such a picture is duplicated by improperly fixed parasites of dysentery vacuoles appearing in the place of the formed parts of the cell. These, again, are duplicated by the ordinary appearance of the smallpox organism as it appears in sections of the skin (Fig. 128). This structure has been, and is still, next to the so-called protozoan inclusions in cancer, the most widely discredited cause of any malig- nant contagious disease. ‘The reason for the skepticism on the part of pathologists generally is that the organism presents no appearance that can be identified with the ordinary cell, its lack of a vesicular nucleus, its highly vacuolated appearance, and its development in cells that are unquestionably pathological and degenerate, being, to them, evidence against its protozoan or parasitic nature. While a great deal of the skepticism is due to traditional conserva- tism on the part of medical men and disinclination on their part to accept any but conclusively demonstrable evidence, it must be stated, with all respect, that there is among them a strong tendency to ignore such evidence as we do have in regard to the nature of these structures, and disinclination to accept such evidence as similar to structures in other protozoa. ‘The difficulties attending the observations on the organism of smallpox are aggravated by the fact that it is apparently an exclusively human disease, and further, that the organism is an intracellular parasite which quickly disintegrates upon leaving its normal environment. One phase of the disease, however—vaccinia— is suitable for experimental study, but at best this is but a mild disorder when compared with variola inoculata of apes or with variola vera of man. Until some means of studying it on an experimental basis is established, we must make the best of the morphological evidence afforded by imperfectly fixed tissues from human beings, or from material in experimental animals with variola inoculata and vaccinia. The cell inclusions in the Malpighian layer of the skin were early seen, interpreted as protozoa, and named Monocystis epithelialis by Pfeiffer, in 1887, but as he found so-called protozoa in all kinds of diseased tissue, his observation did not create much comment nor stimulate research. It was quite otherwise with Guarnieri, in 1892; this skilful investigator inoculated the corneal cells of guinea-pigs and rabbits with vaccine virus and with pustule contents, and found that the peculiar cell inclusions characteristic of smallpox and vaccinia reappeared in each new epithelium inoculated. He found that the structures appear with the greatest regularity in the vicinity of the nucleus, the largest forms appearing around the point of inoculation, while the most distant forms were the smallest. He regarded them as protozoa, naming the form as observed in vaccinia, Cytoryctes vac- 308 THE PATHOGENIC RHIZOPODA cinie, and in smallpox, Cytoryctes variole, but they were much more often referred to in subsequent investigations as the Guarnieri bodies. To Guarnieri, therefore, belongs the credit of placing smallpox and vaccinia among the experimental diseases, and the stimulus given by his work had an immediate effect. The majority of later investigators were opposed to his conclusions, although many, including Pfeiffer, Ruffer, and Plimmer, Clark, Monti, Wasielewsky, and others,believed that the parasitic nature of the inclusions had been demonstrated. The opponents based their criticisms upon the facts that no ameboid movement could be observed, nor division phases, nor cellular struc- tures (Hiickel, Foa, Mann, etc.), and they interpreted the Guarnieri bodies as special secretions or degenerations resulting from a peculiar transformation of a portion of the cell plasm under the stimulus of the vaccine virus. Wasielewsky, in 1901, brought new support to the view of Guarnieri by passing vaccine virus through forty-eight suc- cessive transplantations, the thirty-sixth giving a successful vaccination against smallpox. In each case the same inclusions were present in the epithelial cells and in approximately the same number, indicating that reproduction must have taken place. In 1903 Councilman, in codperation with seven other investigators, published an exhaustive monograph on the pathology and etiology of smallpox, covering all phases of the pathology of the disease, Brinckerhoff and Tyzzer extending the experimental investigations of Guarnieri and Wasie- lewsky to apes, and Calkins working out a tentative life history of the parasite. Howard, in 1905, confirmed, independently, all of the findings of Councilman and co-workers, and identified every stage of life history of the organism. So far as the organism is concerned, the most important discovery of these investigators was made by Councilman, Magrath, and Brinckerhoff, who found that in variola the inclusions are present both in the cell bodies and in the nuclei, while in vaccinia they are present only in the cell bodies. Councilman concluded that the intranuclear position indicates a phase in the life history of the parasite which is absent in the vaccinia cycle, and that this phase is responsible for the greater malignancy of smallpox. Calkins interpreted the parasite asa sporozoan belonging to the group of microsporidia, and, as it now appears, gave an unnecessarily compli- cated account of the lifehistory. Minchin (’06) regards it as more closely related to the haplosporidia, because of the absence of polar capsules and threads. The tentative life history worked out by Calkins was formulated before the observations on the chromidia of rhizopods were made and before the importance of this material of the cell was established. In the light of our present knowledge it is much more probable that the Guarnieri bodies are rhizopods, and that the com- plicated changes which were earlier interpreted as pansporoblast THE PATHOGENIC RHIZOPODA 309 formations are phases in the development of the idiochromidia. Without going into the controversy again as to whether or not these bodies are organisms, a matter, I may add, which is not yet settled to the satisfaction of either pathologists or biologists, I will here give only an interpretation of the questionable structures on the basis of their probable relationship to neuroryctes and the other parasitic rhizopods like nucleophaga, a relationship of which I am fully convinced. The youngest forms of the parasite are small, spherical, and appar- ently homogeneous granules measuring about half a micron. In slightly larger forms a central granule can be detected more easily in the cornea cells of inoculated rabbits than in the human skin. Dif- ferentiation of the organism follows with growth, two substances of the cell indicating differentiation. One of these is distinctly chroma- Fie. 122 Section of the lower part of the epidermis, showing the cytoplasmic stage of cytoryctes in the epithelial cells. xX 1000. toid, and becomes diffused throughout the body of the parasite at first in irregular clumps (Fig. 122), later ina fine network (Fig. 123). Such a structure is to be compared with the chromidiennetz of the rhizopods. As with the chromidia material of the free forms, small, spherical, deeply staining nuclei are formed out of this chromidial substance, the organism then assuming an appearance strikingly like the figure of arcella as given by Hertwig, in 1899 (compare Figs. 46 [p. 118] and 123). These granules are not artefacts, but developmental stages of the organism. The proof of this is given by the fact that they may be distinguished after any of the ordinary differential nuclear stains, but more surely by the fact that their presence is indicated by photographs made with the ultraviolet rays from unfixed and unstained living tissue of the inoculated cornea. These granules were interpreted as gemmules in 1904, and as vegetative spores or merozoites I would 310 THE PATHOGENIC RHIZOPODA similarly interpret them today. The body ruptures and the spores are liberated, to be carried by the blood into new regions of the skin, where the cytoplasmic cycle is repeated. In vaccinia it is apparent that this vegetative cycle is the only phase of the life history, and this, in the same host at least, is limited in extent. In variola, however, the vegetative cycle is repeated many times, but finally the nucleus becomes infected and the parasites, Cytoryctes variole in different stages of multiplication, outside first three figures and outside last two figures of the nucleus, (Alter Calkins.) Fic. 124 Two of the larger cytoplasmic forms of cytoryctes in the epithelium. The two dark bodies in the middle showing reticular structure are the parasites. 1000. like nucleophaga, develop in a more definite manner. Chromidial fragments are formed, varying in size and character, while a residual portion of the chromatin, analogous to the residual nucleus of free living rhizopods, remains unformed and apparently useless (Fig. 123). The many rings, vacuolated structures, etc., which earlier were interpreted as developmental phases of sporoblast and spores, I now believe to be degeneration forms assumed by the parasite, possibly due EXPLANATION OF FIGURES IN PLATE IV. (After Mallory.) The drawings were made with the Abbe camera lucida; projection on to table. Zeiss apochromatic homogeneous immersion 2.0 mm., apert. 130, compensation ocular 6. I'tas. 1 and 2 show numerous large and small scarlet fever bodies (stained light blue) in and between the epithelial cells of the rete mucosum. In Fig. lisa large body in a lymph space of the corium just underneath the epidermis. Several of the bodies suggest fixation while in amceboid motion. Frias. 3, 5, and 6 are coarsely reticulated forms which may be degenerated. forms of the scarlet fever bodies, or stages in sporogony. I'tas. 4, 8, and 9 probably represent stages preceding the radiate bodies. In lig. 9 the bodies lie in a lymph space. It shows also four small forms which have just got free from a rosette. Fras. 7, 10, 11, 12, 13, 14, and 15 show different stages in the development of the radiate bodies. lig. 10 is the earliest stage: there is a distinct central hody and a definite, regular arrangement of granules at the periphery. Figs. 7, 11, and 12 show a little later stage of development; 11 and 12 are optical sections, while 7 is a surface view. Moreover, in Vig. 7 the body lies free in a lymph space in the corium, The segments begin to show a certain amount of lateral separation from each other. Fig. 13 isa still later stage: the segments are increasing in size and are more or less free from cach other, although most of them are still attached to the central body. In lig. 14 the segments are all free and enlarging, although still grouped around the central body. — In Fig. 15 the bodies are still grouped around the central body, which is free and stains deeply with eosin. PLATE. IV ewan (RE ont THE PATHOGENIC RHIZOPODA dll to the toxins, infective material, ete., of the developing pustule, or pos- sibly to ill preservation of the tissues. ‘They are characteristic of the later pustules, and their vacuolated appearance may be ascribed to the same causes as that which produces poorly fixed and stained amebee, or poorly stained Negri bodies. In the latter the better technique of recent methods has shown that what appear as vacuoles in the photographs are actually chromatin fragments (see Fig. 125), and by analogy I would prophesy that when better methods of fixing and staining the intranuclear form of cytoryctes are devised, a similar chromatin distribution will be discovered. ‘The tissues, such as we worked upon four years ago, show many cellular structures like those of the well-fixed and stained Negri body (compare Figs. 121, 122, 124 and 125), and although these were regarded formerly as aberrant forms of the sporoblast structures, many of them were figured and described. Fie. 125 A large cytoplasmic form of Cytoryctes variole. As with neuroryctes, further study with better methods must be undertaken to complete the life history of cytoryctes; the important sexual stages must be found, a hint to this end being given by the changed nuclear phenomena of the intranuclear form (see difference in nuclear processes of vegetative and sexual phases of entameba). In this same category, finally, must be placed the interesting organ- isms discovered by Mallory (’04) in the skin cells of scarlet fever victims, and named by him Cyclasterion scarlatinalis (Plate IV). Also the curious structures described by Prowazek in trachoma, forms 312 THE PATHOGENIC RHIZOPODA similar to cytoryctes and neuroryctes, and all of which, together with the cell inclusions of molluscum contagiosum, Prowazek includes under the name of chlamydozoa or “mantle-covered’”’ organisms. This name represents a point of view held by many protozodlogists that the real organisms are the chromatin granules, while the material about them is only coagulated nuclear material. The entire absence of fortuitous strands of such nuclear material in the cell, apart from the enclosed granules, together with the definite history which corresponds exactly with the idiochromidia formation in other rhizopods, renders this interpretation improbable. BIBLIOGRAPHY. The titles included here are arranged in alphabetical order according to the system adopted in Minot’s Human Embryology, in Wilson’s The Cell in Inhertt- ance and Development, and in other recent works. Each author’s name is followed by the year of publication (abbreviated to the last two digits in all years between 1808 and 1909 inclusive). Thus, BuocHmann, F., 794, refers to Blochmann’s paper in 1894. ABBREVIATIONS. Anatomischer Anzeiger. Archives de Biologie. Archiv fiir Anatomie und Physiologie. 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INDEX OF | Apamt, the cancer problem, 208 Afanassiew, structure of spirochetes, 228 | Anderson, Rocky Mountain spotted | fever, 278 Argutinsky, ring forms in malaria organ- isms, 283 B Bases, Hematococcus bovis, 272 Baeslack, immunity to cancer in mice, 208 Bandi and Simonella, mode of life of spirochetes, 230 Barker, classification, 38 Bashford, Murray, and Bowen, rhythms of growth energy in cancer, 209 Behla, the cause of cancer, 207 the X-body, 211 Bertarelli, rabies problem, 305 Blanchard, Coccidioides immitis, 196 Boggs, Rocky Mountain spotted fever, 278 Bonhoff, spirochete nuclei, 225 Bonnevie, heterotypical mitosis, 207 Borrel, cancer cell-inclusions, 211 division of spirochetes, 227 parasite theory of cancer, 207 spirochetes in cancer mice, 214 Borrel and Marchoux, transmission of spirochetes, 250 Bose, cancer parasites, 211 Boveri, centronuclei, 255 kinoplasmic structures, 29 parthenogenesis, 161 Bowhill and Le Doux, structure of babesia, 270 Brady, classification, 39 Brasil, budding in gregarines, 183 Brauer, parthenogenesis, 161 Brefeldt, classification, 38 Breinl and Hindle, structure of babesia, 270 Breinl and Kinghorn, polymorphism in spirochetes, 229 Brinckerhoff, cytoryctes and smallpox, 308 Axrooks, Balantidium coli, 290, 292 22 AUTHORS. Bruce, trypanosomes and disease, 261 Biitschli, centrosomes in heliozoa, 30 flagella of spirochetes, 224 protoplasmic structure, 21 reducing divisions in infusoria, 166 significance of fertilization, 171 vitality in protozoa, 104 Cc CaRPENTER, Classification, 39 Carter, division of spirochetes, 227 nuclei in spirochetes, 225 transmission by ticks, 230 Cassagrandi and Barbagallo, dysentery problems, 295 Castellani, sleeping sickness, 264 Caullery and Mesnil, cycle, 184 endogamy in actinomyxide, 147 origin of trypanosomes, 233 Celli, melanin and malaria, 201 Certes, Spirocheta balbianii, 219 Christophers, form changes in Leish- man-Donovan bodies, 200 infection of ova in babesia canis, 196 structures and babesia, 270 Clowes, immunity to cancer in mice, 208 Cohnheim, the cause of cancer, 207 Councilman and Lafleur, dysentery, 295 lysis in dysentery, 201 Craig, autogamy in ameba, 141 dysentery problems, 295 malaria problems, 287 Cuenot, conjugation of diplocystis, 189 Cull, conjugation of paramecium, 167 significance of fertilization, 172 life history of D DANGEARD, Classification, 40 gamete formation, 127 nucleophaga amebie, 302 Danilewsky, the hemosporidia, 269 Darling, Histoplasma capsulatum, 278 Defrance, classification, 39 338 INDEX OF Del Cinchon, introduction of quinine, 288 Di Vestea, negri bodies, 303 Dobell, exogamy in copromonas, 153 insertion of flagella, 46 Doflein, Balantidium coli, 290 effect of parasites on nuclei, 203 structure of babesia, 270 of trypanosomes, 258 Dogiel, budding in gregarines, 183 Donovan, organism of kala azar, 238 D’Orbigny, foraminifera, 37 Dujardin, F., classification, 18, 34 sarcode, 21 Dutton, Trypanosoma gambiense, 264 Dutton and Todd, transmission of spirochetes, 230 Dutton, Todd, and Tobey, nuclei in spirochetes, 225 structure of trypano- ; somes, 254 E EHRENBERG, spirocheta and spirillum, 217 Ehrlich, the cause of cancer, 207 Ellis, flagella of spirochetes, 224 Elpetiewsky, exogamy in arcella, 161 Ewing, cancer problems, 207 ring forms in malaria organisms, 283 F Fantuam, Babesia muris, 272 Spirocheta balbianii, 219 Farmer, Moore, and Walker, cause of cancer, 207 : fertilization of epithelial cells, 208 Feinberg, cancer cell inclusions, 211 Fischer, classification, 39 flagella of spirochetes, 224 Flexner, dysentery, 299 Flu, types and structures of crithidia, 242 Fliigge, transmission of germs, 195 Foa, negri bodies, 303 Forde, sleeping sickness, 264 Frenzel, classification, 40 G GaYLorD, the parasite theory of cancer, 207 Spirocheta microgyrata gaylordi, 213 Gleditsch, classification, 38 Goldhorn, division in spirochetes, 227 AUTHORS Goldschmidt, anisogamy, 161 chromidia and sporetia, 118 Mastigina setosa, 120 Gotschlich, typhus fever and protozoa, 278 Grassi, malaria and mosquitoes, 279 origin of hemosporidia, 200 parthenogenesis in malaria, 162 Gray, classification, 39 Greeley, gamete formation, 127 Greenough, cancer cell inclusions, 211 Greenwood, digestion in infusoria, 80 Gregoriew, negri bodies, 303 Gregory, depression periods, 108 Grenacher, centrosome in heliozoa, 30 Griffiths, excretion and urea in infusoria, 83 Gualdo, melanin and malaria, 201 Guarnieri, cytoryctes, 229 variola, 300 Gugliemi, Babesia equi, 272 Guilliermond, fertilization in yeasts, 148 nuclei in bacteria, 221 H HaEcCKEL, monera, 28 heliozoa, 35 Haecker, mitosis in cancer cells, 207 Haller, classification, 38 Hansemann, heterotypical mitosis in cancer cells, 207 Hartmann, autogamy in entameha, 142 Hartmann and _ NAisskalt, origin of hemosporidia, 201 Hartmann and Nagler, exogamy in Ameba diploidea, 150 binucleata, 176 Hartog, function of vacuoles, 83 Hertwig and Poll, rhythms of growth energy in cancer, 209 Hertwig, O., fertilization, 138 idioplasm, 124 Hertwig, R., chromidia in arcella, 119 classification, 38 depression periods, 108 dualism in nuclear materials, 124 fertilization of actinospherium, 148 organotype and cytotype, 203, 208 use of term chromidia, 116 Herxheimer, division of spirochetes, 226 Hintze, Lankesterella, 199, 248 Hoffmann, division of spirochetes, 226 Howard, cytoryctes and smallpox, 308 J JAHN, exogamy in mycetozoa, 150 Jennings, on irritability, 84 INDEX OF AUTHORS Jensen, transplantable mouse tumor, 206 Joukowsky, physiological death, 131 Jones, R., classification, 39 K Karinsky, structure of spirochetes, 228 Keysselitz, endogamy in myxosporidia, 146 exogamy in trypanoplasma, 160 parthenogenesis, 164 reducing divisions, 166 spirochete structure, 219 structure and history of trypano- | somes, 254 King, Rocky Mountain spotted fever, 278 Kinoshita, structure and life history of babesia, 270 Klebs, fertilization as explanation of cancer, 208 gamete formation, 127 Kleine, cultivation of babesia, 274 Koch, division in spirochetes, 227 East Coast fever, 272 infection of ova of ticks, 196 species of trypanosomes, 215 transmission of ticks, 230 Kdlliker, classification, 18, 34 Kossel and Weber, babesia, 274 Krinzlin, exogamy in mycetozoa, 150 Krzysztalowicz and Siedlecki, cycle of Treponema pallidum, 229 flagella in spirochetes, 224 Kuschakewitsch, sporoducts in Gre- garina cuneata, 191 Kutscher, flagella of spirochetes, 224 L Lasse, classification, 57 Lamarck, classification, 39 , Lankester, drepanidium (Lankesterella), 279 Laptschinsky, structure of spirochetes, 228 Laveran, division of spirochetes, 227 the malaria problems, 279 origin of hemosporidia, 200 Laveran and Mesnil, organism of kala azar, 238 spirochetes, 219 chromidia formation garines, 121 crithidia, 242 divison in gregarines, 183 endogamy in actinomyxide, 147 exogamy in ophryocystis, 154 Léger, in gre- 339 Léger, genus trypanosoma, 244 origin of trypanosomes, 233 sporulation in gregarines, 189 Léger and Dubosg, etiect of parasites on nuclei, 203 Leishman, form changes in Leishman- Donovan bodies, 200 organism of kala azar, 238 Leuckart, classification, 34 Levaditi, division of spirochetes, 227 spirochetes in relapsing fever and spirillosis of chicks, 197 Lewis, trypanosomes, 261 Ligniéres, structure of babesia, 276 Lister, chromidia in polystomella, 121 Loesch, ameba coli and dysentery, 295 Léwenthal, endogamy in mycetozoa, Spirocheta microgyrata, 214 Lounsbury, transmission of babesia, 276 Lithe, mode of life of spirochetes, 230 origin of hemosporidia, 199 structures, history, etce., of try- panosomes, 248 trypanosomes, 244 M McCuuwn«, sex differentiation, 126 significance of synapsis, 170 McIntosh, flagella in spirochetes, 224 MacCallum, life history of trypano- somes, 266 MacNeal, structure and history of try- panosomes, 253 MacWeeney, nuclei of spirochetes, 225 Mallory, scarlet fever organisms, 311 Malmsten, Balantidium coli, 290 Manson, environmental effects on pro- tozoa, 194 the malaria problems, 279 Marchiafava and Celli, the malaria problems, 279 Mast, function of trichocysts, 27 Maupas, conditions of conjugation, 172 depression periods, 108 Mesnil, origin of hemosporidia, 200 Metcalf, opalina encystment, 188 Metchnikoff, age, 135 digestion in infusoria, 80 organism of malaria, 279 Migula, units of spirochetes, 228 Miller, Hepatozoén perniciosum, 199, 269 Minchin, conjugation in plasmodium, 287 cytoryctes and smallpox, 308 encystment of trypanosoma, 188 origin of trypanosomes, 247 position of Herpetomonas muscze domestic, 237 340 Minchin, structures, life history, encyst- ment of trypanosomes, 248 transmission of monocystis, 198 Minchin and Fantham, Rhinosporidium kinealyi, 185, 195 Miyajima, cultivation of babesia, 274 Montesano, melanin and malaria, 201 Montfort, classification, 39 Montgomery, sex differentiation, 126 Moore and Breinl, fertilization in Try- panosoma gambiense, 163 reproduction in Trypanosoma gambiense, 188 structure of trypanosomes, 254 Mott, trypanosomiasis, 268 Muhlens, polymorphism in treponema, 229 Musgrave and Clegg, dysentery, 300 N Naauer. See Hartmann and, 150 Negri, hydrophobia, 300 Neresheimer, exogamy in opalina, 154 Nierenstein, digestion in infusoria, 80 Nocard and Motas, babesia and fever, 273 Nocht, malaria organisms, 284 Norman, classification, 39 Nosske, cancer cell inclusions, 211 Novy, life history of trypanosomes, structures, cultivation, etc., 248 Novy and Knapp, structure of spiro- chetes, 225 division, 227 Novy, MacNeal, and Torrey, relation of herpetomonas and trypanosoma, 233 Nusbaum, Schaudinella henleze, 189 Nuttall, mode of life of spirochetes, 230 Nuttall and Graham-Smith, structure and life history of babesia, 270 O OERTEL, the cancer problem, 208 Olive, exogamy in mycetozoa, 150 Opie, Balantidium coli, 290 P Pasteur, silkworm disease, 196. See Preface. Patton, form-changes in Leishman- Donovan bodies, 200 organism of kala azar, 239 structures and history of babesia, 272 transmission of protozoa, 197 INDEX OF AUTHORS Patton, types and structures of crithi- dia, 242 Perrin, Spirocheta balbianii, 219 Pfeiffer, Monocystis epithelialis, 307 Piana and Galli Valerio, Babesia canis, 272 Pianese, cancer cell inclusions, 211 Prandtl, allogromia, 300 Prenant, kinoplasmic structures, 29 Prowazek, chlamydoza, 312 dysentery, 299 endogamy in plasmodiophora, 147 exogamy in trypanosomes, 160 flagella in spirochetes, 255 Herpetomonas muscz domestice, 237 male trypanosomes, 163 negri bodies, 303 structure of spirochetes, 229 structures and life history of try- panosomes, 254 R RECKLINGHAUSEN, fertilization of epi- thelial cells, 208 Remlinger, rabies problem, 305 Reuss, classification, 39 Ribbert, the ‘‘cause”’ of cancer, 207 Ricketts, Rocky Mountain spotted fever, 278 Rixford and _ Gilchrist, immitis, 195 Robertson, exogamy in pseudospora, Coccidioides 154 structure and history of trypano- somes, 247 Rogers, form changes in Leishman- Donovan bodies, 200 systematic position of Leishmania, 234 Ross, malaria and mosquitoes, 279 organism of kala azar, 239 Roux, idioplasm, 124 Ruffer and Walker, cell inclusions in cancer, 211 San Fer.ics, cancer cell inclusions, 211 Sandahl, classification, 39 Sars, classification, 39 Sawtschenko, cell inclusions in cancer, 211 Schaudinn, acute enteritis in moles, 203 autogamy in rhizopods, 141 centrosomes in heliozoa, 31 chromidia function, 116 classification, 40 dysentery problems, 294 INDEX OF AUTHORS Schaudinn, endogamy, 148 exogamy in actinophrys, 153 in rhizopods, 154 idiochromidia in rhizopods, 121 Leydenia gemmipara, 213 malaria problems, 280 origin of hemosporidia, 200 parthenogenesis in malaria, 162 in trypanosoma, 162 sex in protozoa, 126 structure of babesia, 274 the syphilis organism, 218 trypanosomes, structure and life history, 254 yellow fever, 230 Schlumberger, dimorphism in forami- nifera, 114 Schneider, classification, 39 Schoutedan, structure of dimorpha, 30 Schrader, classification, 38 Schréder, fertilization in myxosporidia, 143 reducing divisions in same, 166 Schiider, rabies problem, 305 Schultze, F. E., centrosomes in helio- zoa, 30 classification, 38 Schultze, M., protoplasm, 21 Sergent, life Kistary of trypanosomes, 266 Trypanosoma noctue, cycle, 200 Shibata, intracellular mycorhize, 203 Shiga, dysentery, 299 Siddall, classification, 38 Siebert, flagella in spirochetes, 224 Siedlecki, Caryotropha mesnili, 202 chromidia formation in coccidiidia, 121 Siegel, ‘“‘cytoryctes luis,” 229 Silberschmidt, structure of spirochetes, 229 Simpson, physiological death, 131 species of paramecium, 112 Sjébring, cell inclusions in cancer, 211 Smith and Kilbourne, life cycle of babesia, 270 transmission by ticks, 196 Soudakewitsch, cancer cell inclusions, 211 Spencer on growth, 87 Starcovici, the name babesia, 272 Stengel, Balantidium coli, 290 Stevens, sex differentiation, 126 Stiles, Rocky Mountain spotted fever, 278 spirochetes and bacteria, 232 Stimpson, spirochetes and yellow fever, 231 Stolé, actinomyxide, 147 Strasburger, kinoplasm, 29 Strong, Balantidium coli, 290 Sutton, significance of synapsis, 170 Swellengrebel, spirochete structure, 219 | 341 T THEILER, Babesia parvum, 272 transmission of spirochetes, 230 Thélohan, function of threads in polar capsules, 193 Topsent, classification, 38 Trentepol, classification, 38 Tullock, sleeping sickness, 264 Tyzzer, cytoryctes and smallpox, 308 spirochetes in cancer mice, 214 Vv Van BENEDEN, reduction of chromo- somes, 164 Van Tieghem, classification, 38 Volpino, negri bodies, 303 Von Leyden, the cancer problems, 207 Leydenia gemmipara, 213 Von Mohl, protoplasm, 21 WwW WALDEYER, parthenogenesis of cancer cells, 208 Walker and Boys, classification, 39 Ward, transmission of protozoa, 197 Warming, ‘‘units’’ of spirochete struc- ture, 228 Wasielewsky, cytoryctes and smallpox, 308 Wechselmann and Lowenthal, nuclei in spirochetes, 225 unit structure, 228 Weismann, idioplasm, 124 natural death, 134 constitution of chromosomes, 170 Welch, Plasmodium falciparum, 288 Wenyon, autogamy in ameba, 142 entameba, 298 spirochetes in cancer mice, 214 Williams, Neuroryctes hydrophobie, 303 Williams and Lowden, negri bodies, 303 Wilson, kinoplasmic structures, 29 sex and inheritance, 126 Wilson and Chowning, Rocky Mountain spotted fever, 278 Woodcock, kinetonucleus, 29 trypanosomes, 244 Woodruff, depression periods, 108 renewal of vitality, 131 Woronin, classification, 38 malaria problems, 287 organism of kala azar, 238 Z ZETTNOW, flagella of spirochetes, 224 division of spirochetes, 227 Ziemann, malaria, 284 Zopf, classification, 38 structure of spirochetes, 228 GENERAL INDEX. A ACANTHOCYSTIS, budding and centro- some formation, 31 Acanthonida, classification, 41 Acanthosporidz, classification, 61 Acephaline, classification, 58 Acinetide, classification, 56 Acrasiz, classification, 38 Acrasis, classification, 38 Actinellida, classification, 41 Actinobolus radians, 76 apparent choice of food, 77 Actinocephalide, classification, 60 Actinocephalinw, classification, 60 Actinomyxidia, classification, 68 Actinomyxide, spores, 193 Actinophrys sol, conjugation, 152 kinetic structures, 32 Actinopoda, classification, 40 Actinospherium eichhornii, chromidia, 116, 117 fertilization, 149 Actipylea, classification, 41 Acytosporea, classification, 65 Adelea ovata, exogamy, 159, 183 Adinida, classification, 49 Agpregatide, classification, 59 Allogromia sp., 75, 301 Alveolina, classification, 39 Ameba actinophora, 23 autogamy, 122 budding and chromidia, 139 diploidea, exogamy, 151 idiochromidia, 121 protoplasmic structure and division, 120 proteus, 17 autogamy, 141, 144 idiochromidia formation, 125 tentaculata, 23 vespertilio, 203 Amebea, classification, 39 Angiosporea, classification, 59 Animals and plants, 72 Anisogamy, 126 Anisonema vitrea, 43 Anthorhynchine, classification, 60 ‘Aphrothoraca, classification, 40 122 “ey, Arcella vulgaris, copulation, 119 gametic nuclei, 118 plastogamy, 117 shell material, 24 Arcyria cinerea, exogamy, 150 Articulina, classification, 39 Aspidisca hexeris, 49 Asporscystinea, classification, 62 Assimilation in protozoa, 81 Astasiide, classification, 48 Astomea, classification, 47 Astrorhiza, classification, 39 Astrorhizida, classification, 38 Atoxyl, use in sleeping sickness, 268 Autogamy, 139 in A. limax, 141 in A. proteus, 141 in myxosporidia, 143 Auto-infection, definition, 179 Axiopodia in heliozoa, 32 in classification, 35 B BaBEsrA and transmission, 196 canis, flagellum, 176, 272 genera, species and life histories, 270 Balantidium coli, 290 entozoon, 291 Bedbugs and kala azar, 199 Bertramiide, classification, 68 Bikecide, classification, 47 Binucleata, 176 Bird malaria, 287 Black fever, 277 sickness, 238 Blepharoplast in babesia, 277 Blue fever, 277 Bodo caudatus, 43 saltans, exogamy, 153 Botryoida, classification, 41 Budding, a form of division, 89 in entameba histolytica, 94 in rhizopods, 92 in spherastrum, 92 in suctoria, 95 Bursaria truncatella, size, 19 Bursaridie, classification, 54 344 Cc CaGE infection in mouse cancer, 208 Camptonema nutans, origin of axio- podia, 33 Cancer and protozoa, 204 cell inclusions, 212 Cannopylea, classification, 42 Caryotropha mesnili, 202 schizogony, 183 Caryozoic parasites, 176 Cell autonomy, theory of cancer, 208 division, causes, 87, 88 in paramecium, 88 Centralkorn, centrosome in heliozoa, 30 Centronucleus, see kinoplasmic struc- tures, 29 Centropyxis aculeata, chromidia, 116 Cephalinz, classification, 59 Cephalont, definition, 177 Ceratiomyxa hydnoides, exogamy, 150 Ceratium tripos, 24 Ceratomyxide, classification, 66 Cercomonadide, classification, 47 Cercomonas dujardinii, exogamy, 154 Chalarathoraca, classification, 40 Chiliferide, classification, 53 Chilomonas paramecium, protoplasmic structure, 120 Chilostomellida, 141 Chitin, basis of shells and tests, 24 Chlamydodontide, classification, 52 Chlamydomyxa, classification, 38 Chlamydophora, classification, 40 Chlamydophrys stercorea, chromidia, 116, 213 life history, 294 Chlamydospore, definition, 183 Chlamydozoa, 303 Chloroflagellida, classification, 48 Chloromyxide, classification, 67 Choanoflagellata, classification, 48 Chromatophores, protoplasmic struc- ture, 26 Chromidia at period of maturity, 115 in rhizopods, 116 Chromosomes and inheritance, 89 in trypanosoma, 255 Chromulina flavicans food-getting, 72 Chrysoflagellida, classification, 48 Cilia and classification of infusoria, 49 Ciliata, classification, 52 Cirri, protoplasmic structure, 23 Club-root and cancer, 210 Club-shaped bodies in babesia, 277 Coccidiide, classification, 63 Coccidiidia, classification, 62 Coccidioides immitis, 195 Coccidium schubergi, exogamy, 160 life cyele, 180 life history, 99 Cochliopodium, 23 GENERAL INDEX | Codonecide, classification, 47 | Codosiga cymosa, 20 | Coelosporiide, classification, 68 Coelozoic parasites, 176 Coitus,.mode of transmission of try- panosomes, 265 Collida, classification, 40 Colony formation, 19 Colors in water due to protozoa, 26 Conjugation, 137 Consciousness, 71 Contractile vacuoles, function, 83 Griffiths, Hartog interpreta- tion, 83 Copromonas subtilis, cycle, 188 exogamy, 153 Copromyxa, classification, 38 Cornuspira, classification, 39 Craterium, classification, 38 Crithidia, genus, 241 gerridis, 242 melophagia, 241 subulata, 233 Cyclammina, classification, 39 Cyclospora caryolytica, 183 cause of acute enteritis in moles, 203 Cyrtoida, classification, 42 Cystoflagellata, classification, 49 Cytomere, definition, 183 Cytoryctes variole, 202, 309, 310, 311 Cytozoic parasites, 176 D DactyLopuHorip4, classification, 60 Dallingeria drysdali, exogamy, 154 Death in protozoa physiological and germinal, 130 Dendrocometide, classification, 56 Dendrosomida, classification, 56 Depression periods in protozoa, 108 action of salts and renewed vitality, 131 Desmothoraca, classification, 40 Deutomerite, definition, 178 Dictyostelium, classification, 38 Dictyotic moment in shell formation, 24 Didinium nasutum, food-getting, 74 Didymium, classification, 38 Didymophyide, classification, 60 Diffuse flagella, 45 Digestion in foraminifera, 78 in infusoria, 79 Metalnikoff on acid and alkaline, 80 in mycetozoa, 80 Dileptus, division of distributed nucleus, 92, 93 effects of starvation, 19 Dimorpha mutans, 32 GENERAL INDEX Diniferida, classification, 49 Dinoflagellata, classification, 48 Dinophyside, classification, 49 Diplophrys, classification, 38 Discoida, classification, 41 Disporea, classification, 66 Distomea, classification, 47 Division centre. See Winoplasmic structures, 29 in protozoa, 87 pathological, in paramecium, 133 Doliocystide, classification, 62 Dourine in horses, 196 Dum dum fever, 238 Dysentery in apes, 292 in man, 295 E East Coast fever, 272 Ectoplasm, cortical modifications, 22 Effects of protozoan parasites on host, 201 Eimeride, classification, 63 Eleutheroschizon dubosqui, 182 Enchelinide, classification, 52 Encystment and fertilization, 188 general statement, 18 Endogamy in actinospherium, 149 in myxosporidia, 146 in paramecium, 149 in plasmodiophora, 147 in protozoa, 146 Endogenous cycle of parasites, 181 Endospore, definition, 189 Entameba and dysentery, 295 autogamy, 141 coli, chromidia, 116 muris, 141, 142 Enterozoic parasites, 176 Ephelota butschliana, budding, 95 Epimerite, definition, 178 Estivo-autumnal fever and plasmodium falciparum, 283, 286 Euglena, division, 92 sanguinea, cause of red color in water, 27 viridis, centronucleus type, 30 Euglenida, classification, 48 Euglypha alveolata, 23 Eugregarine, classification, 58 Euplotes patella, division, 91 Euplotide, classification, 55 Excretion in protozoa, 83 Exogenous cycle of parasites, 181 Exospore, definition, 189 F FERTILIZATION by autogamy, 139 by endogamy, 146 by exogamy, 150 345 Fertilization in protozoa, 137 significance of, 171 Fever charts, malaria, 280, 281 Filoplasmodia, classification, 38 Flagella of babesia, 270 of bacteria, 223 in classification, 42 of spirochetes, 223 Food-getting methods, 71 Foraminifera, classification, 38 Fuligo, classification, 38 G Gametocyte, definition, 180 Globigerinida, classification, 39 Glossina palpalis, 264 longipennis, 265 Glugeide, classification, 68 Gonium pectorale, ontogeny, 97 Gregarina cuneata, gamete formation, 121, 191 Gregarinida, classification, 57 Gromia, classification, 38 Gromiida, classification, 38 Growth and reproduction, 86 Spencer on, 87 Guarnieri bodies in smallpox, 300 Gymnamebida, classification, 39 Gymnophrys, classification, 38 Gymnospore, definition, 183 Gymnosporea, classification, 59 Gymnostomina, classification, 52 H Ha.ipHyseEMa, Classification, 39 Halteria grandinella, food of Actino- bolus radians, 77 Halteriide, classification, 54 Haplophragmium, classification, 39 Haplosporidia, classification, 68 Haplosporidiide, classification, 68 Haustoria in parasites, 176 Helcosoma tropicum, 238 Heliozoa, classification, 40 Hematozoic parasites, 177 Hemosporea, classification, 65 Hepatozoén perniciosum, 199, 269, 271 Herpetomonas, 233, 236 donovani, 238 variations in habitat, 175, 199, 240 (Leishmania) donovani, the cause of kala azar, 34 musce domesticz, 235 species of, 236 Heteromastigida, classification, 47 Heteromonadide, classification, 47 Heterotrichida, classification, 53 346 Heterotypical mitosis in cancer, 207 Hexamitus intestinalis, exogamy, 154 Hippocrepina, classification, 39 Histoplasma capsulatum, 278 | Holophyra multifilius, sporulation, 98 | Holotrichida, classification, 52 Hyalopus dujardinii, endogamy, 148 Hydrophobia and protozoa, 293 Hypocomide, classification, 56 Hypotrichida, classification, 54 | I IprocHromipi1a, methods of formation, 118 significance, 122 Idioplasm in metazoa and protozoa, 124 Immortality in protozoa, 106 Indigestion in Paramecium aurelia, 81 Infusoria, classification, 52 Irritability, 84 Hartog on, 84 Isogamy, 126, 153 Isosporidz, classification, 63 Isotrichide, classification, 53 K Ixaua azar, 238 Karyogonad or gonad nucleus, 28 Karyoryctes cytoryctoides, 302 Kkeramosphera, classification, 39 Kinetonucleus, centre of cell activity, 29, 33 Kinoplasm, kinetic substance of the cell, 29 Klosside, classification, 63 Klossiella muris, schizogony, 183 L LaBYRINTHULA, Classification, 38 Lagenida, classification, 39 Lamblia (megastoma) entericum, 291 intestinalis, exogamy, 154 Lankesteria ascidie, 181 Larcoida, classification, 41 Latent bodies in trypanosomes, 259 malaria, 287 Leishmania, 233 Leukocytozo6n ziemanni, 230 Leydenia gemmipara in cancer, 213 | a stage of chlamydophrys, 295 | Licknaspis giltochii, 36 | Lichnophoride, classification, 55 Lieberkitihnide, classification, 54 Life cycle of parasites, 178 Lissoflagellata, classification, 46 Lituola, classification, 39 GENERAL INDEX Lituolida, classification, 39 Lobopodia, in classification, 35 Loftusia, classification, 39 Lysis produced by protozoa, 201 M MacrospHEric and microspheric shells, 114, 115 | Malaria and its causes, 279 problems, 279 Marsipella, classification, 39 Mastigella vitrea, chromidia formation, 119 Mastigina setosa, chromidia formation, 119 Mastigophora, classification, 46 Maturation in protozoa, 137 phenomena, 164 Maturity in protozoa, 113 Melanin and malaria, 201 Membranes, cirri, ete., 50 protoplasmic structure, 22 Menosporide, classification, 62 Merozoite, definition, 99, 180 Metacinetide, classification, 56 Microgromia, classification, 38 Micronucleus of trypanosomes, 255 Microsporidia, classification, 67 Microthoracide, classification, 53 Miescher’s tubules, cysts of cystis, 186 Miholida, classification, 39 Mitrophanow, origin of trichocysts, 27 Molluscum contagiosum and protozoa, 293 Monadida, classification, 46 Monera, 28 Monocystis ascidiz, exogamy, 155 Monopylea, classification, 41 Monostomea, classification, 47 Mosquitoes and malaria, 198, and yellow fever, 199 Multicilia lacustris, 291 Mycetozoa, classification, 38 Myonemes and contractile substance, 52 protoplasmic structure, 23 Myriophrys paradoxa, 37 My sidadee, classification, 67 Myxobolide, classification, 67 Myxobolus pfeifferi, 145 Myxomycetes, classification, 38 Myxosporidia, classification, 66 spore formation, 192 sarco- N Naaana, tsetse fly disease, 261 Nassoida, classification, 41 Negri bodies, 300, 304, 305, 306 GENERAL INDEX Neosporidia, classification, 66 Neuroryctes hydrophobiz, 202, 304 Noctiluca miliaris, division, 91 nucleus in mitosis, 92 Nubecularia, classification, 39 Nuclearia, classification, 38 Nuclei, chromatin and chromidia, 28 in digestion, 82 Nucleophaga amebe, 301 Nummulitida, classification, 39 Oo Opors and taste in drinking water, 27, 73 Oikomonas termo, 70 food-getting, 74 Old age, in Onychodromus grandis, 127 in Paramecium aurelia, 127 in protozoa, 127 Oligosporogenea, classification, 68 Oligotrichina, classification, 54 Opalina intestinalis, encystment, 188 ranarum, exogamy, 154 Opalinide, classification, 53 Operculina, schematic shell structure, 26 Ophryocystis mesnili, 190 exogamy, 154 Ophryodendride, classification, 56 Ophryoscolecide, classification, 54 Orbiculina, classification, 39 Orbitolites, classification, 39 “Organisms” of cancer, 205 Osculosa, classification, 41 Oxytrichide, classification, 54 P PANSPOROBLAST, definition, 143 Paramecide, classification, 53 Paramecium aurelia, life cycle, 104 105 curve of vitality, 107 at depression period, 109 caudatum, a variety, 112 old age, 127 first generation, 129 conjugation, 156 reduction, 166, 168 parasites, 302 Parasite theory of cancer, 209 Parasitism, 174 Parthenogenesis, 161 Parkeria, classification, 39 Pathogenic flagellates, 215 Pedogamy in protozoa, 146 Pelomyxa palustris, size, 18 Peneroplis, classification, 39 Peranema trichophorum, 17 Peronemide, classification, 47 347 Peredinide, classification, 49 Peripylea, classification, 40 Peritrichida, classification, 55 Peritromide, classification, 54 Pheoconchia, classification, 42 Pheocystina, classification, 42 Pheogromia, classification, 42 Pheospheria, classification, 42 Phosphorescence in sea water, 27 Phytoflagellata, classification, 48 Phytomastigophora, classification, 48 Physiology of protozoa, 69 Pileocephaline, classification, 60 Pilulina, classification, 39 Piroplasmosis hominis, 277 Plagiotomide, classification, 54 Plasmodiophora _ brassicz, classifica- tion, 38 endogamy, 147, 148 plant tumors, 202, 209 Plasmodium malariz, vivax, falciparum, 279 vivax, parthenogenesis, 162 Plastids, protoplasmic structure, 26 Platoum, classification, 38 Plectoida, classification, 41 Pleuronema chrysalis, 50 Pleuronemids, classification, 53 Podophyride, classification, 56 Polydinida, classification, 49 | Polymastigida, classification, 47 | Polyphragma, classification, 39 | Polysporea, classification, 66 Polysporogenea, classification, 68 Polystomella crispa, sporulation, 114, life cycle, 123 Polytrichina, classification, 54 Pontomyxa, classification, 38 Porospora gigantea, size, 19 | Porulosa, classification, 40 Primite, definition, 178 | Proteomyxa, in classification, 38 Protogonoplasm, 118 Protoplasmic age of protozoa, 102 | definition, 104 Protozoa, classification, 34 definition of, 17 protoplasmic structure of, 21 size of, 18 Primoida, classification, 41 Prunophracta, classification, 41 Pseudopodia, in classification, 35 Pseudospora, classification, 38 volvocis, exogamy, 154 | Pyrsonympha vertens, 291 | Pyxinia mobiuszi, 177 sp., 17 | Q | QUARTAN fever and Plasmodium malariz, 282, 284 348 Quinine, effects on malaria organisms, R RapviovaRtis, classification, 40 Raphidiophrys elegans, 70 Reducing divisions in metazoa and y protozoa, 164 be in entameba, 165 in heliozoa, 166 Reproduction, kinds of, 90 Rhabdammina, classification, 39 Rheophax, classification, 39 Rhinosporidium kinealyi, 185 Rhizomastigide, classification, 46 Rhizopoda, in classification, 38 Rotalida, classification, 39 Ss SaccaMMIna, Classification, 39 Sarcocystis muris, 186 Sarcode, 21 Sarcodina, in classification, 38 Sarcosporidia, classification, 68 Scarlet fever, 311 organisms of, 311 Schaudinnella henlez, 189 Schewiakovella schmeili, 184 Schizocystis sipunculi, 182 budding, 183 Schizogony, definition, 179 Schizogregarine, classification, 57 Sciadiophorine, classification, 60 Sex differentiation in protozoa, 126 Shells and tests, protoplasmic struc- ture, 22 Shepheardella, classification, 38 Silicoflagellida, classification, 48 Smallpox and protozoa, 293 organisms, 300 Spherastrum, nuclear division, 31 Spheroida, classification, 40 Spheromyxa labrazesi, fertilization, 148, 145 Spherophracta, classification, 41 Spheropylida, classification, 41 Spherozoea, classification, 40 Spirocheta anodontz, 220 balbianii, 222 structure, 220, 22 division, 226 duttoni and transmission by ticks, 197 gallinarum and duttoni, 221, 227 genus, 217 flagella, 223 life history, 228 microgyrata gaylordi, 213 GENERAL INDEX Spirocheta, mode of life and change of hosts, 229 nuclei, 225 relation to bacteria, 231 . species, 219 Spirochetida, classification, 46 Spirochonide, classification, 55 Spiroloculina, classification, 39 Spores, misuse of term, 99 Sporetia. See Idiochromidia, 118. Sporoducts, formation and function, 192 Sporogony definition, 179 Sporont, definition, 177 Sporozoa, classification, 56 Sporozoite, definition, 99, 180 Sporulation, a form of division, 89, 96 in Tillina magna, 97 in flagellates, 98 in sporozoa, 98 Spotted fever, 277 Spyroida, classification, 41 Stemonitis, classification, 38 Stentoride, classification, 54 Stephoida, classification, 41 Stictosporine, classification, 60 Stimuli, effects of, 85 Stylonychia mytilis, 17 Stylorhynchide, classification, 62 Suctoria, classification, 55 food-getting, 74 Surra, disease of horses, 267 Synapsis in protozoa, 170 Synura uvella, 72 Syphilis organism, Treponema pallidum, 226 Syringammina, classification, 39 T TELOspPoRIDIA, Classification, 57 Tentacles and the suctoria, 50, 51 Tertian fever and plasmodium vivax, 281, 282 Testacea, classification, 39 Tetramitus rostratus, exogamy, 154, 155 Texas fever, 272 Textularia, classification, 39 Ticks and the transmission of babesia, 275 Tintinnide, classification, 54 Tokophrya quadripartita, 17 Trachelinide, classification, 52 Trachoma and protozoa, 293 Transmission of protozoa by air, 195 by coition, 196 by contact, 195 by inheritance, 196 by intermediate hosts, 198 Treponema pallidum and syphilis, 197, 226 GENERAL INDEX Trichia fallax, exogamy, 150 Trichocysts and trichites, 27 Trichomonas intestinalis, exogamy, 154 Trichonymphinea, classification, 48 Trichospherium sieboldi, exogamy, 154 Trichostomina, classification, 53 Triloculina, classification, 39 Trophonucleus, vegetative function, 28 Trypanophis grobbini, origin of undu- lating membrane, 43 Trypanosoma agglomeration, 261 borreli, 246 changes in habitat, 175 effects on host in sleeping sickness, 267 equiperdum, cause of dourine, 196 form changes, 257 gambiense, 264 stages in division, 90 genus, 244 grayi, encystment, 188 lewisi, male forms, 163 life cycle, 261 list of species, 250 motile apparatus, 253 noctue, 247 exogamy, 160 nuclei, 255 parthenogenesis, 162, 163 raise, 45, 247 reproduction, 260 theileri, 245 Trypanosomatida, classification, 47 Trypanosomiasis, 267 Tsetse flies, 264, 265 fly and nagana, 199 349 Tsetse fly and sleeping sickness, 199 Typhus fever and protozoa, 278 U ULTRAMICROSCoPIC protozoa, 210, 231 Undulating membranes, 234 Urceolinide, classification, 55 Urea in infusoria, 83 experiments of Griffiths, 83 Urocentride, classification, 53 Uroglena Americana, 20 Urnulinide, classification, 56 Vv VaAcuoLEs, protoplasmic structure, 28 Valvulina, classification, 39 Vampyrella, classification, 38 Vertebralina, classification, 39 Virgulina, classification, 39 Vorticella campanula, food-getting, 73 Vorticellide, clasaiication: 55 Vorticellidine, classification, 55 »'s YELLOw fever, 231 Youth, maturity, and age in protozoa, 110 Z ZOOMASTIGOPHORA, Classification, 46 Zygoplast, 46 Haleish iain tcly ait les ADR Phe heap at siege aa iti akin sein yeeshie Niobe Apt Dr 3 Dreads yeah bet be lant nia | BRCIS Baek epee eR tcbebssyreaor, Sms LAOTIAN gids ; , ? ; eshte Se aT GI : : th fe Malabar a fat 7 We mies nas