Whe gia he, LE ~ Sy SS SS \ \ - re ery Vt.) 1 SYMBIONTICISM AND THE ORIGIN OF SPECIES SYMBIONTICISM AND THE ORIGIN OF SPECIES BY IVAN E. WALLIN, Sc.D. Professor of Anatomy in the University of Colorado School of Medicine BALTIMORE THE WILLIAMS & WILKINS COMPANY 1927 CopyriGcuT, 1927 THE WILLIAMS & WILKINS COMPANY Made in the United States of America Published January, 1927 CoMPoOsED AND PRINTED AT THE WAVERLY PRESS FOR Tue Witiiams & WitKiIns ComMPANY BautiMore, Mp., U. S. A. To JOHAN AUGUST UDDEN My First Teacher In Biology, This Book Is Affectionately Dedicated PREFACE The study of mitochondria in recent years has come to occupy an important position in biology. Two points of view are held in regard to the nature of these microscopic structures, and this circumstance appears to be responsible for the diversity of opinion as to the activities of these bodies. The most commonly accepted view holds that mitochondria are cell-organs derived from the cytoplasm. The other view, that they are microérganisms ‘“‘symbiotically” united to the cell, has attracted only a few adherents, and ap- parently has been looked upon as a fantastic and improbable theory. The bare statement that all living cells of plants and animals contain small bodies that are independent entities capable of a free existence, or in other words, that all cells contain bacteria or microérganisms, is, perhaps, a little shocking to those who hold dogmatic ideas on cell physi- ology. The recent advances in physical chemistry also have served to direct our thoughts and notions on “‘life- organization’ into channels which would belittle the importance of “life” in the organization of protoplasm. Obviously, physical and chemical forces play a role of fun- damental importance in the morphology and physiology of living organisms, but we are still groping in ignorance as to the real nature of living matter, and we are not at liberty to forget or belittle the significance of “life” in the analyses of living bodies. During the past seven years the author has investigated the nature of mitochondria, and has arrived at the unquali- fied conviction that these bodies in the cell are bacterial in nature. The evidence for this conclusion has been published in a series of papers. It has been evident that a large vil Vili PREFACE number of biologists have been skeptical of the results ob- tained in these investigations, and it also appears that many have been opposed to the fundamental conception that mitochondria are bacteria. It was these circumstances that urged the author to seek a rational hypothesis whereby the significance of microérganisms in the cell could be ex- plained. A study of the literature on “micro-symbiosis” revealed a wealth of evidence that supports and emphasizes the significance of bacteria in the origin of species. A principle that is so revolutionary as Symbionticism, not only introduces a new principle in organic evolution, but also inserts a new conception in heredity, development and cell-structure. While it may not be ‘“‘good scientific form” to indulge in speculative hypotheses, the new view- point affects the hypotheses and theories that are held at the present time in major divisions of biology, and the author has deemed it advisable to discuss some of these problems. Obviously it would be most unusual if all the hypotheses that are advanced in this book should in the end prove to be correct. They are based upon some evidence which in many instances is scanty, but in the present state of knowledge it represents the only evidence available. It is obvious that further and more extensive researches on the particular problems may result in modifications and the development of new hypotheses. These theories have been included in this book mainly to indicate the feasibility of Symbionticism, and to stimulate further researches on these fundamental problems. It is pertinent to emphasize that the chief object of this book is to summarize the evidence for the bacterial nature of mitochondria, and to present some of the evidence that demonstrates the fundamental réle played by bacteria in the origin of species. The search for evidence bearing on the theory has carried me into many fields of biological science as well as into the PREFACE ix domains of chemistry and physics. It is an impossible task for any one individual to cover thoroughly the litera- ture even in one of these fields of research, and I must beg the indulgence of my critics in this matter. In my desire to submit these studies in a small and concentrated volume, I have passed over or mentioned only briefly many deserving and creditable pieces of investigation. Obviously, some researches of importance may not have come to my notice. I have made extensive use of Professor Paul Buchner’s monograph “Tier und Pflanze in Intra-zellularer Symbiose.”’ This comprehensive review of the literature on ‘“micro- symbiosis” has shortened my labors by many months. The extensive treatise by Professor Edmund B. Wilson in his recent revision of ‘The Cell in Development and Heredity,” Professor E. G. Conklin’s lectures on ‘‘The Mechanism of Evolution,’”’ Professor Vernon L. Kellogg’s ‘Darwinism Today,” Professor H. F. Osborn’s ‘‘The Origin and Evolu- tion of Life,’ and many other treatises mentioned in the text have served as short-cuts to a better appreciation of the accomplishments in various biological fields. I grate- fully acknowledge my indebtedness to these valuable works. To Professor Arthur I. Kendall, Doctor F. Lohnis and Doctor Montrose T. Burrows, I am especially indebted for sympathetic interest and encouragement in my investiga- tions and for many valuable suggestions. To my colleague Doctor Ralph G. Mills I am indebted for painstaking criticisms of the manuscript. I am, also, greatly indebted to other colleagues at the University of Colorado School of Medicine for opportunities to discuss various phases of my investigations and hypotheses. To my technicians, Mr. Ivan M. Way and Miss Josephine Jones, I am especially grateful for conscientious and reliable assistance in the technical work of the researches. It is a great pleasure to acknowledge my indebtedness to Mrs. Henry 8S. Denison for research funds which were x PREFACE supplied from time to time to the Henry 8. Denison Re- search Laboratories, and for the library facilities that she has so generously provided in the University of Colorado School of Medicine. Lastly, it is a genuine pleasure to acknowledge my in- debtedness to the publishers, The Williams & Wilkins Company, for assuming the responsibility to publish this book, and for pleasant and courteous dealings. Ivan E. WALLIN. University of Colorado School of Medicine, Denver, August 18, 1926. CONTENTS CHAPTER I TERS UH OD IU CTLON: 1 fated eis ater Soe cree By RR EIST Cea ae eee eee eee 1 CHAPTER II HisTory OF MITOCHONDRIAL RESEARCH..............cccccceccccceces 10 CHAPTER III Tue BacTeriaAL NATURE OF MITOCHONDRIA..............2cececeecees 25 CHAPTER IV LAE oOEHAVIOR OF MITOCHONDRIA. -ncydeines eee eee eee eee 42 CHAPTER V SS SNUB TON TICIS Mc sts crcle’ sta ae (areca he oars else ea Coe ah aah AsO eace ST oe TEC nae COTE 58 CHAPTER VI INET GR OSIWEEBYORES core ceria ceete che ee Pete TAS eee RU ACC La ana aad en A rea 67 CHAPTER VII ANPANATIYSIS OF SXMBIONT FRACTIONS icici cic creo eiiicaele 92 CHAPTER VIII SYMBIONTICISM AND THE ORIGIN OF SPECIES............0.ececcceccees 104 CHAPTER IX SYMBIONTICISM IN RELATION TO HEREDITY AND DEVELOPMENT........ 115 CHAPTER X SYMBIONTICISM AND ORGANIC EXVOLUTION......... 0... 0c ccecccceceees 131 LD PNG Caney ofl Oa G Zola eet nt Ns ee ae ed ae TS hei FRR A UM et et EEE 148 Qu F +f } x1 CHAPTER I INTRODUCTION The earliest indications of a recognition of the essential principle embodied in the theory of organic evolution—that the more complex organisms have evolved from simpler—is, perhaps, to be found in the writings of ancient scholars. These early writings do not state a definitely formulated theory of evolution, but they contain hazy references that similar forms of life may have had a relationship in origin. Such deductions were reasonable conclusions drawn from observations of the more superficial resemblances of many animal groups. Since these earlier times, the study of plants and animals has developed into two major fields of scientific research, botany and zoology, each with a number of distinct subdivisions. During the development of these sciences, particularly zoology, there gradually crystallized a conception of organic evolution. The similarity of animals - was found to be not only superficial, but the minute struc- tural details exhibited a close resemblance in large groups of animals. Through a large series of observations and re- searches by a host of pioneer investigators, data accumu- lated which prepared a firm foundation for the theory of organic evolution. Although Linnaeus upheld the separate creation theory in the origin of species, the introduction of his binomial system of nomenclature served in great measure to emphasize the relationships of plants and animals. It was perhaps Lamarck who first clearly stated the theory of organic evolution as we understand it today. In the earlier stages of the postulated theory, philosophic speculation occupied the center of the stage. Nevertheless, we gained the principles of ‘“The Struggle for Existence’’ and 1 2 SYMBIONTICISM AND THE ORIGIN OF SPECIES “The Survival of the Fittest’’ from these earlier observations and debates. To Darwin we are indebted for a lasting demonstration of the method of scientific procedure, and for the first extensive attempt to analyze the factors involved in organic evolution. To Darwin, also, is due the credit for the first comprehensive and acceptable attempt to explain the manner of the origin of species. The extensive investigations in all departments of biology, the critical analyses and the illuminating discussions which have followed in the wake of Darwin’s dissertations, have served to clarify our conceptions and to crystallize the problems involved in organic evolution. It has become apparent that evolution is an exceedingly complex process, dependent upon a great number of factors. There appear to be three major features of organic evolution controlled by three cardinal principles. The concensus of opinion of biologists today credits Darwin with having established one of these principles. The other two principles remain to be established. Besides these three cardinal principles, there apparently are a great number of factors influencing the operations of the major principles. These factors are the various influences that have been long recognized as the modifiers of protoplasmic reactions. What then are the three major features of organic evolu- tion? A clear and concise statement of these features is not found in the literature, so it becomes necessary to attempt to formulate it here. The conception of organic evolution embodies, first of all, a conception of the origin of new forms. This conception is embodied in the term “origin of species.”” This, however, is not the sum total of organic evolution. It is well known that along with the ever increasing number of kinds of plants and animals, many that were abundant in earlier geologic times have since disappeared; other forms, apparently, are disappearing today. The kaleidoscopic character of life upon the earth INTRODUCTION 2 is the second major feature of organic evolution and, ap- parently, is controlled by a separate principle. This prin- ciple operates to control the ultimate character of the organisms present in any stage of the progress of evolution; in other words, it determines the retention or destruction of species on the earth. The third major feature of organic evolution is concerned with direction or progress. This feature has been spoken of as the principle of progressive evolution. Biologists are quite agreed that there is a “something” directing organic evolution toward an ever more complex and specialized end. There are many ex- amples of individual groups (genera) of animals which do not follow the general law, but exhibit regressive characters. However, regressive forms are explainable exceptions, and do not destroy the general principle of progressive evolution. A great amount of confusion has existed in the ranks of the evolutionists. This confusion, it appears, has been largely due to a lack of proper evaluation of the cardinal features involved in organic evolution. The discussions have centered chiefly about Darwin’s explanations. In Natural Selection, Darwin has established one of the cardinal principles that is operative. Natural Selection determines the character of life at any period in evolution; it is the principle that controls the retention or destruction of formed species. Darwin, apparently, recognized the insufficiency of Natural Selection to produce new species and introduced other factors to fill this gap in the explanation of organic evolution. These subsidiary factors have not been ac- ceptable to a large group of biologists. Modern writers have recognized the insufficiency of Darwin’s hypothesis to explain the origin of species. The ‘unknown factor’ in organic evolution has been especially emphasized by Osborne, Bateson, Kellog, and other recent writers. This “unknown factor” is especially concerned with the origin 4 SYMBIONTICISM AND THE ORIGIN OF SPECIES of species. It is the factor that causes permanent variations in the structure of animals and plants. Another unknown factor of evolution is the principle determining the direction of organic evolution, or the goal toward which evolution is proceeding. This factor has Fria. 1 been recognized, but very little has been attempted or accomplished in determining its nature. It is possible that this particular feature of organic evolution has been and still is a stumbling block to the layman’s appreciation of the reality of organic evolution. From a common ancestor INTRODUCTION 5 various diverging stocks have arisen, but only one stock forms the channel through which organic evolution proceeds. The diagram in figure 1 may serve to make the principle more clear. The various lines both solid and dotted rep- resent new species that have arisen from the common stock A. The dotted lines represent species that have arisen but are off of the line of progressive evolution. These forms may persist for longer or shorter periods and may give rise to new and more complicated species, but they are not in the direct line of evolution and therefore are not enduring. The solid lines of the diagram represent the species which are in the line of progressive evolution. The question arises: why has organic evolution proceeded along the lines that it, apparently, has chosen? ‘The factors involved in Natural Selection may account for the ultimate destruction of the aberrant species and stocks, but is Natural Selection sufficient to determine the course that organic evolution has taken? Does Natural Selection supply an element of progress, a something that has directed evolution to a more and more complex end? This is a mooted point, and it appears to me, as it has to many others, that Natural Selection, by itself, is not sufficient to determine the direction of organic evolution. If we accept Natural Selection as the cardinal principle controlling the direction of organic evolution, then it is necessary to recognize a preordained purposive factor in Natural Selection that carries us beyond our powers of comprehension. Natural Selection can only deal with that which has been formed; it has no creative powers. Any directing influence that Natural Selection may have in organic evolution, must, in the nature of the process, be secondary to some other unknown factor. In attempting to determine the cardinal principle involved in the origin of species, it appears reasonable to assume that a number of factors combine to inaugurate those changes in an organism that shall lead to the origin of new species. 6 SYMBIONTICISM AND THE ORIGIN OF SPECIES Of all the factors that play a réle in such a process, one factor, whatever it may be, is crucial or fundamental. The remaining factors, obviously, would occupy varying degrees of importance in the completion of the process. The fundamental factor in the origin of chemical compounds consists of the presence of two or more chemicals in close spacial relationship. In order that a chemical reaction, resulting in a new compound, shall take place it is also neces- sary that various extrinsic factors be properly satisfied. Obviously, these extrinsic factors are not necessarily the same for different chemical combinations, but the funda- mental factor, two or more chemicals in close proximity, is invariably constant. Exceptions to these statements, ap- parently, may be found in the inorganic world. It has been claimed that it is possible to produce a more complex mole- cule from a simpler one by the introduction of extrinsic factors alone. It is possible, also, that such a process has been operative in inorganic evolution, but, if so, it must be emphasized that this is the exception and not the rule. In our experimental laboratories, as well as in nature’s labora- tory, the presence of two or more chemicals appears to be essential in the production of a new and more complex molecule. Does the origin of species differ fundamentally from the origin of chemical compounds? Are there funda- mental factors and principles utilized in organic evolution which are absent in inorganic evolution? In recent years we have come to realize that the gap between the organic and the inorganic world is narrowing and it appears that the time is not far distant when it may completely disappear. Those activities and properties which only a few years ago were classified as ‘‘vitalistic’’ are today recognized as but the ultimate expression of chemical and physical forces. The vitalistic school, which at one time dominated biological thought, has all but completely disappeared. ‘There is no evidence at hand to indicate that organic evolution is INTRODUCTION ‘4 fundamentally different from inorganic evolution. Inor- ganic chemical reactions are dependent on the same set of extrinsic factors that govern organic chemical reactions. It appears, also, that the fundamental principle involved in the origin of species is primarily of the same general nature as it is in the origin of chemical compounds. Thus, just asitis necessary for two or more chemical compounds to be brought together if a new compound is to be formed, it may be necessary for two or more forms of life to unite in the pro- duction of a new species. While we lack specific data on some phases of plant and animal behavior, it appears from general observations, that higher plants and animals exhibit a greater degree of sta- bility than do the lower forms. The lowliest forms of life—the bacteria—appear to exhibit the greatest degree of variability. These lowly forms which, apparently, are neither plant nor animal, appear to respond more readily to environment than do the complex forms of life. It has been suggested, and it appears likely, that the bacteria are the primordial organisms from which all higher organisms, both plant and animal, have sprung. The nature of the process by which this phase of organic evolution may have occurred is indicated in the phenomenon symbiosis. Notwithstanding the fact that symbiotic rela- tionships have been studied for many years, the fundamental principle embodied in the phenomenon does not appear to have been recognized. This, probably, is due to the fact that the first recognized examples of symbiosis dealt with those cases in which both symbionts were complex animals or plants, and the life relationship was looked upon more in the nature of a biological curiosity, than the expression of a fundamental principle. A more universal type of symbiosis is represented in those relationships in which one symbiont consists of a microérganism. It is this latter type which may be designated ‘‘microsymbiosis” that appears to offer the solution to the problem of the origin of species. 8 SYMBIONTICISM AND THE ORIGIN OF SPECIES Microsymbiosis appears to be a universal biological phe- nomenon. The cells of all normal plants and animals contain minute bodies which have been named “mito- chondria.”’ In a series of researches the author has been able to demonstrate the bacterial nature of these bodies. Their universal presence in the cell coupled with the known properties of bacteria, appear to indicate that mitochondria represent the end adjustment of a fundamental biologic process. The establishment of intimate microsymbiotic complexes has been designated ‘“‘Symbionticism’”’ by the author. Symbionticism, then, is proposed as the funda- mental factor or the cardinal principle involved in the origin of species. It is a rather startling proposal that bacteria, the organ- isms which are popularly associated with disease, may represent the fundamental causative factor in the origin of species. Evidence of the constructive activities of bacteria has been at hand for many years, but popular conceptions of bacteria have been colored chiefly by their destructive activities as represented in disease. This destructive con- ception has become so fixed in the popular mind that the average person considers bacteria and disease as synony- mous. Bacteria occupy a fundamental position in the world as it is constituted today. It is impossible to conceive of this world as existing without bacteria. The cyclic rotation of essential food elements is dependent on bacterial activity. Thus, fixed nitrogen which is practically limited in amount in the world and is, also, the pivotal element in living matter, is made available through bacterial activity. Those kinds of bacteria which produce disease in man and animals, the pathogenic bacteria, represent only a small fraction of all the known varieties of bacteria. The activities in nature that are dependent upon bacteria are numerous and diver- sified. It is this diversity of bacterial properties, that lends support to the theory of Symbionticism as the causa- tive factor in the origin of species. INTRODUCTION 9 The basis for the postulation of the theory of Symbionti- cism rests upon the nature of mitochondria. Our first task, then, in attempting to establish the theory consists in reviewing the evidence for the bacterial nature of mito- chondria. It also becomes necessary to seek in nature for further evidence of the manifestations of the principle, for, if mitochondria represent the end adjustment of a biologic process, then it appears that it should be possible to find in nature examples representing various phases of this mani- festation in actual operation. A large number of such examples have been described in biological literature. The morphologic and physiologic responses exhibited in many of these symbiotic associations furnish concrete evidence of the manner in which the principles of Symbionticism may be operating in the origin of species. CHAPTER II History oF MITOCHONDRIAL RESEARCH Interest in mitochondria dates back to 1890 when Altmann published his treatise on “The elementary organisms and their relationship to the cell.’”’ Earlier observers had rec- ognized granulations in the cell, but the presence of these granules was only occasionally observed and consequently suggested bacterial contamination. The special methods of fixation and staining developed by Flemming, Altmann, and Benda made it possible to demonstrate these minute bodies consistently. This ubiquity of the granules in the cells of plants and animals led Altmann to formulate an hypothesis concerning their nature and significance in relation to the cells of all organisms. Altmann named these granules “‘bioblasts”’ and believed that they represent the ultimate units of living matter, and that the cells in which they are situated are otherwise lifeless passive ele- ments. This revolutionary conception of the cell was attacked by contemporary investigators (Verworn, and others) who clearly showed that Altmann’s conception was erroneous. Along with the rejection of this conception of the cell, the idea of the ‘“‘bioblast’’ as the elementary or primordial organism was also rejected. While Altmann’s conception of the nature of these cell granules was not accepted, his work ‘served to stimulate an active and increasing interest in these structures. The constant presence of the granules in the cell pointed strongly to some significant functional relationship. With a few exceptions which shall be discussed later, the granules came to be looked upon as originating from the cytoplasm and consequently genetically a part of it. The extensive re- 10 HISTORY OF MITOCHONDRIAL RESEARCH i searches that followed Altmann’s postulation of the ‘Bi- oblast Theory” have brought to light many interesting and instructive activities and properties of these granules. The granules have been given various names, but recently the term ‘‘mitochondria” suggested by Benda has come into general usage, although the term ‘‘chondriosome”’ is used extensively. Mitochondria have various shapes, globular, rod-like, filamentous and irregular. Various attempts have been made to classify mitochondria according to their shape, but as E. V. Cowdry (18) has intimated in his ex- tensive review of mitochondrial literature, the shape of mitochondria is of no particular significance. These classifi- cations have not entered into common usage. With only a few exceptions, mitochondria have been demonstrated in all groups of plants and animals. It appears probable that when a proper technique is used there will be no exceptions. It is not the purpose of this historical sketch to give a detailed account of the literature on mitochondria. The interested reader who desires to follow this in greater detail is referred to Duesberg’s review of the literature up to the year 1912 (in German) and to Cowdry’s review up to the year 1918. The sketch given here is selective and mainly intended to emphasize the salient features of mitochondria that have been advanced in the literature, and which have a direct bearing upon the principles of Symbionticism. A number of significant features and properties of mito- chondria have been brought out in the literature since the pioneer researches of Altmann. In 1897 Benda studied the formation of the spiral filament in the spermatozoa of the mouse and found that mitochondria are transformed into this structure. His observations have been confirmed by a number of investigators. Meves (712), Held (12), Romeis (13), Fauré-Fremiet (’13), Duesberg (’15), Levi and others have observed that the mitochondria or their products enter the egg with the sperm on fertilization. On the basis of ye SYMBIONTICISM AND THE ORIGIN OF SPECIES these observations the idea was advanced that mitochondria play a rdle in heredity. Meves in particular has advanced this theory. This conception finds a certain amount of support in the views held by Wilson and Conklin who recognize that the cytoplasm may play a réle in hereditary transmission. In 1894 Altmann expounded the doctrine ‘‘Omne gran- ulum ex granulo” fashioned after the well-known aphorism of Virchow: ‘‘omnis cellula e cellula.’”” Numerous studies have since been made in an attempt to determine the manner in which mitochondria increase in the cell. Duesberg, Meves and many others have observed what they regard as mitochondrial division, and believe with Guilliermond that ‘the mitochondria result from the division of the preéxisting mitochondria of the egg, and that none of them arise ‘de novo’ in the cytoplasm.’’ Chambers (’15), on the other hand, observed that mitochondria suddenly appear in the cytoplasm and is against the idea of ‘‘mitochondrial con- tinuity.”’ It must be emphasized that other granules besides mitochondria are supposed to be present in the cytoplasm in many instances. While there is no occasion to question Chambers’ observations, there nevertheless remain various possibilities in the interpretation of the phenomenon. It is possible that the granules which were observed to arise “‘de novo”’ in the cytoplasm have no rela- tionship with the mitochondria. Cowdry has found that the mitochondria sometimes go into ‘‘solution”’ in the cell. My own observations (Wallin, ’22) on the mitochondria of lymphocytes, confirm Cowdry’s observation. Under cer- tain conditions of the cytoplasm, whatever they may be, mitochondria are miscible with the cell cytoplasm. In such cases the mitochondrial dye stains the entire cyto- plasm. In fixed material, obviously, it is impossible to determine if the mitochondrial material could again collect into granules. Chambers’ observation on the living cell HISTORY OF MITOCHONDRIAL RESEARCH 13 would tend to show that such may be the case. In a changing chemical system such as cytoplasm is, one may expect solution and precipitation phenomena to occur. ‘This is in agreement with observed phenomena in colloidal chem- istry. ‘There is no available evidence to show that a ‘‘solu- tion” and “precipitation”? phenomenon in connection with mitochondrial material would necessarily exclude the pos- sibility of division by fission when this material is in the physical state of granules or bodies. Cowdry is inclined to believe that true mitochondria arise de novo in the cyto- plasm. He bases his opinion on the assumption that mito- chondria have a “phospholipin-albumin” composition, and that it does not seem reasonable that such a complex would actively divide like microérganisms. The consensus of opinion of modern cytologists, as well as recent researches (Horning, ’25; Takagi, ’25; Causey, 25; Horning, ’26; and others) favor the idea of mitochondrial continuity. In 1899 Benda investigated the mitochondria of the developing muscle cell, and believed that the mitochondria differentiate into the myofibrils of the muscle fibre. Meves later arrived at the same conclusion. Duesberg in 1909 and 1910 conducted extensive investigations of the subject. He was able to follow the transformation changes and noted the diminution of mitochondria parallel with the increase in fibrils. ‘The conclusions arrived at by Benda, Meves, and Duesberg have been confirmed by von Kurkiewicz, Leplat (12), Levitsky (10) Favre et Regaud (’10), Hoven (’10), Prenant (’11), Amold (12), Schafer (12), Luna’ (’18), Briick (’14) and many others. Against this conception of the nature of myofibril-formation are the opinions of Heiden- hain (’11), Levi (11), Gurevitsch (13) and Cowdry. Cowdry bases his opinion on some slight variations of staining in his preparations of chick embryos, and on the impossibility of mitochondria, assumed to be composed of phospholipin- 14 SYMBIONTICISM AND THE ORIGIN OF SPECIES albumin, being transformed into myofibrils which contain tyrosin. The conceptions embodied in Benda’s work that mito- chondria enter into histogenetic differentiations, apparently, stimulated a number of investigators to study mitochondria in relation to various histogenetic activities. This point of view was further strengthened by Regaud’s ‘‘eclectosome theory,’’ which held that mitochondria behave somewhat like plasts, choosing and selecting substances from the sur- rounding cytoplasm, and transforming them into diverse products. Meves, in 1910, investigated the origin of con- nective tissue fibrils. He found that mitochondria leave the cell and become filamentous, and he believed that they enter into the formation of the fibrils. Against this conception is, apparently, the work of M. R. Lewis (’17) who studied con- nective tissue fibril formation in tissue cultures. Cowdry criticizes Meves’ work from two points of view. He assumes that the chemical composition of the mitochondria of all cells is the same, and that the assumed phospholipin- albumin structure is incompatible with collagen fibril forma- tion. On the further assumption that the mitochondria of all cells are alike and the fact that filamentous or thread-like mitochondria are present in cells which do not contain fibrils, Cowdry holds that the development of filamentous mitochondria in Meves’ preparations have no significance. Firket (11) investigated the origin of epidermal fibrils and concluded that they arise from the differentiations of mitochondria. Duesberg later confirmed this work of Firket. Meves, in 1907, investigated the origin of fibrils in nervous tissue. He concluded that the mitochondria of the nerve cells differentiate into the neurofibrils. A number of investigators have also confirmed his work, of whom we might mention Hoven and Arnold. Marcora, Levi, and Gurevitsch, on the other hand, reject the con- ception that neurofibrils are formed from mitochondria. HISTORY OF MITOCHONDRIAL RESEARCH 15 The investigations of Regaud, Fauré-Fremiet, Lowschin and others on the chemical nature of mitochondria cast a cloud of doubt on the ‘‘diverse transformation activities”’ that had been attributed to mitochondria. Thus, Cowdry (18), in summing up his criticism on fibril formation, ex- presses the sentiments of cytologists in general when he remarks “that it is somewhat illogical to suppose that sub- stances of such diverse chemical constitution are all formed through the transformation of a single substance, a phospho- lipin, combined perhaps with a small fraction of albumin.” The influence that these chemical investigations have had on subsequent mitochondrial research has been unfortunate. It is pertinent to analyze minutely the evidence on which the ideas on the chemical constitution of mitochondria have been based. ‘This may be facilitated by considering the collected evidence as it is given by Cowdry in his critical review of the literature. After pointing out that three individuals, Regaud, Fauré-Fremiet and Léwschin arrived at the same conclusion independently, he tabulates the evidence briefly as follows: (1) Mitochondria are almost completely soluble in alcohol, chloroform, ether, and dilute acetic acid. They are rendered in- soluble by chromization. They are not doubly refractile and they do not stain with either Sudan III or Scharlach R. They are only sometimes blackened with osmic acid. From the above evidence, apparently, it is assumed that because mitochondria in general are sensitive to these chemicals they all have a like constitution. However, it is well known that mitochondria vary in chemical constitution at different periods in the life of the cell. Thus, to quote from another section of Cowdry’s treatise, ‘‘It is well known that the mitochondria undergo definite chemical changes in the course of spermatogenesis. Regaud (1910, p. 294) clearly showed that their resistance to acetic acid grows _ Us, 16 SYMBIONTICISM AND THE ORIGIN OF SPECIES greater and greater; in fact, the structures which they form, nebenkern and spiral filament, were known and recognized long before the mitochondria in the earlier stages of sperma- togenesis were brought to light.’’ Assuming that all mito- chondria responded in a like manner to these various chem- icals this would not be sufficient evidence to establish a more or less definite chemical constitution of these microscopic bodies. In some of my own experiments (Wallin ’22a), various bacteria were subjected to these chemicals. It was found that some strains of bacteria responded in a manner similar to the usual response of mitochondria. Bowen’s extensive researches on the nebenkern of certain insects demonstrated that chemical transformations occur in the mitochondrial body during its complicated changes. These chemical variations become apparent in the staining reactions. (2) It is said that part of the mitochondrial substance is not soluble in these fat solvents and it is supposed that this portion is albumin (see also Bullard, 1916, p. 26), for formalin and bichro- mate, which are used as fixatives for mitochondria, are energetic coagulants of albumin. Miuillon’s reagent is the only color-test for protein which can be satisfactorily applied to material in section (the xanthoproteic reaction may also be used, but it is less satis- factory because it is more destructive). I learn from Dr. R. R. Bensley that the mitochondria do not give a definitely positive Millon reaction in comparison with the strong Millon reaction which is given by such cytoplasmic structures as the zymogen granules. Even if there were a change in color in the mitochondria it might not be of sufficient intensity to be appreciated in filaments of such extreme fineness as mitochondria (0.2 micron in diameter) embedded in a colored cytoplasm. I have obtained no success with the xanthoproteic reaction. Mitochondria do not give any of the color reactions of polysaccharides. This section, apparently, is intended to contain the evidence for the partial albumin constitution of mitochon- HISTORY OF MITOCHONDRIAL RESEARCH Wi dria. In the first sentence the word ‘supposed’ gives a clue to the finality of the evidence. (8) Artificial mitochondria have been made by Loéwschin of lecithin in different salt and albumin solutions (resulting in the formation of lecithalbumin), which apparently present the same form and solubilities as true mitochondria. They form granules, rods, and filaments which, he claims, multiply by division. He ‘embedded them in glycerin-gelatin, fixed them, and found that they stained in the usual way by the various mitochondrial methods. The production of rod-like, coccoid, and variously-shaped “precipitation bodies” in solutions is a common phenomenon in colloidal chemistry, and has been obtained in various ways with different kinds of reagents. Certainly this is not a phenomenon limited to solutions containing a phospholipin- albumin complex. (4) The temperature solubility of mitochondria may also be significant. It has been discovered by Policard (1912d, p. 229) in the case of animal tissues, and by N. H. Cowdry (1917, p. 220) in plants, that the mitochondria are soluble at a temperature from 48° to 50°C., while the other parts of the cell remain practically unaffected. Phosphatids have a low melting-point also. Because mitochondria disappear at a temperature of 48° to 50°C., and the melting point of phosphatids is also low, does not necessarily imply that the two are identical in constitution. In some experiments that the author (Wallin, ’22a) made on the effects of temperature on various bacteria, it was found that some strains of bacteria ‘‘dissolved”’ at this temperature. (5) Apparently the specific gravity of mitochondria is somewhat greater than protoplasm (Fauré-Fremiet, 1913, p. 602). This is determined by the centrifuge method. If they are thrown down they are said to be of high specific gravity. If the protoplasm is 18 SYMBIONTICISM AND THE ORIGIN OF SPECIES in the physical condition of a “gel” rather than a “sol” as in the nerve cell, the distribution of the mitochondria is unaltered by centrifuging (Key). There is no reason to believe that the mitochondria themselves are different. At any rate, where the method is applicable (i.e., in egg cells) the mitochondria are heavier than protoplasm, in which respect they conform to what we know of phosphatids and differ sharply from oils and neutral fats, which rise to the surface and float instead of being thrown down. Granting that the specific gravity of mitochondria is greater than that of the cytoplasm of the cell, such a cir- cumstance would not constitute evidence that they are related to phosphatids merely because the latter are heavier than cytoplasm. Regards the probability that the mito- chondria may be different in different varieties of cells, Cowdry (’18, p. 85) remarks “In some varieties of cells the constitution of the mitochondria apparently differs, slightly but noticeably, from that of the mitochondria in other cells, though in cells of the same kind their composition is very constant.’’ The staining reactions of mitochondria when the Giemsa stain and the Kull method are used, demonstrate clearly that the mitochondria of a single cell are not all alike. (6) Mitochondria act as solutes for various substances. They are often pigmented and assume the most brilliant hues. Pre- nant (Asvandourova, 1913, p. 293) has actually styled them “chromochondria” on this account. This solution of other materials in mitochondria is particularly frequent in plant cells. It may or it may not be significant from the point of view of their constitution. The evidence here does not appear to have any specific bearing on a phospho-lipin-albumin constitution. (7) There seems to be a certain correspondence between varia- tions in the histological picture of mitochondria and the variations HISTORY OF MITOCHONDRIAL RESEARCH 19 in the phospholipin content of the same organ on chemical analy- sis. Thus Mayer, Rathery, and Schaeffer (1914, p. 612) have been able to alter the mitochondria experimentally in liver cells. In stages with more mitochondrial substance, chemical analysis showed an increase in phosphorized lipoid; in stages with less, a diminution. Fauré-Fremiet (1912b, p. 347) has extracted from the ovaries and testes of Ascaris a phosphatid with properties identical with those of mitochondria in the cells of these organs. The evidence given here is limited in application. The experiments of Mayer, Rathery and Schaeffer were based on chemical analysis and, apparently, demonstrated that there was a relationship between the number of mitochondria and the amount of phosphorized lipoid present. This work was done on the liver. Is there any evidence that the same would hold true in other tissues? The evidence is not given. Furthermore, it is at least possible that the methods used to reduce the number of mitochondria at the same time affected the lipoid constituents of other portions of the cell. The work of Fauré-Fremiet also is not conclusive in that he did not demonstrate that the phosphatid which was extracted came from the mitochondria. (8) Russo (1912, p. 215) has apparently been able to increase the number of mitochondria in the oocytes of the fowl by the injection of lecithin. R. Van der Stricht (1911, p. 455) found that there are two different kinds of eggs in the cat, one containing much vitellus and the other containing only a small amount; and further, that, following intraperitoneal injections of lecithin, the relative number of female offspring increased noticeably. In the normal condition 62 per cent are males, while after treating in this way only 23 per cent are males. That is to say, the ad- ministration of lecithin increases the amount of deutoplasm in the eggs, increases the number of eggs with much deutoplasm as contrasted with those with small amount, and in this way increases the percentage of females in the offspring. While this is of great interest in the determination of sex, and will be discussed in that 20 SYMBIONTICISM AND THE ORIGIN OF SPECIES connection, it is also of importance as an indication of a possible relationship between the amount of mitochondria and the phos- phatid lecithin. The “‘evidence”’ here deals with possibilities and not with pertinent facts. In summing up, we find that only one piece of investiga- tion, that of Mayer, Rathery and Schaeffer has definite bearing on the problem. It relates only to the mitochondria of the cell of a single organ,—the liver. While we can neither deny, nor affirm the phospholipin-albumin constitu- tion of mitochondria, there is no reason to conclude that mitochondria have the same constitution in all kinds of cells, and at all periods in the life of the cell. We certainly are not justified in rejecting the work that has been done on the histogenesis of fibrils on the basis of an incompatibility with the assumed chemical constitution of mitochondria. We must frankly confess that the chemical constitution of mitochondria is not known. It is interesting to note that Cowdry himself recognizes the variability of the chemical constitution of mitochondria, in other parts of his thesis (p. 85). He suggests that this variability may be more apparent than real: “it may be due to factors other than a difference in the mitochondria—to changes in the water content, for example.’ This, it appears to me, is a weak substitute for the rational interpretation suggested by such variations. Mathews (15), in his text book on Physiological Chem- istry, states that the phospholipins or phosphatids are “among the most important substances in living matter. For they are found in all cells, and it is undoubtedly their function to produce with cholesterol, the peculiar semifluid, semisolid state of protoplasm. The latter holds much water in it, and does not dissolve. Indeed it might be said that the phosphatids with cholesterol make the essential physical substratum of living matter.’’ Let us then assume that the HISTORY OF MITOCHONDRIAL RESEARCH 21 mitochondria are phosphatids, would this phosphatid con- stitution be incompatible with fibril formation? In the light of Mathews statement, and we have many reasons to accept the statement, mitochondria contain one of the essentials in the physical substratum of living matter. If we assume a phosphatid constitution of mitochondria then it certainly appears logical that mitochondria are proto- plasmic in nature; in other words, they possess one of the essential components of living matter. If mitochondria are living structures, we would naturally expect them to exhibit differentitation into many and diverse structures. However, such argument based on assumptions is not suffi- cient to replace evidence gained by observation and experimentation. Various other histogenetic activities have been attributed to mitochondria. Meves in 1908, stated that ‘‘with the specialization of the embryo into different organs and tis- sues, primitively similar cells assume special functions which find expression in characteristic structures or differentiations. All these products, no matter how heterogeneous they may be, arise through the metamorphosis of one and the same elementary plasma-constituent, the chondriosomes’”’ (quoted from Cowdry). Meves’ hypothetical statement has found a rather wide confirmation in the researches of subsequent investigators. The following list taken from that given by Cowdry will serve to indicate the range of these described activities: Anthocyanin (Guilliermond), Apparato reticulare (Hoven), Aqueous humor (Mawas), Batonnets of Heiden- hain (Policard), Carotine (Guilliermond), Cerebrospinal fluid (Grynfelt and Euziere), Chlorophyl (Guilliermond), Chromoplasts (Guilliermond), Ciliary apparatus (Saguchi), Connective tissue cell granulations (Renant and Dubreuil), Granulations (Meves), Epidermal cell secretion (Saguchi), Fat (Altmann), Glycogen (Arnold, Alexieff), Goblet cell mucous (Grynfelt), Hemoglobin pigment (Ciaccio), Leuco- we SYMBIONTICISM AND THE ORIGIN OF SPECIES plasts (Lewitsky), Lipoid in nerve cells (Cowdry), Liposomes (Fauré-Fremiet), Mammary gland secretion (Hoven), Mel- anin (Asavadourova), Mucorine (Moreau), Pancreatic zymogen (Hoven), Parathyroid secretion (Bobeau), Parotid secretion (Regaud and Mawas), Pigment, (Prenant), Poison-gland secretion (Torraca), Prepucial-gland secretion (Altmann), Prostate secretion (Akatsu), Retineal pigment (Luna), Sebaceous-gland secretion (Nicholas, Regaud and Favre), Starch (Guilliermond), Submaxillary gland secretion (Regaud and Mawas), Suprarenal pigment (Mulon), Supra- renal secretion (Mulon), Thyroid-gland secretion (Gryn- felt), Tyrosin (Asavadourova), Urea in the kidney (Oliver), Venom (Grynfelt), Vitellus (Loyez), Yolk (Coghill). In many instances of the above named activities, the researches on which they have been based have been confirmed by other investigators, and in some instances the reverse has occurred. It would be quite beyond the scope of this treatise to re- view and analyse all these investigations. However, the researches of Levitsky (’10), Pensa (712), Guilliermond (12a; ’12b), and a host of other investigators on plast formation deserve more than passing notice. A number of varieties of plastids are found within the cells of plants. These plastids are concerned with the production of specific products in the plant. Guilliermond, in particular, has conducted extensive investigations on the origin of these plasts. He has apparently demonstrated the transformation of mitochondria into the various kinds of plastids. His work has been accepted in standard text books. Ap- parently, no serious refutation of Guilliermond’s work has appeared. Cowdry is not inclined to accept these inter- pretations on plast formation, and states that ‘‘the work which has been done so far is extremely suggestive, but it is not conclusive.” It appears that the asswmed fixed phos- pholipin-albumin constitution of mitochondria has in- fluenced Cowdry’s criticisms. The living properties of HISTORY OF MITOCHONDRIAL RESEARCH 23 plasts has been emphasized in recent years, and has formed the basis for a pertinent and significant theory advanced by Merejkovsky in 1920. On the basis of the relationship of plant plastids to the independent plast-like organisms, the blue-green algae, Merejkovsky has advanced the theory that all the higher green plants are symbiotic complexes. A further consideration of Merejkovsky’s theory will be given in a subsequent chapter. The relationship of mitochondria to secretory processes has been investigated extensively. The results in these researches have led to opposing views. In some instances mitochondria have been shown to enter into secretory activity. In other cases it appears that they are unrelated to this cellular activity. The solution of the problem of the relationship of mitochondria to secretion has a significant bearing on the theory of Symbionticism, and will be dis- cussed at greater length in the chapter on ‘““The Behavior of Mitochondria.” In 1918 Portier published an extensive treatise entitled: “Les Symbiotes,”’ in which he brought forth evidence to support his theory that mitochondria are bacterial organisms symbiotically combined with the cells of all higher organisms. Many of the views and the fundamental evidence that Portier submitted have not been acceptable to cytologists, bacteriologists, and biologists in general. He quoted an extensive literature which supports his views and we shall have occasion, in a later chapter, to draw upon these ex- amples to illustrate the various degrees of microsymbiosis which may be found in nature. The crucial evidence sub- mitted by Portier was unacceptable to bacteriologists in particular. This evidence had to do with the actual demon- stration of the independent nature of mitochondria. Portier planted tissues in culture media and obtained bacterial growths in the media. An analysis of these experiments revealed possibilities of error which were not taken into 24 SYMBIONTICISM AND THE ORIGIN OF SPECIES account by Portier. (The author published a critical analy- sis of Portier’s experiments in 1923 (Wallin, ’23).) The nature of the organisms cultured in Portier’s experiments, apparently, had none of the properties ascribed to mito- chondria; they not only resisted the action of acetic acid, alcohol and ether, but Portier maintained that the organisms retained life after being kept in absolute alcohol, for example, for very long periods. Such properties are incompatible with the known properties of living matter, or protoplasm. The Paris biologists who examined Portier’s preparations were convinced that the growths obtained represented contaminations. It is evident from this brief review of mitochondrial literature that misleading assumptions have served to color the interpretations in connection with mitochondrial re- search. The solution of the central problem in this field of investigation—the nature of mitochondria—gives promise of the most fundamental and far-reaching modifications in our conceptions of the cell. Certainly, the most generally accepted views on the nature of mitochondria have had no real basis for their pronouncement; nor have these views led the way to any real advance in a better appreciation of cell physiology. CHAPTER III Toe BactTerRiAL NATURE OF MITOCHONDRIA Although a great number of investigators have attributed diverse properties to mitochondria, the specific conception that mitochondria may be living bodies does not appear to have been seriously considered. On the contrary, this con- ception in Altmann’s work was not accepted and again it was rejected in Portier’s “Symbiotes.” The outstanding morphologic characteristic of mitochondria is their resem- blance to bacteria. While morphologic resemblance, con- sidered by itself, does not necessarily imply relationship, it does, at least, suggest that possibility. The possible relationship between mitochondria and bacteria is further suggested by some of the properties exhibited by mitochon- dria, particularly their evident mode of multiplication by simple fission. So, also, the secretion activities that have been attributed to mitochondria by so many authors are indeed suggestive of the properties of living bodies. When the literature up to the year 1918 was carefully examined, it became apparent that the characteristics at- tributed to mitochondria which differentiated them from bacteria were assumed rather than proved. ‘The literature did not contain any reference to experiments that were intended to demonstrate specific differences in the two groups of bodies. It is evident that the necessity for experi- mental and specific determination of the relationship be- tween these two groups of structures is essential before we can proceed intelligently to develop a logical conception of the nature of mitochondria. In the early part of 1919, I began a series of investigations on the nature of mitochondria. These investigations were 25 26 SYMBIONTICISM AND THE ORIGIN OF SPECIES at first directed toward testing experimentally the reactions of bacteria to those agents that had been used in determining the “characteristics” of mitochondria. The complete details of these and the subsequent investigations may be found in the original papers, a list of which is given in the bibli- ography at the end of this book. The following resumé contains the chief results obtained in these researches. The properties that had been specifically attributed to mitochondria and had been assumed to be distinct from those of bacteria are in the nature of reactions which will be considered under the following classifications: Staining properties, reactions to various chemical and physical agents, and thermal responses. The assumed chemical constitution of mitochondria was largely based on the sum total of these various reactions. Staining properties. While most of the mitochondrial stains have not been considered specific for mitochondria, one stain, Janus green B (vital stain) has been placed in this category by Cowdry. A number of the mitochondrial stains were applied to bacteria and it was found that many strains of bacteria were stained like mitochondria by these methods. The Janus green B vital method was also applied to various strains of bacteria. Some varieties were very poorly colored by this method, but others, particularly the pneumococcus and Bacterium coli were stained in a homo- geneous manner like mitochondria. It is evident from the results of these experiments that the staining reactions do not indicate any fundamental differences between mito- chondria and bacteria. Mitochondria were also subjected to bacteriological stain- ing methods. This was done on smear preparations of tis- sues from fetal and young animals. They were treated like ordinary bacterial smears. The following bacteriological stains were used: Loeffler’s methylene blue, pyronin-methyl green, the Giemsa stain and Gram’s method. The mito- PLATE I MirocHoNDRIA OF VARIOUS CELLS Fig. 1. Human spinal ganglion cell (after Cowdry). Fig. 2. Dog thy- reoid cell (after Takagi). Fig. 3. Dividing insect cell showing mitochon- drial division (after Bowen). Fig. 4. Exeretory duct cells of testis of fish (after Duesberg). Fig. 5. Guinea-pig pancreas cells (after Bensley). Figs. 6and 7. Cells from growing root-tip of barley. Starch grains forming in mitochondria in figure 7 (after Guilliermond), THE BACTERIAL NATURE OF MITOCHONDRIA ya chondria in the smears were distinctly stained by these methods. Gram’s stain, however, did not bring them out as sharply as the other methods. These two sets of staining experiments further indicate the danger of attributing specificity to staining reactions. These results do not belittle the value of various stains, but they indicate rather the limitations of staining reactions in determining the chemical constitution of small bodies that cannot be isolated for direct chemical analysis. Chemical reactions. A number of investigators have observed that mitochondria are sensitive to certain chemicals. These chemicals are ether, alcohol, and acetic acid. Two of them are used extensively in histological technique. and this circumstance, perhaps, explains why mitochondria were not recognized at a much earlier date. It is usually stated that these chemicals dissolve mito- chondria. This may not necessarily be the case, for the “dissolution” is usually determined by staining reactions. It is obvious that the reagent may alter these bodies so that they do not stain, and consequently they would not be readily detected in the cytoplasm. The effects of alcohol, ether and acetic acid on bacteria have been determined. Various strains of bacteria were placed in shallow cups built up on microscopic slides, and the various solutions of acetic acid and alcohol were then in- troduced. Ether was used in concentrated form. The chemicals were allowed to remain for atime and then evap- orated, leaving all solid matter on the slide. After floating a thin layer of celloidin over the slide and permitting it to harden, the preparations were stained. Microscopic ex- amination of these preparations showed a variety of reactions of the various strains of bacteria in the different solutions. In some cases, the organisms remained intact, but were unstained. In some instances, an amorphous mass is all that remained on the slide; it was stained in some specimens, 28 SYMBIONTICISM AND THE ORIGIN OF SPECIES but not in others. In other cases the organisms were well preserved and deeply stained. These experiments failed to demonstrate any significant differences in the reactions of bacteria and mitochondria to ether, alcohol and acetic acid. Reactions to physical agents. Mitochondria are known to be sensitive to mechanical manipulation, desiccation and slight variations in the physical environment. They fragment readily. This sensitiveness of mitochondria is usually spoken of as fragility. It has been assumed that bacteria do not possess such a fragility and that this char- acteristic differentiates them from bacteria. Guilliermond (19), in particular, has stated that there are no bacteria known that are as fragile as mitochondria. Experiments were devised in which the tissues of fetal, young and old animals were smeared on glass slides, the film dried, fixed and stained by various mitochondrial and bacteriological methods. It was at once apparent in these preparations that the fragility of mitochondria varies in the different tissues, and with the age of the animals. In the smears from adult animals mitochondria were seldom visible either in the intact cells in the preparation or in the cyto- plasm of the ruptured cells. In the tissue smears from young animals, the mitochondria were present in the intact cells, but seldom in the cytoplasm of ruptured cells. The smears from fetal tissues invariably contained mitochondria in great numbers in the fragments of ruptured cells as well as in those still intact. These experiments seem to indicate that fragility of mitochondria increases directly with the age of the animal. Fragility in bacteria was not investigated experimentally. Bacteriological literature contains ample evidence that bacteria vary considerably in fragility. One microérgan- ism, in particular, exhibits a degree of fragility properties that, apparently is as great as that exhibited by mito- THE BACTERIAL NATURE OF MITOCHONDRIA 29 chondria. This organism (there is some question in regard to its true bacterial nature), which is associated with the disease Rocky Mountain spotted fever, has been investi- gated recently by Wolbach who has taken cognizance of its fragile nature. A number of related forms, spoken of collectively as “‘Rickettsia bodies,” also, are characterized by their fragile nature. Fragility is thus not a specific characteristic of mito- chondria, but in known microérganisms it appears to be a property acquired in the development of a symbiotic existence. Thermal responses. A number of investigators have found that mitochondria are apparently destroyed when the tissues containing them are exposed to a temperature of 48° to 50°C. for a comparatively short period. It is well known that microérganisms exhibit a wide range of reactions to temperature. Some organisms live constantly in hot springs where the temperature is not far below the boiling point; others thrive in cold regions where the temperature may be far below zero. Experiments were made in which some ten strains of bacteria were placed in vials containing physiological salt solution and incubated at a temperature of 49°C. for half an hour. The organisms were examined before incubation to determine if the physiological salt solution affected their morphology. ‘They all appeared to be intact. After the incubation, a drop from the bottom of the vial was removed with a pipette, smeared on a slide and allowed to dry. A celloidin film was then floated over the dried smear to fix it on the slide. In these experiments it was found that some of the strains of bacteria were apparently unaffected. Other strains, however, had ‘‘dissolved”’ or disappeared; amorphous masses, stained in some cases, were all that remained of the organisms. These experiments failed to demonstrate any fundamental 30 SYMBIONTICISM AND THE ORIGIN OF SPECIES difference in the reactions of bacteria and mitochondria to temperature. Chemical constitution. The chemical nature of mito- chondria has been presumed to be a phosphatid combined with albumin. The reasons for this assumption were dis- cussed in the previous chapter. While the evidence, so far as it goes, points to the presence of phosphatids in the mitochondria of the liver cells, it is probable that they are also present in other mitochondria. There is no evidence, however, to indicate that the phosphatid-albumin complex is the only constituent of mitochondria. Certainly the chemical determinations that have been made on mitochondria and bacteria are not sufficient to justify the conclusion that the two groups of structures are fundamentally different in chemical constitution. So far as the reactions of mitochondria to physical, chemical, and thermal agents have been the basis for determining their nature, the responses of bacteria to these same agents would rather suggest a similarity of the two groups of structures. In a series of studies on ferment action, Joblin and Peter- sen (’14a, b, c) have determined the presence of lipoids in bacteria. It may not be amiss to quote their opinion in this connection. ‘Bacteria do, however, contain fats and lipoids in varying amounts, which because of their marked effect on surface tension, would for purely physical reasons tend to become concentrated at the periphery of a colloidal system such as the bacterial protoplasm. With or without a morphologically distinct limiting membrane we can reason- ably assume that the external surface of the bacterial cell is potentially lipoidal.”’ The results of these experiments and the analysis of mitochondrial literature failed to reveal any evidence that excluded the possibility that mitochondria may be bacterial in nature. On the other hand, many properties that have THE BACTERIAL NATURE OF MITOCHONDRIA 31 been attributed to mitochondria seem to find their most logical explanation in a conception of their bacterial nature. Obviously, it became necessary to attempt experimental and specific determination of the nature of these bodies. It is evident that, if mitochondria could be induced to grow independently of the cell, there could be no question regard- ing their bacterial nature. The problem at the beginning was beset with a number of difficulties. Bacteriological literature contained a con- siderable number of references to experiments on growing bacteria in culture media planted with tissues from pre- sumably healthy, normal animals. These growths had been thought to arise from extra-cellular, non-pathogenic, symbiotic microédrganisms. There was nothing to indicate that they had not developed from the mitochondria nor- mally present in the cells of the explanted tissues. It was also presumed that these symbiotic microérganisms were acquired by the animal after birth. Hence, to avoid this uncertainty, it was deemed advisable to choose for experi- mentation those tissues in which the possibility of con- tamination would be reduced to the minimum. The tissues of the fetus and the new-born, at least theo- retically, should be uncontaminated by extraneous micro- organisms and consequently were chosen for the proposed culture experiments. The problem of a proper culture medium offered, perhaps, the greatest difficulty. It appeared that it would be neces- sary to use a medium which possessed the essential at- tributes of cytoplasm, inasmuch as mitochondria normally live in a cytoplasmic environment. This idea suggested the use of some kind of living protoplasm as a culture medium, but on further reflection it was obvious that any positive growth in such a medium, would not furnish conclusive evidence on the point at issue. At best, it would permit of various interpretations which would only lead back 32 SYMBIONTICISM AND THE ORIGIN OF SPECIES again to the original status of the problem. It was decided to test out various bacteriological culture media and to devise new media. Experiments were then made in which liver tissue from fetal and new-born rabbits was planted in a number of the more commonly used culture media. The results of these experiments were chiefly negative. A human blood medium was tested, resulting in a positive bacterial growth. The organisms which developed as a surface growth had some unusual characteristics. It was difficult to procure suffi- cient quantities of human blood to make this medium on a large scale, and satisfactory sterilization of the medium was extremely difficult. Special media were then devised on the principle of the meat-infusion medium, in which rabbit liver replaced the meat. ‘The media were adjusted to various hydrogen-ion concentrations, ranging from pH 6.4 to pH 8.8. When bits of liver tissue from fetal and new- born rabbits were planted in these media, surface organisms developed in a number of the culture tubes. In practically all of the tubes that did not contain the surface organisms, a peculiar clouding developed in the culture medium about the planted liver tissue. When a part of this clouded me- dium was removed from the tube, smeared on a slide, dried, fixed, stained and examined microscopically, it did not contain any visible bodies that had the appearance of bacteria. When wet preparations of the clouded material were examined microscopically, small coccoid bodies could be seen. The clouded culture media and planted tissues were removed from a number of culture tubes, fixed in a mitochondrial fixative, embedded in celloidin, sectioned and stained. Microscopic examination of these preparations revealed myriads of coccoid bodies in the clouded medium. It was evident from the results of these experiments that the coccoid bodies of the clouded medium were fragile, and disappeared in the smear preparations from the clouded medium. THE BACTERIAL NATURE OF MITOCHONDRIA 33 In all these culture experiments the possibility of con- tamination was constantly kept in mind and guarded against. All instruments and vessels that were used were thoroughly sterilized. The fetus or new-born rabbit, after decapitation was saturated with 95 per cent alcohol. The instruments employed in opening the abdomen and removing the liver were always sterilized in the flame immediately before use. The liver was quickly removed to a sterile petri dish, cut into pieces by inserting a sterile scalpel under the lid. Pieces of the liver tissue were then planted in the media by the usual bacteriological technic. The ‘‘clouded growths’? obtained in these experiments appeared to be the more significant, since they developed consistently. It appeared possible, however, that these coccoid bodies of the clouded medium might be the result of some precipitation of a colloidal nature induced by the planted liver tissue. This possibility was further suggested by the failure of all attempts to subculture them. How- ever, the clouded medium was examined with the dark field microscope in the hope of determining if the coccoid bodies exhibited true movement. In a number of such examinations true movement was observed. In some in- stances, the minute bodies were seen to move across the entire microscopic field. ‘These movements were not con- fused with Brownian movement, which was abundantly present in the preparations. These observations strength- ened the idea that the coccoid bodies might be living organisms. The experiments completed up to this time, while not conclusive, certainly enhanced the probability of demon- strating the bacterial nature of mitchondria. An analysis of the nature and extent of growth, particularly the deep clouded growth, appeared to indicate that the media were deficient in some essential food material. It was sug- gested by Dr. A. I. Kendall’ that possibly the (limited) 1 Personal communication. 34 SYMBIONTICISM AND THE ORIGIN OF SPECIES growth observed was dependent upon food materials present in the planted liver tissue. When this material is exhausted, growth necessarily ceases. The pertinence of this suggestion was strengthened by the observation that the extent of cloudiness about the planted tissue bore a direct relation- ship to the size of the tissue fragment explanted. The problem then resolved itself into discovering what food material might be deficient in the culture media. It ap- peared likely that it was some nitrogenous foodstuff. Vari- ous sugars and salts had been tested with practically no indication of variations in the results. Nitrogenous food had been supplied to the media from two sources: the infused liver and peptone; but it appeared probable that the nitrogen from these sources was not in a form available for mitochondrial utilization. This naturally led to an analysis of the possible sources of nitrogen for mitochondria in the living cell. Obviously, nitrogen compounds in the living cell have two sources of origin: those brought to the cell in the form of amino acids by the blood, and the waste products of nitrogenous metabolism, chiefly in the form of urea. On the basis of these speculations, a new series of culture media was devised in which various amino acids and urea were used. ‘These chemicals were added to the rabbit liver infusion medium in varying dilutions. The preliminary experiments consisted in testing the various amino-acid and urea media. Of the amino acids, only tryptophane appeared to satisfy the conditions neces- sary for free surface growth of mitochondria. It is possible that certain combinations of other amino acids might have supported growth; a few were tested with negative results. The urea medium also satisfied the conditions for inde- pendent mitochondrial growth. In general, it appeared that growth was more luxurient in the urea media than in that containing tryptophane. Attention was again turned to the deep clouded medium THE BACTERIAL NATURE OF MITOCHONDRIA 35 of the earlier experiments. A number of specimens were subcultured into the new media. In a large number of the subculture experiments, the coccoid bodies of the clouded media developed definite surface growths. Deep clouded growths that were several weeks old did not develop into surface growths so readily as did younger cultures. These results in subculturing the deep clouded growths removed the last vestige of doubt concerning the bacterial nature of the coccoid bodies. There is no occasion to confuse the coccoid bodies ob- served in these experiments with the ‘‘artificial bacteria’’ produced by Fischer (’99), Loéwschin (713) and others. Precipitation of colloids may be produced in solutions by various methods, the precipitate assuming various forms and characteristics. Some of these may have a morphologi- cal resemblance to the coccoid bodies and to bacteria. Evidence is lacking, however, that the precipitates can be subcultured upon the surface of solid media and caused to multiply, properties shown to be possessed by the coccoid bodies and known to be characteristic of most bacteria. A large number of culture experiments were then made, in which liver tissues from fetal, new-born and adult animals were planted in the tryptophane and urea media. When tissues from the fetus and new-born were used, surface growths developed in practically all cases with either media. In a few experiments in which adult tissue was used surface growth appeared in nearly all of the tryptophane media tubes, but in none of those containing urea. The morphologic nature of the organisms that developed in the culture experiments was puzzling. The constancy with which surface growths appeared, and the precautions used in the experiments appeared to rule out contamination, nevertheless, the morphology of the organisms seemed to point to it. The mitochondria of the liver cells are mostly globular bodies. A few ovoid forms are usually seen in the ~ 36 SYMBIONTICISM AND THE ORIGIN OF SPECIES cell, and occasionally a distinct rod-shaped body may be observed. While many of the surface growths in the culture experiments were pure cultures of cocci, the ma- jority, perhaps, consisted of diplococci, clumped or agglu- tinated cocci, and a type of coccus that I have called “‘ring- forms.”’ In a few instances, rod-forms predominated in the surface growths. Subcultures of these organisms fur- nished a clue to the interpretation of this variation in mor- phology. In some instances, where the initial growth con- sisted of a pure culture of cocci, the subcultures were found to contain all the varieties of forms mentioned above (see plate III, facing p. 51). An extensive study of this pleo- morphism was made, and it became evident that slight variations in the culture medium were sufficient to bring about remarkable variations in the morphology of the microérganisms. These experiments will be considered more fully in the next chapter. It was obvious that the results obtained so far did not constitute conclusive evidence that mitochondria are bac- terial in nature. There was, after all, no direct evidence that the organisms grown in the culture media had origi- nated from the liver cells. It was also possible that the tissues used for planting might contain congenital con- taminations or infections. This possibility did not appear likely, for, when the normal unincubated control tissue (a part of the same liver used for planting) was minutely examined there was no indication of any organism other than the mitochondria. It again became necessary to seek for some method that would conclusively demonstrate the source of the variable organisms in the cultures. A new set of experiments was performed in which liver tissue was planted in the same kind of media previously used, but the tissue was permitted to incubate for only a short time (from two to twenty-four hours in the different tubes), when it was removed and treated with a mito- THE BACTERIAL NATURE OF MITOCHONDRIA on chondrial fixative. When these incubated tissues were examined microscopically, it was found that the mito- chondria had changed in morphology within the cell. ‘The pleomorphism here observed was identical with that noted in the case of mitochondria grown on the surface of the media in the previous experiments. Figures 1 to 12 on plate II are camera lucida drawings of some of the organisms that developed on the surface of the cultures. Figure 13 is a camera lucida drawing of a piece of normal unincubated liver tissue from a rabbit showing the normal appearance of the mitochondria. Figures 14 to 18 are similar drawings of liver tissue from the same rabbit as shown in figure 13, after being incubated in urea-liver media at 37.5°C. for two, four, six, eight and six hours, respectively. It is evident, after an examination of these figures, that the mitochondria changed in morphology when the tissue was incubated. It is also evident that the organisms that had developed on the surface of the media are identical in morphology with the pleomorphic mitochondria within the incubated liver cells. The vagaries of contamination in bacterial work are a veritable ‘“‘bug-a-boo” to the investigator. The bacteri- ologist has learned that error from contamination often leads him astray in the search for truth. Sometimes he traces the source of his contamination to the glassware used, and which he had sterilized in the usual manner. In other cases, the contamination, apparently, crept into the media during the manipulations of planting. It seems to. me, that error from contamination is ruled out in this last set of experiments. I have attempted to find some expla- nation other than a mitochondrial origin for these organ- isms. The tissues during explantation might possibly become contaminated, but it is impossible to conceive of such contaminating organisms penetrating to the interior of cells in the deeper part of the tissue and multiplying to 38 SYMBIONTICISM AND THE ORIGIN OF SPECIES such numbers in so short a time. Again the constancy of the organisms in different experiments is against the prob- ability of contamination from the air. These facts, appar- ently, admit of no other interpretation than that mitochondria are living organisms, symbiotically combined with the cells of plants and animals. PLATE II All illustrations are camera lucida drawings made at the same magnification. Fia. 1. Organisms developed on surface of culture medium when new- born rabbit liver was planted in a rabbit liver medium (D), pH 6.6. Fie. 2. Organisms grown from new-born rabbit liver in rabbit liver medium (D), pH 7.4. Fig. 3. Organisms grown from new-born rabbit liver in rabbit liver medium (D), pH 8.2. Fig. 4. Organisms grown from new-born rabbit liver in rabbit liver urea medium (D), pH 7.4. Fia. 5. Subculture of organisms of figure 2 into a different rabbit liver medium (C), pH 6.0. Fic. 6. Organisms grown from new-born rabbit fat in nutrient agar tryptophane medium, pH 7.5. Fie. 7. Subculture of clouded medium into rabbit liver urea medium (D), pH 8.2. Fig. 8. Subculture of clouded medium into nutrient agar tryptophane medium, pH 7.5. Fria. 9. Subculture of clouded medium into nutrient agar urea medium, pH 6.5. Fria. 10. Subculture of clouded medium into rabbit liver medium (F), pH 7.5. Fig. 11. Subculture of an upgrowth into nutrient agar, pH 6.5. Fig. 12. Subculture of an upgrowth into rabbit liver medium (F), pH 7.93 Fria. 13. Normal liver of a three-week-old rabbit (K*). Fra. 14. Liver of rabbit K* after 2 hours incubation in rabbit liver urea medium (F), pH 7.5. Fra. 15. Liver of rabbit K* after 4 hours incubation in rabbit liver urea medium (F), pH 7.5. Fia. 16. Liver of rabbit K* after 6 hours incubation in rabbit liver urea medium (F), pH 7.5. Fia. 17. Liver of rabbit K* after 8 hours incubation in rabbit liver urea medium (F), pH 7.5. Fig. 18. Liver of rabbit Sc. after 6 hours incubation in rabbit liver urea medium (I), pH 7.5. II PLATE rt} a THE BACTERIAL NATURE OF MITOCHONDRIA 39 The major portion of these culture experiments have been made with liver tissue of fetal and new-born rabbits. Other tissues of fetal and new-born rabbits, guinea pigs, dogs, and cats have also been tested. Growths similar to those described have been obtained from fat, kidney and suprarenal of the above named animals. ‘There is no reason to believe that the mitochondria of the liver are funda- mentally different from the mitochondria of other cells, and that they alone are bacterial in nature. The question has been raised, and it deserves consideration here: What grounds are there for calling mitochondria “bacterial in nature’’ or bacteria; why are they not to be considered merely a part of the cytoplasm which has properties of self-perpetuation and independence under proper artificial conditions? ‘This is a pertinent question, and it strikes at the root of the theory that I have advanced in explanation of the fundamental significance of mito- chondria. The complete answer to the question can not be given in a few words, for in the attempt to answer this question one must deal with indefinable matter. In the first place, if we seek a definition of bacteria, we are unable to find an adequate one in the text-books on bacteriology. Indeed, we have a mental conception of a type of organism when we use the word “bacterium.” Morphologically, we differentiate bacteria from unicellular plants and animals by the absence of an organized nucleus. This does not constitute a definition of bacteria, however, nor would a catalogue of their physiological and morphological attri- butes constitute such a definition. A definition would have to go farther and include a statement of what they are in relation to the earth and the life that it contains. There has been, in recent years, a growing conception of what bacteria really are. The suggestion that they are primordial organ- isms indicates such a trend. Again, the difficulty of dis- tinguishing between some of the lowly, supposedly, pro- 40 SYMBIONTICISM AND THE ORIGIN OF SPECIES tozoan microdrganisms and the bacteria, led Haeckel to propose the term “‘protista’’ to include a lowest order of living organisms, a distinct kingdom of living things. In this connection too, the suggestion has been made that the “bacteria” are the primordial organisms from which all higher forms of life have sprung. (So far as I know, no one has ever attempted to expound a concrete theory of the nature of this early evolution. The evidence which will be presented in a later chapter of this book, establishes a basis for a better understanding of the nature of such an evolu- tionary process.) The second factor that complicates the question of the evidence or basis for calling mitochondria bacteria, con- cerns our knowledge of protoplasm and particularly cyto- plasm. The nucleus has long been looked upon as the con- trolling factor in the cell. The center of vital activity is supposedly located in the nuclear portion. Bits of cyto- plasm removed from the cell do not continue to grow and function as an independent entity. The remarkable anal- yses of chromosomes in relation to heredity, that have re- cently been made by students of genetics would appear to exclude any possible independent participation of cyto- plasmic bodies. On the other hand, Conklin and Wilson believe that the cytoplasm participates in hereditary trans- mission. It might be argued that parts of the cytoplasm should be capable of independent existence. Such argu- ments might proceed along various lines. It might be assumed that the cytoplasm as such (not the formed bodies that it contains) may have such properties of self perpetu- ation. Or it might be assumed that formed bodies of the cell having these properties are a part of the cytoplasm. This raises a subtle question which must be left for the future to determine. Mitochondria, apparently, represent the end result of the development of an absolute micro- symbiosis. What is the nature of the intimate relationship THE BACTERIAL NATURE OF MITOCHONDRIA 41 of mitochondria to the cell? Do the mitochondria become a part of the cell cytoplasm, or do they retain their indi- viduality? The fact that they may be induced to exist independently of the cell would appear to indicate that they do retain their individuality, but this does not neces- sarily indicate an absolute independence. Again, the observations of Chambers in which he apparently saw mitochondria disappear by mixing with the cytoplasm, has a bearing on the question. It will require special investigations to determine the intimate physical relation- ship of mitochondria to the cytoplasm. The possible reactions of microsymbionts in the cytoplasm of the host cell is discussed in later chapters. The evidence for calling mitochondria bacteria, rests upon the following attributes: Their general behaviour in the cell is similar to that of known microérganisms which live symbiotically in the cells of higher organisms; for example, the root-nodule bacteria of legumes. When grown inde- pendently in artificial culture media, they behave in all observed particulars like bacteria. They divide like bac- teria. They are similar to bacteria in structure and shape. They exhibit no cultural characteristics foreign to bacteria. CHAPTER IV THe BEHAVIOR OF MrrocHONDRIA One of the more characteristic attributes of mitochondria that may be gathered from a perusal of the literature is their property of changing form or pleomorphism. As early as 1897, Benda showed that the mitochondria in spermato- genesis transform into the spiral filament of the sperma- tozoon. Later investigators have traced the ‘‘transfor- mation”’ or differentiation of mitochondria of various tissue cells into a large variety of end-products (myofibrils, con- nective tissue fibrils, secretion droplets, prosecretions, pig- ment granules, plastids, etc.). Many of these so-called transformations have been questioned on the basis of an assumed phospholipin-albumin constitution of mitochondria, which also presupposed a “‘passive” nature of these bodies. The relationship of mitochondria to secretory processes appears to have partially weathered the storm of criticism, and is still accepted by a large number of investigators. Altmann was the first to find that the granules (mito- chondria) are related to secretory processes in the wax glands of the ear and the submaxillary gland. Regaud and Mawas (’09) confirmed Altmann’s work on these glands, and further showed that the granular or coccoid mito- chondria arise from the bacillary or filamentous forms by a process of fragmentation. O. Schultze (11) investigated the problem and concluded that the secretory granules originate from mitochondria. Debeyre (’12) investigated the submaxillary gland of the rabbit, and with the aid of vital stains (Janus green and neutral red) he was able to observe the transformation stages of mitochondria into secretion granules. Guieysse-Pelissier (’11) examined the 42 THE BEHAVIOR OF MITOCHONDRIA 43 submaxillary gland of the mouse, and was of the opinion that the secretory granules originate from the mitochondria. Levi (’12), on the other hand, advanced the idea that mito- chondria are permanent cell-organs, and while they may be involved in the secretory process they do not transform as such into secretion granules. In a series of papers, Moulon (10a, ’10b) has described the transformation of mitochondria into secretion in some of the cells of the suprarenal gland. The rod-shaped mitochondria with blebs become globular, later agglutinated masses, and finally the entire cell is filled with the mito- chondrial product which he calls a “prosecretion.” Takagi (’22) described a similar chain of events of mitochondria in the thyroid gland of the dog. Ma (’25) concluded that mitochondria are concerned in thyroid secretion in the albino rat. Key (25), however, does not believe that mitochondria are responsible for the specific activity of the cells that contain them. Cowdry’s general discussion of the relationship of mito- chondria to secretion may be considered as reflecting the opinion of those who are against the idea that mitochondria are concerned in secretion. He says: “‘I incline strongly toward Regaud’s eclectosome theory (1919a, p. 919), ac- cording to which mitochondria play the part of plasts choosing and selecting substances from the surrounding cytoplasm, condensing them and transforming them in their interior into infinitely diverse products; but I would venture to emphasize the fact that in all this the mitochon- dria may be acting in an entirely passive manner as a vehicle, taking up materials by virtue of their phospholipin constitution, or on account of physical forces acting on their surfaces, or for other reasons, and that the optically homogeneous ground-substance of the cytoplasm may be the active and essential agent in this as in so many other vital manifestations.” It is difficult to understand why 44 SYMBIONTICISM AND THE ORIGIN OF SPECIES one should assume that mitochondria are ‘‘passive’’ bodies, if one accepts the statement that they have selective and secretory properties. These properties of selecting sub- stances from the environment and elaborating “infinitely diverse products” is one of the outstanding characteristics of bacteria and all living matter. In discussing the mechanism of secretion it becomes necessary to consider briefly, ideas recently advanced on the functional significance of the Golgi apparatus. This cell structure was first demonstrated in gland cells by Negri (99). Numerous investigations have been con- ducted upon the Golgi apparatus, but until recently no acceptable idea as to its function has been advanced. Nassonov (’23) after painstaking investigations arrived at significant conclusions regarding its activities. He believes that secretion is intimately connected with the Golgi ap- paratus and has offered various suggestions regarding the probable nature of the mechanism through which it acts. More recently Bowen (’24) has presented other evidence in support of Nassonov’s conclusions. Bowen quotes oppos- ing views of investigators as to the réle played by mito- chondria in secretion, and states: “In view therefore of the conflicting statements of fact and the known sources of error, it appears probable that the origin of secretory granules from mitochondria, ergastoplasm, and related cytoplasmic elements must be looked upon as unproved and of doubtful utility as a working hypothesis.”’ While space will not permit an extensive discussion of these apparently conflicting views on secretion, it is perti- nent to direct attention to other investigations which appear to offer a solution of the difficulties. ‘Two papers in partic- ular were published in 1925 which are significant in this connection. Hirschler (’25) emphasizes the fact that mito- chondria and the Golgi apparatus are similar in staining reactions. Karpova (’25) concludes from observations on THE BEHAVIOR OF MITOCHONDRIA 45 living as well as fixed material, that there is nothing to indicate a fundamental difference between the two struc- tures. While she admits that she has not observed mito- chondria transforming into dictosomes (Golgi bodies), nor does the literature refer to any such observations, she maintains that the apparent identity in chemical nature points to a similarity in origin. It would be premature to draw definite conclusions as to the relationship of these two groups of cell structures on the evidence at hand. However, it is proper to suggest the probability that mitochondria and the Golgi bodies are at least related structures, and that in some instances secretion may be derived directly from the mitochondria, while in other cases the mitochondrial bodies first transform into a reticulated apparatus before secretory activities begin. Evidence has been presented by the author (Wallin, ’24) that the mitochondria in a single cell are not all alike. When using the Giemsa stain, some mitochondria stain blue while others are stained red. Karpova emphasizes the fact that there is a greater difference between the mito- chondria and the chloroplasts derived from them than there is between mitochondria and dictosomes. In this connec- tion it should be further emphasized that there is no good reason for assuming that the secretory mechanism is the same in all kinds of cells. On the contrary, it is well known that there are gross variations in the behavior of the cells of different secreting glands. Does this not at least allow for variation also in the internal secretory mechanism. Ludford (’25a) discusses the réle of mitochondria and the Golgi apparatus during secretion in the epididymis, and says: “At the onset of secretion the mitochondria increase in numbers, and there is a diminution after secretory ac- tivity, so that they contribute to the production of formed bodies seems practically certain, but the exact nature of their contribution is a matter for theoretical discussion 46 SYMBIONTICISM AND THE ORIGIN OF SPECIES rather than practical demonstration.’”’ In another investi- gation on the sebaceous gland cells, Ludford (’25b) says: ““Mitochoncria do not appear to be directly concerned in the formation of the secretion.” From an analysis of the literature on secretion, it is evident that the internal mechanism of secretion varies in different glands and in different animals. Investigations have demonstrated that in some glands the mitochondria are concerned in secretion. The recent experimental in- vestigations of Takagi (’25) in which he used various means to stimulate secretory activity in the submaxillary gland of the cat demonstrate conclusively that mitochondria play a role in submaxillary secretion. Cramer and Ludford (26) demonstrated a relationship of mitochondria to the functional state of the thyroid gland. Horning (’26) found mitochondria in the food vacuoles of paramoecium, indi- cating a digestive function. The manner in which mito- chondria enter into secretory activity appears to vary in different kinds of cells. In some cases the mitochondria appear to transform directly into the secretion; in other mstances the secretion appears to be related to the mito- chondria, but not in the nature of a transformation. In still other cases the Golgi apparatus appears to be directly concerned in the secretory process. ‘The question that presses for solution in connection with the mechanism of secretion is: What is the relationship between mitochondria and the Golgi bodies? While some investigators maintain that the Golgi bodies are chemically related to mitochondria, others hold that they are distinct and of a different nature. The basis for these opinions rests on the staining reactions of the two structures. It is pertinent to emphasize the fallacy of attributing specificity to staining reactions. It is conceded that slight variations in staining reactions can be detected between mitochondria and the Golgi apparatus; but what THE BEHAVIOR OF MITOCHONDRIA 47 significance should be attached to these slight variations? It has been shown that mitochondria vary in their staining reactions. So far as the researches on these two groups of structures have been carried, there is no conclusive evidence to show that mitochondria and the Golgi bodies may not be related structures. When one contemplates the modi- fications that mitochondria undergo in the formation of the nebenkern, a transformation of mitochondria into a net- like structure, or a laminated structure, appears to be a simple process. Other variations in the behavior of mitochondria have been recorded in the literature. It has been observed by a number of investigators, and I believe it is generally ac- cepted, that the amount of mitochondria varies with the age of the cell. Young growing cells contain a larger number of mitochondria than those that are old and senile. It has also been observed by Romeis (’13), Torraca (14a, ’14b), and others that the mitochondria increase in numbers in regenerating tissues. Of particular interest are the modifications attributed to mitochondria in abnormal or pathological tissues. The researches in this field have been fairly numerous, but the results have not led to any partic- ular fundamental conception of the nature of mitochondria. Observations on the mitochondria in pathological tissues have indicated, in some cases, an alteration in the number of mitochondria, and in other instances, pleomorphic modi- fications have been mentioned. The observation of Goetsch (16), are particularly significant, in as much as he correlates the number of mitochondria present in the thyroid with various diseased conditions of the gland. Goetsch found a profound reduction of mitochondria in colloid goiter. In cases of adenoma with hyperthyroidism and exophthalmic goiter he found an increased number of mitochondria. Quite recently, Seecof (’25), has found definite mitochondrial changes in the thyroid running parallel with experimental 48 SYMBIONTICISM AND THE ORIGIN OF SPECIES hyperplasia and hypoplasia of the gland. Other patho- logical tissues have been investigated by various authors, but the results, apparently, have not been so definite. Experimental modifications of mitochondria have been induced by various methods. Ciaccio in 1913 observed the changes in the mitochondria of various tissues after phos- phorus poisoning. The following year Mayer, Rathery and Schaeffer investigated the effects of phosphorus poison- ing on the mitochondria of the liver. They found pleo- morphic modifications of the mitochondria as well as destruction of these bodies. In 1916, W. J. M. Scott investigated the effects of experimental phosphorous poison- ing on the mitochondria of the pancreas in white mice. Scott’s results are particularly interesting when compared with the behavior of mitochondria in culture media. He found that the first change observable in the mitochondria in mild poisoning is a loss of the bleb-like swellings normally present in pancreas mitochondria and a thickening and shortening of these bodies. When the poisoning is carried further, the mitochondria clump together like agglutinated bacilli. In more severe cases the agglutinated forms appear to fuse and completely lose their identity. In some of the stages described by Scott, he found globular bodies which appeared to contain a vacuole. In some instances, only a peripheral ring stained with the dye used, the center appear- ing clear. These forms, apparently, are identical with the pleomorphic mitochondria, to which I have previously referred as “ring forms.”’ Nicholson, in 1924, used various means to modify experi- mentally the mitochondria of the thyroid. The blood supply to the gland was interfered with in one set of experi- ments. After three days the mitochondria which, nor- mally, are filamentous were shorter and more granular. After the tenth day they had become spherules. In another set of experiments, about three-fourths of the THE BEHAVIOR OF MITOCHONDRIA 49 gland was extirpated, and the effects on the mitochondria noted in the remaining portion of the gland which pre- sumably was called upon to increase its activity. The mitochondria were examined at various periods after the operations. It was found that they had not altered in morphology, but had increased considerably in numbers. Nicholson also tested the effects of various gases and chemi- cals. In some instances the mitochondria exhibited pleo- morphism. The investigations of Bowen (’19-’22) and others, on the behavior of mitochondria in spermatogenesis have brought to light some interesting activities of the mitochondria of germ cells. It has been shown that the mitochondria in the male germ cells of certain insects come together and fuse into a single body, the nebenkern. This body is not homogeneous in structure, but is composed of at least two distinct materials which Bowen was able to distinguish by means of staining reactions. In later spermatogenetic activity, the nebenkern becomes peculiarly laminated. - These activities, that have been so clearly described and illustrated by Bowen, appear, on first analysis, to be quite unrelated to any comparable process in the bacteria. Lohnis, in his monograph on the ‘‘Life Cycles of Bacteria,”’ however, has described and illustrated processes that are identical with the early changes described by Bowen. According to the conclusions of Léhnis which are based not only upon his own observations, but also correlated with those of many other investigators, bacteria possess a life cycle. One step in the process consists of the fusion of a number of bacteria into an amorphous mass which Léhnis has named the “symplasm.” Later, individual bacteria become separated from this mass or symplasm, and again become independent organisms. While many bacteri- ologists have not accepted this work of Lohnis, I know of no definite evidence against it. 50 SYMBIONTICISM AND THE ORIGIN OF SPECIES J. Terrill Scott (’26), working in this laboratory, has studied the responses of mitochondria to variations in the hydrogen-ion concentration. When liver tissues were in- cubated in physiological saline solutions of different pH values for short periods of time, there was a variation in the behavior of the mitochondria. He also found that the staining reaction of the incubated mitochondria varied under these conditions. Jessen (’25), produced degen- eration of mitochondria in kidney cells by the injection of bacteria, diphtheria toxin and distilled water. In the experiments described in the preceding chapter, it was noted that the cultured mitochondria did not always have the same morphology as those in the normal liver tissue. This peculiarity was noticed in the first surface growths that were obtained. A large series of subculture experiments were made from the initial growths in order to study more minutely the pleomorphic possibilities of the cultured mitochondria. The first subculture experiments were made with a coccus which developed in a rabbit liver medium eight days after it had been planted with liver from a new-born rabbit. When the organisms were subcultured into beef heart media of various hydrogen-ion concentrations from pH 7.1 to pH 7.5, the growths in these media varied in size according to the pH value of the medium. It was possible to trans- plant any of these subcultured organisms into a medium with a particular pH value and obtain organisms of a uni- form size. After these cultures had been kept a little more than a year in cold storage, they were found to have lost their specific response to the hydrogen-ion concentration of the medium. The size would vary in media of different pH values, but the variations were erratic in character. A large number of subcultures were made from the initial growths that developed in the urea and tryptophane media. The highly specific response obtained in the earlier subcul- PLATE III Feb. 23, 1924 2733 O'-21, Rabbit fat planted in nut. agar pH 7.5—+4+ ihe 988g ts | Subcultures ‘ March it toctie March 12,'24 April 1,'24 | O’- 1. Ote21 int bbit liver ¢ H 5.2 @. 2.2%. [G7 as ov-aa tate sabbat iver © ae S.2l+] QU QPeh | ot test | ‘é) me Se " " " " " H 5.6 Sa *e e e ¥ a : o% ! " ” ” " wenn es i oe rise + STA QOgee ! eee” " = d'- 8. PE 5.0 t "e fem's%, m09tO “gg | ow. 6 ” " " " " peE6.2t+ & hes es ms, @., y °s e om 5 7 e¢ a“ co’. 7. " ” " ” o pH 644 a = os he “ ” " " “ pH 6.8 ; re 9, ” ” ” " n pH 7.0 : 6-10. “ " " ” " pH 7.2 t a + r?) -li. " ” ”“ ” " pz 7.6 t ie ¢'-12. rs i ¥ . “pH 7.8 t ¢. oo -Si0 Ses30 “g: ! 2 " « e . + e * : —- i ee a tae, MGT cadch ae t 2 Mea a. a oof ca eee o’-14, n n " “ “pH 8.4 ; co -i6: " w ” " " pH 8.6 : } oe > ee cia " # ” " ” pH 8.8 t cf ly (me ee " u = ye 0.0 ¢ to 18, 2 “ * ‘urea pH 7.0; + e-aige =.) " " ” tryp. pH 7.0 + ; March 12, 1924, March 13,124 | 0-20. O'-21 into nutrient agar pH 6.5 t) _0 -2l. ” " P ’ " " } pH 7 «0 - O's 22 . “ " " ” pH 7 . 5 + March 13, eg March 14,'24 April 1, '24 * = 232: Ca 23 oN dh into nutrient agar pH 6.5 + ale : ue ” “ “ * pH 7.0 +4 eri 3 san L o -25 “ ” ei a: " pH 7.5. + ee thet Pye as ov. 26 of 19 ” “ " ae 4 ae eau vag ee ay Ooi e. * urea ae ARS ne. 5 *e 2 “ tryp. pm 7.5, + fel echt PROTOCOL OF SUBCULTURE EXPERIMENT Original upgrowth (0'-21) developed after four days incubation. Char- acter of organisms shown by means of camera lucida drawings at right. THE BEHAVIOR OF MITOCHONDRIA 51 ture experiments, however, were not again observed, that is, the production of a definite size of organism in a given hydrogen-ion concentration of the medium. The results that were obtained in the later subculture experiments, however, appear to be more significant in relation to the known behavior of mitechondria as indicated by the experi- ments of the investigators quoted above. Plate III is a reproduction of the protocol of a subculture experiment. The character of the organisms that developed in each subculture is shown by means of the camera lucida drawing at the right. The initial growth from which these subcultures were made consisted of a pure culture of cocci, represented in the upper part of the plate. The original smear of the initial growth was carefully searched, but the only type of organisms that could be seen were single cocci. A study of this protocol reveals the peculiar and delicate pleomorphic response of cultured mitochondria to slight variations in the culture media. Another feature of their behavior is represented by the drawings illustrating con- ditions observed April 1, 1924. The first vertical column of drawings was made from smears obtained twenty-four hours after subculturing. The second vertical column represents the same subcultures after incubating for twenty days. In many instances, it is evident that the organisms underwent a second pleomorphism during the ageing of the medium. In another set of experiments, the hydrogen-ion concentration of the medium was determined after the organisms had shown a similar second pleomorphism, and it was found that it had changed from an original pH 7.5 to pH 8.8. The subcultures illustrated at the bottom of the plate under date of March 13, 1924, also show an interesting reaction. The subcultured organisms of 02-5, which were almost wholly diplococci, were again transplanted after two days into the original kind of medium—nutrient agar. The 52 SYMBIONTICISM AND THE ORIGIN OF SPECIES organisms that developed in the second subcultures (02— 23—02"-25) were single cocci, more or less similar to the original organism. Smears of these growths eighteen days after transplanting also showed secondary pleomorphism as a result of the ageing of the medium. In this instance, the organisms decreased considerably in size. So also when the organisms of 02-19 were subcultured into the original kind of medium (nutrient agar) the growth con- sisted of single cocci. A large number of subculture experi- ments of this type were made with similar results. It does not appear that this change in morphology was related to the nutritive properties of the substratum because the gross indications of multiplication were identical in all cases. The subculture experiments demonstrate the extremely sensitive and pleomorphic character of mitochondria. ‘The responses were not only obtained in media of various hydro- gen-ion concentrations, but in media with different com- position. ‘These results introduce a new factor in the inter- pretation of the results obtained in the experiments of Mayer, Rathery and Schaeffer, Scott, Nicholson and others. This does not invalidate the interpretations that have been made, namely, that mitochondria are perhaps the first structure in the cell to show a response to abnormal con- ditions, but the fact that mitochondria are living organisms gives to these findings a new significance. The reality of the bacterial nature of mitochondria intro- duces a new factor in cytological inquiry which gives promise of rich rewards. ‘The possible relationship of mitochondria to cell functioning has been indicated in numerous re- searches. These indicated activities have been questioned on the basis of an incompatibility with an asswmed “passive” nature of mitochondria. The living, bacterial nature of mitochondria introduces a new and firm basis for, at least, a provisional acceptance of the various activities that have been attributed to mitochondria. Obviously, further in- THE BEHAVIOR OF MITOCHONDRIA Do vestigations in this field, and from the newer point of view, are necessary to establish the full significance of mito- chondria in cell activity. The possible relation of mitochondria to the etiology of disease is immediately indicated by their nature. The researches that have been made in pathology in this con- nection certainly give promise of ultimate fundamental discoveries. ‘The earlier researches on mitochondria in this field were abandoned when Altmann’s “bioblasts”’ were rejected. ‘The reéstablishment of the fundamental part of Altmann’s conception—namely, the bacterial nature of mitochondria, should again serve to stimulate investi- gations on the relationship of these ever-present micro- organisms to disease. Another feature of the possible behavior of mitochondria in their relationship to the cell may be discussed in a hypo- thetical way. It appears obvious that there must be a mechanism in the cell that controls mitochondrial growth. Mitochondria, being organisms, would have a tendency to grow indefinitely in a cytoplasmic medium, which is being constantly supplied with proper nutriment. When patho- genic microdrganisms invade the cell, various types of responses may be observed. In some cases, the pathogenic organism is of such a nature that it rapidly destroys the invaded cell. In some instances, however, the invading organism is not especially toxic in its reactions, but grows rapidly resulting in the mechanical rupture of the cell. The organism of Rocky Mountain spotted fever produces such a result in the endothelium and muscle cells of blood vessels. The resulting haemorrhagic conditions are responsible for the “‘spotted’”’ feature of the disease. The bacteria (Bacillus radicicola) in the root nodules of legumes, on the other hand, have the same type of rela- tionship to the nodule cells that mitochondria have to cells in general. When the Bacillus radicicola has entered the 54 SYMBIONTICISM AND THE ORIGIN OF SPECIES root hairs of the legume, certain cells are stimulated to growth and modification into nodule cells. Growth of these cells continues up to a certain point, when not only the cell ceases to grow, but bacterial growth also ceases. When the cells have reached this stage, they are no longer capable of resisting other microérganisms. When one examines an old nodule, microscopically, one usually finds the cells loaded with a great variety of microérganisms, and very few if any Bacillus radicicola. It appears evident that the root nodule cells control the growth of the contained bacteria. It further appears probable that the microérganisms in turn exercise an influence upon cell reproduction or division. Eventually, in the growth of the tissue, a state of equilib- rium is attained in which the cell and its symbiotic organ- isms come to a state of rest. Obviously, all living tissues may not develop the same kind of growth mechanism. Many cells in the body stop growing at a certain period in adult life, others, the epithelial cells of the skin, for example, continue to reproduce so long as life is maintained. The complex changes that chromatin material undergoes in mitosis or cell division, does not appear, on first analysis, to be dependent on influences emanating from the cyto- plasm. Obviously, this remarkable series of progressive changes in the chromatin must be dependent upon some stimulus, extrinsic to its own substance. It is conceivable that mitochondria produce chemical substances, or perhaps, physical influences which ultimately become sufficiently strong to inaugurate mitosis. The observations that mito- chondria are more numerous in young, growing cells than in old or senile cells, appear to support the hypothesis that mitochondria may be the controlling influence in cell di- vision. However, it would be difficult at present to attempt to definitely correlate the number of mitochondria with cell division. While many observations have been made in which mitochondria appear to be more numerous in young, THE BEHAVIOR OF MITOCHONDRIA fay growing cells than in mature or senile cells, other obser- vations have been made in which there appears to be no such relationship. The observations of Sokoloff (25) on neoplastic tissue, also, appear to indicate that the number of mitochondria is unrelated to cell division. Under abnormal conditions, it is also conceivable that chemical or physical influences may modify the cell in such a manner that the equilibrium is upset, or in other words, the mechanism controlling mitochondrial growth is de- stroyed. In such an event, the mitochondria would grow and increase so long as food materials are present. Their influence on cell division would, perhaps, be retained, re- sulting in a continuous growth of cells. There is a certain amount of evidence that points directly to the probability of the presence of such a growth mechan- ism in the cell. Long ago, Engler (’82), Winkler (99), Ernst (’02), Wigand (’84), and others, observed an up- growth of “bacteria” in intact, but dying algal cells. Some of these observations were made before mitochondria had been discovered. It is quite possible that these ‘‘bacteria”’ were the mitochondria of the normal algal cell which began to grow when vitality ceased in the host cell. ‘The author has obtained similar results in the mitochondrial culture experiments represented in plate II (p. 38). The tissue represented in figure 14 is a small bit of liver tissue that had been incubated only two hours. A comparison of this figure with figure 13, which is the normal unincubated tissue from the same animal, reveals at once a decided increase in the number of mitochondria during two hours incubation. It is hardly probable that this increase in mitochondria was dependent upon the food materials present in the medium, but rather that the growth resulted from the food present in the cell which became available as the cell died. While the piece of liver tissue that was incubated was not more than 6 to 10 mm. in diameter, it does not appear probable 56 SYMBIONTICISM AND THE ORIGIN OF SPECIES that the food materials of the medium penetrated to the interior of cells in the deeper part of the block in two hours time in sufficient quantity to explain the degree of mito- chondrial growth that took place. It should be mentioned that the medium had the consistency of a rather firm jelly. Another property of mitochondria has been revealed by the culture experiments. This concerns the utilization of urea by the mitochondria of the fetal rabbit liver. The fact that mitochondria may utilize urea suggests a possible mechanism for destruction of urea in the fetus. J. Whit- ridge Williams (717), investigated urea elimination in pregnancy, and found that it did not deviate appreciably from the normal. E. C. P. Williams (’23), Hellmuth (’23), and others, have investigated the blood urea during preg- nancy, and have found a reduction below the normal. This would appear to indicate, in conjunction with the urea culture experiments, that the mitochondria of the fetus not only utilize the urea produced in the fetal metabolism, but may even extract it from the mother’s blood. If it should be found that mitochondria are the stimulating influence in cell division, as it appears possible, then we, apparently, have a tentative explanation for the rapid growth of the fetus. The mitochondria have a special predelection for urea and grow rapidly in its presence, the cells in which they occur undergo frequent divisions with a consequent rapid enlargement of the fetus as a whole. If this property of mitochondria proves to be true in the fetus, then they must change in regard to the character of the food utilized in the adult. That such a change of diet actually occurs has been indicated by the culture experiments. Dr. K. 8. Chouke, working in this laboratory, is investi- gating the development of the capacity for urea elimination from the kidneys in rabbits. His experiments to date ap- pear to indicate that urea elimination first begins at birth and is gradually increased after birth. THE BEHAVIOR OF MITOCHONDRIA 57 The interest and significance attached to this hypothetical mechanism of cell division becomes more apparent when we realize the bearing it has on the analysis of such uncon- trolled growth as we find in neoplastic diseases. Obvi- ously, it will require experimentation and more extensive analysis to determine the reality and nature of this prob- able growth mechanism. While this hypothesis of the mechanism of cell division may appear to be highly specu- lative, the variation in mitochondrial content of young and senile cells, together with the above mentioned responses of mitochondria in dying cells, gives reasonable grounds for advancing the hypothesis. CHAPTER V SYMBIONTICISM When a biological phenomenon has been observed and the universality of its occurrence has been established, it is within the province of science to correlate it with other biological processes. In attempting to analyze natural processes and principles, it becomes apparent that our knowl- edge has not advanced sufficiently so that it is possible to make final analyses of natural phenomena, nor ascribe absolute causes to biological activities; nevertheless, our intellectual horizon is broadened if we can reduce a phe- nomenon to the category of a principle. The universal presence of microérganisms within the cells of all plants and animals, obviously, points to the consummation of a biologic process or principle as funda- mental in significance as that of reproduction. This principle has been named “‘Symbionticism” by the author (Wallin, ’23). Just as sexual reproduction is dependent upon other biological principles or factors (fertilization, cell division), so, also, Symbionticism is the end result of a more primitive factor or principle. This more primitive principle has been named ‘‘prototaxis”’ by the author and has been defined as ‘‘the innate tendency of one organism or cell to react in a definite manner to another organism or cell.”’ Prototaxis may be positive or negative. Negative prototaxis is the repulsion of one organism or cell by an- other. Negative prototaxis can lead to no permanent mani- festation or alteration in an organism and consequently it may not be readily detected. Positive prototaxis is the affinity of one organism or cell for another organism‘ or 58 SYMBIONTICISM 59 cell. This may result in various kinds of vital relationships. These relationships have long been known and recognized by the terms: conjugation, symplasm, cell fusion, parasi- tism, and symbiosis. Prototaxis, on first analysis, may appear to be identical and synonymous with ‘‘chemotaxis.”’ It is possible that chemotaxis is the essential force in prototaxis, but doubt- less other forces play a réle in the operation of the principle. It would be quite impossible to analyze and enumerate the factors that enter into prototaxis. It appears probable, however, that such forces as surface tension, light, tem- perature, moisture, and probably electrical potential are involved in the process. Still other modifying factors may be concerned in the operation of the principle. Our present purpose, however, is not an analysis of the fac- tors involved in prototaxis, but rather a record of its occurrence. The more familiar expressions of positive prototaxis are to be found in those life relationships that are known as parasitism and symbiosis. It is evident that these two vital relationships are closely akin. The conceptions that have developed in connection with these associations have been based more or less upon the injurious or beneficial results derived therefrom. These conceptions have also involved the idea of purpose. The term “parasitism” implies some benefit to the parasite with either a harmful or indifferent response on the part of the host. Symbiosis is applied to those cases where mutual benefit is derived. It is evident that there is no sharp distinction between parasitism and symbiosis. Considered from the point of view of prototaxis, parasitism and symbiosis are merely different end responses in the expression of one and the same biological principle. The following diagram may serve to clarify the relation- 60 SYMBIONTICISM AND THE ORIGIN OF SPECIES ships of the various end results in the expression of the principle of prototaxis: Temporary Conjugation Permanent Symplasm Incomplete Tntrseenolee oe (Symbion- Ashok [ ticism) Positive eymaioee (Incomplete Extracellular Absolute? Cell fusion Prototaxis (in tissues) Intracellular Parasitism Extracellular Negative Conjugation has been observed, chiefly, in the protozoa or one-celled animals. It consists of a temporary, in some cases permanent, union of two individuals of a species. In the paramecium, for example, two individuals come to- gether and remain attached for a short period of time. During this attachment certain internal changes occur, and an interchange of some materials of the two conjugat- ing individuals takes place. Conjugation in the bacteria has been described by Lohnis (’21) and others. On account of the small size and the mode of life of the bacteria, conjugation in these forms is not so readily detected as in the larger protozoa. Ldéhnis has submitted a number of photographic illustrations of bacteria in the state of con- jugation to support his claim that bacterial conjugation does occur. SYMBIONTICISM 61 “Symplasm” is a term proposed by Léhnis and Smith for another phase of bacterial behavior. It represents a stage in the various life-cycles of bacteria. Briefly, it may be described as a clumping together of a number of bacteria of a single strain in which the boundaries of the individual organisms are lost. The bacteria coalesce and resolve into an amorphous mass; this constitutes the symplasm. Later, in the completion of the life cycle, individual bacteria are reformed by a dissolution of the symplasm. Léhnis has collected a considerable amount of evidence from the litera- ture to support his own researches on this subject. Alm- quist (’22), and others, also, have observed the symplasm stage in the life cycles of certain bacteria. Cell fusion represents another type of expression of proto- taxis. The end result in this type of activity may be repre- sented by two kinds of cell relationships. In one case, cells appear to “flow together’ and produce a true syncytium. The author observed a type of fusion in the branchial epi- thelium of the lamprey larva in which the cell boundaries completely disappeared (Wallin, 718). The other type of cell relationship is found in certain tissues in which the cells have protoplasmic bridges extending from one to another. The interesting experiments of Galtsoff (’25) on ‘“Re- generation after dissociation” of the cells of the sponge, in which “‘the regeneration is primarily the process of coming together of cells, accompanied by their further differentiation,’’ demonstrates another phase of the same principle. In symbiosis two dissimilar organisms become intimately associated. This type of vital relationship was, apparently, first observed by Reinke in 1872, who named the phe- nomenon ‘‘consortism.”’ In 1879, De Bary observed and recorded the same phenomenon, and introduced the term “symbiosis,” which has since been adopted into common usage. 62 SYMBIONTICISM AND THE ORIGIN OF SPECIES Since the observations of Reinke and De Bary, the num- ber of recorded cases of symbiosis has steadily grown. In most cases of symbiosis early described the two symbionts were multicellular plants or animals. It is perhaps be- cause of this circumstance that symbiosis has been looked upon more or less as a digression in plant and animal behav- ior. That symbiosis may represent the end result of a biological principle appears to have escaped detection. The kind of symbiosis in which one symbiont is a micro- organism is not so well known. It is this type of symbiosis, which we designate “‘microsymbiosis,”’ that is of particular significance in the theory of Symbionticism. Various classifications of symbiosis have been devised by different authors. These have been based for the most part upon the physiological responses of multi-cellular symbionts. While such classifications may be valuable from one point of view, they are of little significance in connection with prototaxis. Based upon the morpholgic relationship of the symbionts, symbiosis may be divided into intracellular and extracellular types. Symbiosis may be incomplete or absolute (facultative or obligate) de- pending upon the degree of physiological dependence of the two symbionts. In incomplete symbiosis, the two symbionts are capable of more or less independent existence and reproduction. In absolute symbiosis, they are incapa- ble of independent existence and reproduction. In this grouping the author has particular reference to microsym- biosis. It might be well to use the term ‘‘macrosymbiosis” to designate that type of association in which the two symbionts are multicellular organisms. In macrosymbio- sis, the two symbionts are not so intimately associated, and appear to have little or no significance in the modifi- cation of species. The term ‘symbiosis’ is used in bacteriology in a differ- ent sense than in general biology. Certain bacteria, ap- SYMBIONTICISM 63 parently, grow only in the presence of certain other bacteria. One organism produces extrinsic conditions that are essential for the life of another. Their relation- ship is more in the nature of an indispensable association and has nothing to do with prototaxis. It is possible that bacteria may also play the réle of relative or host sym- biont. The D’Herelle phenomenon would allow of such a possibility. Mitochondria represent one of the symbionts in the expression of a positive prototaxis resulting in an absolute symbiosis. The universal presence of mitochondria in the cells of all plants and animals constitutes evidence that the development of such relationships is not a digres- sion from normal biological behavior, but rather the result of a fundamental principle. The universality of positive prototaxis resulting in absolute symbiosis, or Symbionti- cism, forces us to recognize this as of fundamental impor- tance. Just as reproduction insures the perpetuation of existing species, the author believes that Symbionticism insures the origin of new species. In absolute symbiosis, the adjustment responses of the two symbionts have been completed, making it difficult to recognize their exact nature. On the part of the bac- terial symbiont, it appears, however, that one response has been the development of fragility. While it is quite impossible in most cases to recognize the complete modifi- cations that have occurred in the host cell in absolute symbiosis, modifications are specifically indicated in many of those that are as yet incomplete. It is recognized that the ideas on symbiosis embodied in this book are at variance with those usually conveyed by this term. It is perhaps unfortunate to utilize an estab- lished terminology with a new meaning as it may lead to confusion. Nevertheless, the author believes that in this 64 SYMBIONTICISM AND THE ORIGIN OF SPECIES instance it lends itself to greater ultimate clarity to enlarge and re-define the significance of a term, rather than intro- duce an entirely new word to replace the old. A number of authors (Buchner, Nuttall, Cleveland, Meyer and others) have recently discussed the propriety of the term “symbiosis” in connection with particular life relationships. These discussions have been based upon the original definition of symbiosis as given by De Bary, in which the idea of mutual advantage is implied. Other writers (Geddes, Thomson, Farmer, Wallin, Castellani and others) appear to have sensed an underlying principle in the various types of life relationships, and do not draw a sharp distinction between symbiosis and parasitism. If we should retain the narrower conception of ‘mutual advantage”’ in symbiosis, then it follows that we must limit the term to those chance relationships in which “benefit”? happens to be an accompaniment to both sym- bionts. It is often most difficult to determine the nature of the advantage gained by a symbiont. From the recent discussions on symbiosis one gathers the impression that there is an element of choice exercised by the symbionts. Thus to quote Meyer (25), From the experiments and the findings reported, it is easy to assume that the mollusks derive some benefit from the intracellu- lar bacteria as anabolists or catabolists of metabolic waste products, but, what possible benefit can the microérganism derive from the association? . . . . The recent literature on “symbio- sis’ has been carefully searched for other examples, but only theories have been found which are not established on any scien- tific basis. The function of the microscopic “symbiotes” and their benefit to the host are explained, but little or nothing is said regarding the possible advantages to the microérganisms. . . Buchner’s suggestion that the intracellular organisms are benefited by being protected within the host from the drastic atmospheric influences of heat, cold, desiccation, etc., is a trifle unreasonable. SYMBIONTICISM 65 It appears obvious that the end responses in life relation- ships are not the fundamental cause for these associations, but rather the chance outcome which is dependent upon the physico-chemical properties and reactions of the sym- bionts concerned. Certainly, one can not recognize a display of choice in microérganisms which would lead them to seek out certain hosts on account of advantageous quali- ties; nor can we recognize such properties in the host. The cause for the development of a life relationship is unrelated to the responses accompanying the association. It is these circumstances that led the author to introduce the term ‘“‘prototaxis’” to explain the ‘‘cause’’ for the develop- ment of life relationships, and the term ‘‘Symbionticism”’ to designate the fundamental tendency and object of proto- taxis. In attempting to explain the nature of the development of symbiotic complexes, Meyer states: During an early period, the bacteria, fungi, etc., now found as harmless entities within the insects, are probably parasites producing pathologic conditions and disease. Acquired im- munity later becomes inherited, and the microérganisms are gradually gotten under control. The insects do not rid them- selves of the invaders on account of the fact that transmission from generation to generation is established with remarkable precision. Still later, the invaders lost all of their harmful effects, and since they secrete enzymes that prove serviceable to the hosts, the conflict ends in mutual adaptation; just as in plants (orchids and their mycorhizas) the symbiosis between the tissues of the host and the microérganisms is a phenomenon of parasitism, infection or disease, which has finally become essen- tial to the existence of the animal. In a similar manner Cleveland (’26) suggests that “Such structures as mycetocytes, bacteriocytes and mycetomes of insects may be the survival of profound pathological changes.” 66 SYMBIONTICISM AND THE ORIGIN OF SPECIES While it might be true that some symbiotic complexes may have arisen in this manner, there is no evidence at hand to indicate that this is the usual manner of develop- ment of symbiotic complexes. On the basis of our knowl- edge of microbial behavior, we must admit that there exists a wide range of reactions of living organisms to each other. We are justified in concluding that in many instances the physico-chemical properties of the symbionts entering into a relationship are of such a nature that the terms “‘parasi- tism,”’ “infection”? and ‘‘disease,’’ as they are commonly understood, could not be applied to the relationship. The evidence for and the nature of the development of symbiotic complexes are discussed at more length in the chapters following. CHAPTER VI IMIcROSYMBIOSIS Long before it was discovered that bacteria enter into symbiotic relations with plants and animals, it was known that single-celled plants, particularly the algae, may be symbiotic guests in the tissues of certain animals. Due to their larger size and more distinctive characteristics, the algae are more readily detected in symbiosis than bacteria, and consequently they are better known in microsymbiosis. The morphologic and physiologic responses that are indi- cated in these symbioses vary within wide limits. The “degree” of symbiosis also varies. This variation extends all the way from an occasional presence of the symbiont to absolute symbioses. The study of microsymbiosis has been pursued by a number of investigators during the past twenty-five years. More recently Paul Buchner (1921) has rendered an invaluable service to investigators by his comprehensive and analytical review of the literature. A considerable portion of the data which will be catalogued in this chapter has been taken from Buchner’s treatise. The reader who desires more detailed descriptions than will be found here is referred to this comprehensive mono- graph (in German). The study of microsymbiosis, perhaps, had its beginning in the interest aroused in the nature of the chlorophyl that occurs in a number of lowly animals. Quite a few of these animals are familiar to the general biologist on account of their green coloration (amoeba viridis, fresh water sponges, hydra viridis, and other coelenterates and marine worms). For a long time the green color was not associated with chlorophyl. Later, discussion arose as to its nature and 67 68 SYMBIONTICISM AND THE ORIGIN OF SPECIES origin. Siebold in 1849 declared that if the green material of animal cells is not identical with chlorophyl, it is, at least, very closely related to it. Max Schultze, a little later, by means of chemical tests, definitely established the similarity of plant and animal chlorophyl. The question then turned to the origin of chlorophyl in the animal cell. A number of investigators (Geddes, Lankester, Kleinen- berg, McMunn, and others), declared for an animal origin of the chlorophyl in the cells of animals. The bearing that this interpretation had upon the much discussed “‘dif- ferences” between plants and animals, stimulated a large number of biologists to investigate the problem. Cien- kowsky, in 1871, was the first investigator to present evidence of the “‘parasitic’’ nature of the chlorophyll bodies. He described the cellular nature of these bodies with their nuclei, starch granules, and cell membrane. He also observed that the chlorophyl bodies continued to live after the death of the animal host. R. Hertwig, Brandt and later Geddes, brought forth evidence in favor of the sym- biotic nature of the chlorophyl bodies. They were shown to be symbiotic algae. Brandt named the green forms “Zoochlorellae’”’ and the yellow-brown bodies, ‘‘Zooxan- thellae.”’ Microsymbiosis in which single-celled plants are the de- finitive symbionts, apparently, has not persisted in the higher groups of animals. It would appear that nature stopped experimenting in this direction long before the higher mammalia came into existence. Still it appears probable that the fruits of these ‘“experiments’’ have not been entirely lost to the higher animals. The similarity of chlorophyl and hemoglobin is suggestive of a possible origin of hemoglobin from chlorophyl or a closely related sub- stance. This possibility is particularly enhanced when it is viewed in the light of Symbionticism. However, on account of their greater familiarity and the known physio- MICROSYMBIOSIS 69 logical responses accompanying their invasions into animal cells, algal symbioses furnish important evidence of the proposed mechanism by which Symbionticism operates in the origin of new species. Furthermore, that type of symbiosis in which algae are the definitive symbionts lends itself to experimental analysis more readily than most cases of bacterial microsymbiosis. So far as Symbionticism is concerned, there is no fundamental difference in bacterial and algal symbiosis. Both types of symbionts, as well as symbiosis in which other types of organisms are definitive symbionts, will be considered in the following review. The cases of symbiosis in which bacteria are the definitive symbionts, and which will be discussed in this chapter, obviously, represent only those instances in which the bac- terial nature of the symbionts have been readily recognized. It has been suggested in a few instances, that the bodies in question are mitochondria. Such confusion is rather to be expected when we realize that mitochondria and bacteria in general are one and the same thing, but showing wide variations in morphology, physiologic response, staining reactions, and responses to physical and chemical agents. Symbiosis in the protozoa, or one-celled animals, has been studied mainly in the fresh-water forms, although a few marine species are known to contain symbionts. The majority of described cases of symbiosis in this phylum deal with the more readily detectable algal symbionts, of which there are numerous examples. Buchner divides the protozoa into three groups from the standpoint of algal symbiosis. The first group contains algal symbionts in whatever locality they may be found. The second group he calls “facultative algae breeders.’”’ This group is made up of those species which contain algal symbionts in certain localities, or under certain conditions. The third group is made up of all remaining protozoa, and is characterized as “‘algal enemies;” they never harbor algal symbionts. 70 SYMBIONTICISM AND THE ORIGIN OF SPECIES The “‘facultative algae breeders’ are interesting and signifi- cant in our study of microsymbiosis, in-as-much-as they, apparently, furnished evidence of the influence of extrinsic factors in the development of symbiosis. It is possible that the particular species that fall in this group are repre- sented by varieties in different localities, and that they do not show any distinctive morphological features that dif- ferentiate them from the type species. Such ‘“‘varieties”’ may have chemical and physiological variations which could only be detected by experimental methods. It is also possible that the microsymbiont may have different properties in different localities. It appears more probable, however, that the absence of the algal symbionts in certain localities is due to extrinsic factors, such as temperature, oxygen, light, pressure, ete. Certain cases of symbiosis, which will be mentioned later, furnish evidence of the influence of extrinsic factors. The hereditary transmission of the algal symbionts is a simple process in the protozoa, where reproduction consists of simple fission of the parent organism. When the cyto- plasm of the protozoan host divides, the algal symbionts are distributed more or less equally between each of the daughter animals. In the encystment stage of the proto- zoan host, it has been observed by Penard (’90) and Leidy (79) that in a number of the Foraminifera the algae are retained within the body of the encysted animaf. Gruber (04) also found algae within the cysts of Amoeba viridis that had been sent from America to Germany. In some protozoa, however, the algal symbionts are expelled from the body of the host before or during encystment (Acan- thocystis aculeata), but the symbionts are retained in the wall of the cyst. A number of investigators have observed that when the host is in a state of starvation, the algal symbionts are digested by the host animal. Greef (’75) observed a spon- MICROSYMBIOSIS 71 taneous expulsion of the symbionts in Acanthocystis, in the vegetative stage and a subsequent re-entrance of the sym- bionts into the animal body. Seasonal variations in the occurrence of algae in a host have been described by Balbiani, Roux, and Wesenberg- Lund. Roux observed that Stentor polymorphous har- bored algae in the spring of the year, while in August, September and October, the animals did not contain any algae. The experiments of LeDantec (’92) are significant in connection with a study of the physiological responses under the influence of extrinsic factors. He placed Par- amecium bursaria in the dark, andfound that after twenty- four hours the algae had become brown in color. When kept there for a longer period, the algae were found to be in a state of digestion. When the animal is later returned to the light and kept in sterile water, the algae do not re- appear; it is necessary for a reinfection to take place. An interesting example of the apparent influence of extrinsic factors on symbiosis is to be found in the well known luminiferous Noctiluca milearis. This animal is quite widely distributed, and usually does not contain any algal symbionts. M. and A. Weber (’90-’91) found this animal in the Bay of Bima on the Island of Sumbawa (Indian Ocean) so numerous and so filled with green algae that the surface of the water appeared green. They could find no evidence of digestion of the symbionts. Zooxanthellae, or ‘‘yellow algae,’ have been described in a large number of protozoa. These forms have been ob- served to produce “spores” while in the symbiotic state. These spores are apparently retained in the cytoplasm of the host as small yellow or brown granules. Zooxanthellae in the free-living state are often provided with two flagella at one end of the body. These flagella are left behind when the yellow algae enter the body of a host. Bacterial symbionts in the protozoa have been described 72 SYMBIONTICISM AND THE ORIGIN OF SPECIES only in one genus, Pelomyxa. ‘The bacteria are rod-shaped and vary in size in the different species. In Pelomyza vivipara they are grouped close to the nucleus, reminding one of the oft-observed arrangement of mitochondria. In other species they are scattered throughout the cytoplasm just as mitochondria often are. These symbiotic bacteria measure 10-15 micra in length in some species, while in other species they may be from 40-50 micra long. The physiologic significance of these symbionts has not been determined. In the Sponges, algal symbiosis is fairly common, particu- larly in the fresh-water species. More than one species of algae may be present as symbionts. Some sponges con- tain only single-celled algae within their cells; others harbor the more complex multicellular forms. In some species of sponges, the symbionts are represented by a varying algal flora. ‘The physiologic and morphologic responses in these symbiotic relationships do not appear to have been investigated. No reference to bacterial symbionts in the sponges has been found. Symbiosis in the Coelenterates has been widely known and studied through familiarity with the fresh water form Hydra viridis. Buchner gives a table of the species in which symbiosis has been recognized. The list includes a large number of species of Hydrozoa, Scyphozoa, Anthozoa and Ctenophora. The definitive symbionts in all these symbiotic relationships are zoochlorellae and zooxanthellae (green and yellow algae). In the more simple Hydrozoa the symbiotic algae are located chiefly in the entoderm. There is a variation, however, in their distribution within this layer. The symbionts are usually absent in the lower part of the entodermal cavity where the cells are vacuolated. They are present, in most cases, in the ento- derm of the tentacles. In the higher coelenterates the symbionts are more widely distributed. They are also MICROSYMBIOSIS 73 present in the ectoderm in some species. In some cases there appears to be a definite localization of the symbionts in a particular part of a tissue. Thus, in the canals of the so-called liver of the Portuguese man-of-war the algal symbionts are only present in the upper cells of the canals. These cells do not have the typical entoderm-cell appear- ance, while those in the lower half of the canals suggest secretory and absorptive functions. The symbionts are also found in other parts of this animal. When the animal reproduces by budding, the daughter forms receive the algal symbionts in a passive manner. The number of symbionts present in a cell varies considerably in different species. The usual number is four to eight, but in some cases it runs up into the hundreds. An idea of the intimacy of the relation between the algae and the host in some instances is suggested by the ease with which the former can be eliminated when the host is kept for a time in a dilute solution of glycerine. Gly- cerine is known to be highly toxic to many bacteria and other low forms of life with a notable exception of certain filterable viruses whereas it has little or no effect upon higher forms. It is supposed that this effect is produced largely through its influence upon osmotic pressure within the cell. The distribution of the symbionts in a hexacoral (Adamsia diaphana) described by K. C. Schneider (’02) is also in- teresting and indicative of the specificity of certain cells to the symbiont. The symbionts are found in special cells in the septa which produce a bulging at that point. These special cells have a morphology which differs from that of the other cells of the septum. This appears to be the lowliest animal in which there has been found to be a distinct morphologic response in the host to the presence of the symbiont. The behavior of the algal symbionts in sexual reproduc- 74 SYMBIONTICISM AND THE ORIGIN OF SPECIES tion varies in different Coelenterates. In a number of species, the symbionts do not enter the egg. In such cases, the later larval stages become infected from the surrounding water. In the hydroids, it has been observed by a number of investigators that the algal symbionts are carried from one generation to another within the ovum. Hamann (’82) studied egg formation in Hydra viridis and observed the algal symbionts transferred to the egg. He believed that there was no active movement of the algae into the eggs, but that they were carried passively in the “food stream”’ from the entoderm to the egg in the formation of the yolk. Such an interpretation, it appears to me, is a rather super- ficial one. The experiments of Hadzi (’06) on the influence of light on egg infection are mteresting and significant. Hydra viridis was kept in the dark during ova formation. The ova developed under these conditions, but more slowly and they were colorless. Out of twenty eggs produced in this manner, only one developed into a polyp. This polyp was also colorless, and lived but a short time. He also tested the influence of colored light on egg development. In red light the eggs developed into normal polyps, and contained the algal symbiont. In blue and violet light a few individuals developed into polyps, while in weak green light no development was observed. Beijerinck (’04) discovered a free living alga that was strikingly similar to Brandt’s zoochlorellae (symbionts). He inoculated some of these algae into a colorless Stentor polymorphus. Vacuoles formed around the algae, and in a short time they disappeared apparently as a result of digestion. Experiments of a similar nature have been made by various investigators with identical results. These experiments indicate that there is a distinct “speci- ficity’’ of the organisms concerned in symbiosis. They also indicate the presence of chemical or unseen differences in organisms with similar morphology. MICROSYMBIOSIS 75 Symbioses in the turbellarian worms have been described in the following groups: Acoela, Rhaddocoela and Alloeo- coela. The definitive symbionts in all cases are green and brown algae. The thorough and extensive researches of Keeble (10) on the two marine species, Convoluta roscof- fensis and C. paradoxa, represent one of the more extensive and complete analysis of microsymbiosis that has come to my notice. The observations and experiments of Keeble were conducted over a period of ten years. His results have been confirmed by other investigators. In his book “Plant-Animals, a Study in Symbiosis’ he describes im detail the habitat and habits of these two species. He also describes a number of experiments determining the reactions of the worms to various extrinsic influences. These are valuable studies in animal behavior, but since they have no direct bearing on our problem they will not be further considered. Convoluta roscoffensis contains green algae and C. para- doxa harbors yellow-brown algae. In the adult condition C. roscoffensis ingests no solid food. Kept under abnormal conditions, Keeble found that the animal may become cannibalistic. C. paradoxa, on the other hand, is a vora- cious feeder throughout life. The adult C. roscoffensis depends upon the carbohydrate food supplied by the sym- biotic algae within its tissues. Obviously, the time must arrive when there is nitrogen deficiency, and instead of obtaining this from the outside, it digests the algal sym- bionts that had previously nourished it. Soon after the destruction of the symbionts, the animal dies. In the early stages of development, C. roscoffensis contains no algae, but feeds like most animals upon other animals and plants. Then for a time it receives food from two sources—from ingested plants and animals and from its own green cells. Ultimately, it depends upon the algae alone, and thereby brings about its own destruction. 76 SYMBIONTICISM AND THE ORIGIN OF SPECIES The two worms are remarkable among the turbellarians in possessing no excretory system. ‘The nitrogenous waste products are stored in the tissues of the animal. In the cells of young worms which do not yet possess any algal symbionts, crystalline bodies can be seen. These cells remind one of the “urate cells’ of insects. After the algae have invaded the tissues of the worms, these crystals dis- appear, and in adult life they are absent. ‘‘Comparative cultures of the free stage of the infecting organism have demonstrated that the algae flourish better when supplied with nitrogen in the form of uric acid than when it is sup- plied with a nitrate (potassium nitrate).”’ The transmission of the symbionts in the reproduction of the host does not follow in the manner that one might expect in such cases of obligate symbiosis. The egg cell itself does not contain any symbionts, but the egg capsule was found to harbor the algae. It is only in a later develop- mental stage that the offspring become infected. The host in one of these symbiotic associations (C. roscof- fensis) exhibits definite physiological and morphological responses to the invasion of the symbiont. The cessation of food ingestion from the outside apparently develops into a fixed physiological habit. The failure in the develop- ment of an excretory system appears to be an evident mor- phologic variation associated with the anticipated presence of uric acid-consuming symbionts. Keeble says ‘‘the con- clusion forces itself upon us that the green and yellow- brown cells in the bodies of their respective hosts obtain access to and utilize the stores of waste nitrogen compounds accumulated therein. Or, to put the same idea in another way, green cells and yellow-brown cells constitute the excretory organs of C. roscoffensis and of C. paradoxa respectively.” Keeble suggests the possibility that the ultimate death of the animal may not be due to nitrogen starvation, but to ‘fan aggravated attack of ‘uric acid trouble.’ ”’ MICROSYMBIOSIS riz The responses shown by the algal symbionts are equally interesting and significant. The free-living algae possess a nucleus, besides pyrenoid and chloroplast. After the algae have become symbionts they gradually lose their nuclei. ‘A parallel suggests itself between the green cells of C. roscoffensis and the red blood corpuscles of the higher vertebrates. As the red discs are enucleate, partial cells budded off from the nucleate red cells, so may the green cells be regarded as enucleate, partial cells budded off from the outermost, nucleated green cells; and as the red blood corpuscles are of limited life and specialized (respiratory) function, so are the green cells of C. roscoffensis of limited life and specialized photosynthetic function.” This state- ment, perhaps, does not convey the full significance of the status of the algal symbionts. The morphological and physiological responses indicated in algal symbionts will be discussed in more detail in another chapter. Algal symbionts in the annelid worms, apparently, have never been observed. Bacterial symbionts, however, have been described by a number of authors. These symbionts have been called ‘‘bacteroids,’’ and they have been credited with connective tissue fibrillae formation. Cerfontaine (90) discovered these bacteria in the coelom of the dew worm, and believed that they are the cause of the rapid putrefaction that takes place in these animals after death. Cuenot (’92) and K. C. Schneider (’02) investigated these organisms and confirmed Cerfontaine’s diagnosis of their bacterial nature. Trojan (19) investigated the bacteria and observed their relationship to fibrillae formation, but believed that they were ‘‘chromidia”’ derived from the nucleus. Schneider, also, observed these bacteria in Nereis, Sigalion and in marine polychaetes. Buchner emphasizes the difficulty of distinguishing between bacteria, mitochon- dria and mitochondria-like cell-inclusions. In the Bryozoa, yellow algae have been described in 78 SYMBIONTICISM AND THE ORIGIN OF SPECIES Zoobothrium pellucidum by Brandt (’83). Brandt, also, described yellow-brown amoeboid algae in the echinoderms (Echinocardium). Geddes (’79) described brown amoeboid bodies in various echinoderms. Holothurian larvae also are supposed to contain yellow algae, present as clumps. Brandt observed that when the larva dies the color disap- pears at once. The bodies can be demonstrated after death however by means of iodine. Semon raised the question whether they are algae or pigment cells. Vogt and Jung (’88-’89) have described algae in the crinoids. De Negri (’76) claimed that Elysia viridis, a mollusc, con- tains real chlorophyl. Brandt investigated this form, and found symbiotic algae present in the contractile canal system. In the land snail, Cyclostoma elegans, there is a peculiar group of cells located between the kidney and the stomach that supposedly functions as a ‘‘storage kidney.”’ Mercier (13) investigated this structure, and demonstrated the presence of bacteria in the cells. In the early stages of development the cells contain no bacteria. A peculiar type of inclusion (like a cyst) develops in the cells, and at the same time bacteria appear in the cytoplasm. The “eyst”’ contains uric acid and xanthin. It enlarges until there is only a thin layer of cytoplasm around it. The bacteria also increase in number. They vary in morphol- ogy; some are straight rods; others slightly S-shaped, and the remainder are filamentous. Presently, phagocytes wander in amongst the cells and later penetrate them. Vacuoles then develop in the phagocytes and the bacteria can now be seen clumped in the vacuoles, apparently, in a state of digestion. The uric acid cyst dissolves and is absorbed. Mercier found this structure in animals ob- tained from various localities. He was not able to find a mature female with eggs, and consequently was unable to study the manner of transmission of the microsymbionts. MICROSYMBIOSIS 79 More recently, Meyer (’25) has investigated Cyclostoma elegans and a number of related species. He was able to cultivate bacteria from the animals, but was not convinced of their relationship to the bacteria in the ‘‘purinocytes.”’ In the small group of the Ascidia (Molgulidiz) urate- storage kidneys are also found. These storage cells con- tain a fungus in place of the bacteria, according to Buchner. They produce both mycelia and spores. Microsymbiosis is very widely distributed in the insects, being found in all the orders of this group. The definitive symbionts are bacteria in a large number of cases. We shall discuss here only a few of the more representative cases, and such relationships as have a more intimate bearing on Symbionticism. All of the species in the family Blattidii harbor symbion- tic bacteria. Blochmann in 1887 was the first to describe bacterial symbionts in these insects. In Blatia germanica and Periplaneta orientalis he found that the central part of the fat bodies was composed of cells devoid of fat. These cells contain small rod-shaped bodies which were of un- doubted bacterial nature. When he failed in his attempt to cultivate them, he concluded that this was evidence of their intimate dependence upon the insect organization. Cuenot (92), Prenant and Henneguy (’04) investigated these cells in the fat bodies, and believed that the rod- shaped structures represent metabolic products. Mercier (06) was able to cultivate them outside of the body and definitely established their bacterial nature. Frankel, a pupil of Buchner, investigated a large number of species, and demonstrated the presence of symbiotic bacteria in all. Buchner proposed the name “‘Bacteriocytes” for all cells containing bacteria. The bacteriocytes vary in shape and arrangement in different species. The bacteria also vary in size in the bacteriocytes of different species. Philiptschenko (’07) studied these insects, and showed that 80 SYMBIONTICISM AND THE ORIGIN OF SPECIES the bacteriocytes in young animals also contain glycogen. In older animals he found only fat and uric acid concre- tions. Mercier investigated the bacteriocytes after starvation, and found morphologic variations in the bacteria, apparently due to inanition. In an animal that was infected with a yeast-like parasite, the bacteriocytes were not present. Blochmann, Mercier, Buchner, and Frankel have shown that the bacteria are transmitted from one generation to another in the egg cell. Heymons (’95) studied the bac- terial symbionts in embryonic development, and demon- strated their presence in all stages. Symbionts in ants were first observed by Blochmann (’84—’87). In the midgut of Camponotus ligniperda, pecu- liar cells filled with bacteria-like rods are constantly pres- ent. So also in Formica fusca there are two cell-groups of similar appearance near the ovaries. He also observed the bacteria-like rods in the ova. Strindberg (’13) believed the rod-shaped structures to be mitochondria. Buchner in- vestigated these bodies and concluded that they were fungi. The cells in which they are present he calls ‘‘mycetocytes.”’ The development of the ova and the infection of the egg have been clearly described by Buchner. After the ova have entered the oviduct and the nurse cells have come into position, the symbiotic rod-shaped fungi begin to enter the follicle cells. A little later they penetrate the ova as well as the nurse cells. When the ovum has increased to about three times its original size, the follicle cells contain but few symbionts, but the ovum is so packed with them that the cytoplasm of the egg cell is obscured. The host-cell plasm now increases in amount, and at the same time appears to have assumed a control over the growth of the symbionts, for they increase no further. Buchner believes that this is a chemical regulation. A little later the yolk begins to accumulate in the egg cell. During this process MICROSYMBIOSIS 81 the symbionts form a closely packed group at one pole of the egg. This process reminds one somewhat of the nebenkern formation, as described by Bowen and others. The nucleus of the ovum develops a peculiar activity during the early stages of the symbiont migration into the egg. Parts of the nucleus appear to be thrown off into the cytoplasm so that the ovum appears to become multinu- cleate. Buchner believes that these bodies originate from the nucleolus. Mycetocytes are also to be found in the wood-boring insects. Buchner investigated some of these forms, and gives a comprehensive description of their relationships and the mode of transmission of the symbionts. In Lito- drepa panicea there are peculiar rounded outpocketings of the foregut, which are composed of mycetocytes with a few compressed entodermal cells. These mycetocytes con- tain symbiotic yeast cells, which are oval in outline and usually contain a large vacuole. Escherich (00) was able to cultivate them in artificial culture media. The eggs of the host do not contain the yeast symbionts, but Buchner found them clinging to the surface of the egg capsule. Buchner discusses the physiological significance of the symbiotic yeasts in these animals. A primitive type of gland organ is indicated by the outpocketings of the gut tube. The digesting food mass or chyme lodges in these pockets. He believes that the yeast cells of the myceto- cytes produce enzymes which are secreted into the gut lumen, and act as ferments digesting the food for the insect. Such an interpretation, he mentions, is strength- ened by a similar utilization of microérganisms in the digestion of cellulose in the herbivora. In these higher ani- mals, the cellulose-digesting bacteria are not intracellular symbionts. They are free in the lumen of the gut tube. It is important to note that a large number of insect 82 SYMBIONTICISM AND THE ORIGIN OF SPECIES species, if not all of them, harbor free bacteria in their intestines. It is a difficult matter to determine definitely the physiological significance of bacteria in such small animals. Buchner describes the free bacterial symbionts in one insect, the olive fly, (Dacus olae), which deserves consideration. The description of the symbiosis in this species was made by Petri (’09). In the larva of this form which feeds on the olive, there are four large outpocketings in the midgut. These pockets are lined with a flattened epithelium. The lumen of the pockets is completely filled with bacteria. When the larva transforms into the pupa, some radical changes develop in the gut tube. With the cessation of feeding, the bacteria leave the pocketsin masses, and pass through the remaining part of the midgut and the hind-gut to the outside. A few bacteria remain in the alimentary canal. They pass forward into the foregut. During the transformations of the pupal stage, an unpaired dorsal, gland-like outpocketing develops in the pharynx. In this structure the bacteria find a haven. They persist in this situation in the imago, or adult. Two strains of bacteria have been recognized in the diverticula of the olive fly. Paoli (08) discovered two strains of bacteria in the irregularly-arranged tubercules on the branches of the olive tree, which he thought were identical with the symbionts in the olive fly. There still remains a large number of symbiotic relation- ships in the insects that have been described in literature. In a few instances the symbionts are associated with dis- tinct organs. For example, in certain lice, a ‘“mycetome,”’ richly supplied with tracheal tubes, is present in the ab- domen. Certain insects harbor distinctive bacteria (luminous); these cases will be left for later consideration. In a number of insects and insect-like organisms (ticks and other arachnoids), small bacteria-like bodies are often present. While the nature of these bodies is not definitely MICROSYMBIOSIS 83 known, it has been demonstrated that they are capable of producing disease in man and some of the higher animals, when introduced through bites or other forms of injury. These organisms are known collectively as ‘Rickettsia bodies.” The Rickettsia bodies of the wood tick (Derma- centor venustus) are well known through the researches of Wolbach (719), and many others. The microdrganism appears to be an occasional parasite of the wood tick, since all individuals do not seem to harbor them. In so far as can be learned, they produce no injurious effects in the larva, nymph or adult wood tick. They have been demonstrated in the egg of the host. The particular disease in man associated with the microérganism is known as “Rocky Mountain spotted fever,’’ and is more or less limited to certain sections of Montana. The host, how- ever, is widely distributed over the Rocky Mountain region. Dr. F. E. Becker, working in this laboratory, has been able to demonstrate structures indistinguishable from these Rickettsia bodies in wood ticks that were collected near Boulder, Colorado. He also has examined the tissues of a number of guinea pigs upon which the native wood ticks have been placed. In the tissues of these guinea pigs he was able to demonstrate the Rickettsia-like bodies, but in a different situation from that affected by the Montana strain. The Montana strain, which produces Rocky Mountain spotted fever, localizes in the peripheral blood vessels. The Colorado strain, according to Becker’s find- ings, appears to select the intestinal tract. It appears probable that the Rickettsia bodies are symbiotic micro- organisms in the wood tick, that vary in their specific reactions in different localities. These organisms are extremely delicate, and difficult to demonstrate on a mic- roscopical slide, due to their great fragility. In the examples of microsymbiosis that have been re- viewed in the preceding pages, a large number of the defin- 84 SYMBIONTICISM AND THE ORIGIN OF SPECIES itive symbionts were algae, fungi or yeasts. Recorded instances of bacterial symbiosis have not been so numerous as one might expect. Various reasons may be given for this apparent scarity of bacterial symbionts. Unless the bacterial symbiont possesses definite stability, or has dis- tinctive characteristics, it would not be detected readily within the cell. Furthermore, when bacteria are present in an intracellular position, they are usually called ‘‘mito- chondria” and their bacterial nature has not been generally recognized. Certain bacteria, however, have physical characteristics as distinctive as have algae or yeast cells which may readily be recognized in microsymbiosis. Among these bacteria are the ‘phosphorescent’ or luminiferous forms which have been observed in symbiotic relationship in a large number of animals. The biological relationships exhibited by these symbionts in respect to their hosts, furnish perhaps one of the most significant chapters in microsymbiosis. On ac- count of their luminiferous properties, these organisms can be “followed and watched,’ not only in the development of the host, but also in experimental investigations. This can not be done so easily nor with the same degree of ac- curacy and certainty with microsymbionts that do not have such outstanding characteristics. We are again indebted to Buchner for an extensive review of the literature on ‘Light Symbiosis,” and for original observations in this important branch of microsymbiosis. The question of the nature of the material that is lumi- nous in plants and animals was formerly the subject of con- siderable polemic discussion. Certain kinds of decaying woods are known to emit light of a continuous nature. Bacteriologists long ago demonstrated that the emission of light in these cases is associated with bacteria, apparently feeding on the decaying wood. In the common “‘ight- ning bug” or “firefly,’’ on the other hand, the light appears MICROSYMBIOSIS 85 to be intermittent in character. An anatomical investiga- tion of the “lightning bug” reveals a definite body or organ in the abdomen that is associated with the production of light. This organ is supplied with branches from the tracheal respiratory system, as well as with blood vessels. It has been thought that the cells of this organ contain a material of fatty nature, sometimes called ‘‘noctilucine”’ or “luciferin,’? which when oxidized produces light. The intermittent character of the light was supposed to be pro- duced by contractions and relaxations of the tracheal tubes under the influence of nerves. Dubois (’86, ’87) found that the eggs in two familiar families of beetles, while still in the oviduct and before fertilization had taken place, were luminous. He also noticed that after the egg had been laid it retained luminescence a much longer time than did segregated bits of the luminiferous organ. Dubois saw minute granulations in the cytoplasm of the luminif- erous organ, as well as in the egg cell. These granula- tions which he observed increasing by division, he also found to be associated with luminescence. He named the granules “‘Vacuoloids,” and considered them as elementary and highly significant parts of the living substance which might be bearers of a number of other functions. Regarding the finer structure of the luminferous organs in the beetles, there are two types of cells entering into the formation of the organ, a deeper opaque and a superficial layer. The opaque layer is made up of cells filled with urates, which performs a secondary function in the organ, acting as a condensor and reflector. The superficial layer of cells is transparent and represents a modified portion of the chitin cells. According to M. Schultze (’65), Dubois and others, it is only the superficial cells that contain the luminous substance. Pierantoni, in 1914, demonstrated the presence of two kinds of bacteria in the cells. One variety is rod-shaped and about 10 micra in length. The 86 SYMBIONTICISM AND THE ORIGIN OF SPECIES other variety is a form of coccus. He was able to cultivate them in an agar-peptone medium in which they were col- lectively colored light yellow and were opalescent. The bacteria that developed in Pierantoni’s culture ex- periments did not exhibit luminescence in the culture media. Buchner remarks in this connection that the sceptic finds, in this circumstance, grounds for interpreting the cultured bacteria as contaminations. He also points out that a doubter might be inclined to call these bacteria ‘plastids,’ ‘‘vacuoloids,’ or ‘‘mitochondria.’’ However, Buchner refers to a similar condition of affairs in connec- tion with his own experiments on some parasitic luminous bacteria in the wood louse. When these bacteria were cultivated in artificial culture media they did not exhibit luminosity. When the cultured organisms were inoculated into a wood-louse the luminosity returned. Buchner believes that the symbiotic bacteria cultivated by Pieran- toni might have responded in like manner. The cells containing urate crystals do not belong essen- tially to the luminiferous organ. They are absent in this organ in some species, and are present in the fatty tissues of some insects which are not luminous. It was mentioned above that symbiotic bacteria may be associated with these cells. There is considerable variation in the tracheal supply to the luminiferous organs in different species. M. Schultze described the morphology of the trachea in the Lampy- ridae. He found the finer tracheal tubules extending into the cells. It has been shown by various authors that oxygen is utilized in the light-producing process in these forms. The experiments of Molisch in this field are con- elusive. When air is denied to light-producing bacteria there is no luminosity, but when a bubble of air is brought in contact with the bacteria they again emit light. Bei- jerinck (’89) mixed a bouillon culture of luminiferous MICROSYMBIOSIS 87 bacteria with a leaf extract containing chlorophyl. When the mixture was kept in the dark for some time, the bacteria showed no luminosity, but if the mixture was then exposed to sunlight for only a minute the bacteria became luminous. The light from a burning match was sufficient to restore the luminosity of the organisms after they had been kept in the dark for some time. By means of these experiments it was possible to detect very minute quantities of oxygen liberated by the chlorophyl. Molisch utilized the luminif- erous bacteria to test for the complete absence of oxygen in anaerobic culture media. Very small amounts of oxygen in the media were sufficient to produce luminosity in the bacteria. In the beetles, whose luminosity appears to be inter- mittent, Dubois, Verworn and others, have observed that the light does not entirely disappear between ‘‘flashes.” Dubois experimented extensively with these beetles, and found that when he checked the flow of blood by bending the prothorax, the ‘flashes’? did not occur. When. he pressed on the abdomen and forced the blood along more rapidly, the “flashes” coincided with each compression. The tracheal tubes, apparently, also bring oxygen to the cells containing the luminiferous bacteria. The nature of the mechanism, that is responsible for the ‘‘flashes’’ in insects is not definitely known. Luminosity in the tunicate, Pyrosoma, was observed centuries ago. Buchner, recently, has investigated these forms, and has traced particularly the luminiferous sym- bionts in the more or less complicated development of the host. Various interpretations have been given by different authors regarding the nature of the luminosity. Buchner has demonstrated cells that contain the light-producing microédrganisms which are comparable to the myceto- cytes. He believes that the microdrganisms are fungi, although he thinks it is quite possible that they are “bac- 88 SYMBIONTICISM AND THE ORIGIN OF SPECIES teroid’’ stages of light-producing bacteria that have adapted themselves to an intracellular life. It is not clear from Buchner’s discussion why he considers these forms fungi. So far as their morphology is concerned they might as well be bacteria. Although it is immaterial whether they are called fungi or bacteria, they are nevertheless intracellular microsymbionts. The most remarkable instances of luminiferous organs associated with known microsymbionts occur in certain cephalopods. Pierantoni (’17, 718) and Chun (710) have described the development of the organs in Rondeletia minor, Sepiola intermedia and Pterygioteuthis maculata (fig. 2). The luminiferous organ in the adult has the general appearance of an eye. It possesses a “tapetum” (dark screen) in the deep peripheral part of the organ. Next to the tapetum nearer the surface is a contractile reflector. In the central part of the organ and superficial to the reflector is a gland-like structure containing the luminiferous bacteria. A lens is present in front of the luminiferous body, and is covered by transparent epithe- lium. The development of this organ is remarkable in that it utilizes accessory structures in its formation. There are accessory “nidamental glands’ found in these animals which later become the luminous element in the light organ. Bacteria are associated with these glands, but they are usually not luminiferous in the earlier stages of development. Modifications of these glands take place, and they come into association with the ink bag, which ultimately forms the tapetum. Between the ink bag and Fic 2. DIAGRAMMATIC SECTIONS THROUGH THE LIGHT ORGANS OF Rondeletia minor (A), Sepiola intermedia (B) AND Pterygiotheuthis maculata (C) Ep = epidermis, Le = Lens, Lu = luminous part, P = pigment (in A and B, ink bag) Re = reflector. A and B from Pierantoni, C from Chun (after Buchner). MICROSYMBIOSIS 89 the accessory “‘nidamental gland,’”’ muscular tissue grows in from the sides and is transformed into the contractile reflector. Superficial to the region of the ‘“nidamental gland,’ connective tissue grows in, and becomes trans- formed into the lens. The ‘‘nidamental gland” portion contains the luminiferous bacteria, whereas other symbio- tic bacteria, which are not luminous, are associated with the formation of the lens. In one species the luminiferous bacteria do not appear in the organ until after it has com- pletely formed. Here is an example of an organ, in which the whole of its complicated structure is essentially a re- sponse to bacterial influence, and the entire structure ap- parently is designed to utilize symbiotic bacteria. There are other instances of organ formation, associated with symbiotic luminiferous bacteria, but these examples sufficiently illustrate the physiological and morphological responses to such organisms. Harvey (’20) has written a monograph on “The Nature of Animal Light” in which various aspects of “bioluminescence” are considered. This treatise abounds in observations and experiments on the physiology and chemistry of light production. In a num- ber of organisms the light producing materials, luciferin and luciferase are present in the cytoplasm of the luminif- erous organ cells, usually in the form of small bodies or granules. The probable genetic origin of this granular material is indicated in the symbiotic relationships that have been discussed and will be considered more fully in a later chapter. Microsymbiosis is not limited to associations with ani- mal hosts. There are a number of well-known microsym- biotic relationships in plants. The physiologic and mor- phologic responses in these symbioses are as significant as those in which an animal is the relative symbiont. We shall only discuss at this time one of these symbioses—the root nodules and their symbionts in the legumes. 90 SYMBIONTICISM AND THE ORIGIN OF SPECIES The presence of bacteria in the root nodules of the legumes was first established by Winogradsky. These symbiotic bacteria (Bacillus radicicola) have been the ob- ject of intensive research by a host of investigators. It has been demonstrated that they have the faculty of fixing atmospheric nitrogen, and rendering it available for use by the host plant. The microérganism is present in the soil. It enters the root hairs of the host plant and comes to lodge in some of the root hair cells. Its presence stimu- lates these cells to special growth, and an organ,—the root nodule—is formed. The invading organisms are minute rod-shaped structures. After asojourn for some time in the root nodule cells, they become transformed into rather large bacilli and branched Y-shaped forms. In the older cells of a nodule, the bacteria have become still more modi- fied. In sections of the nodule stained by a mitochondrial method, the author found large globular forms in the oldest part of the nodule (Wallin, ’22b). Lohnis (’21) had pre- viously observed these forms, and believed that they rep- resent “regeneration bodies,’ but the true significance of these globular forms is not known. In the young root nodules no other organisms but the Bacillus radicicola are present in the cells. As the nodules mature, other micro- organisms begin to invade the cells of the nodule. In an old nodule, very few if any Bacillus radicicola can be found in the nodule. They appear to have been replaced by a variety of parasitic microdrganisms. In recent years botanists and bacteriologists have demon- strated the presence of nodules containing bacteria on the roots of a number of plants other than the legumes. The physiological significance of these symbiotic relationships is not known in most instances. There are also a number of plants that contain extracellular symbiotic bacteria in the leaves and other parts of the plant. Psychotria bac- teriophila is an example of such a plant in which it has been MICROSYMBIOSIS 91 shown that the microsymbionts are transmitted in the seeds from one generation to another. The examples given above, while far from exhausting the list, suffice to illustrate the prevalence of microsymbiosis in nature. It is obvious that a phenomenon which is so common must be of some fundamental significance, and can not be dismissed as a biological digression or mere curi- osity. An analysis of the responses of microsymbionts given in the next chapter reveals the fundamental impor- tance of these associations. CHAPTER VII An ANALYSIS OF SYMBIONT REACTIONS The entrance of a microérganism into the tissues of a plant or animal may result in various types and degrees of response. In many instances, there appears to be no response in either the host or the microérganism. In other cases, the host organism responds in both a physiological and morphologic manner. The resistance of the host may be slight, resulting in “disease,” or it may be active and bring about the destruction of the invading micro- organism. ‘There may be a morphologic response in the host, resulting in new tissue formation. When such a tissue production is injurious to the host, we may recognize it as a neoplastic disease (tumors, crown gall, etc.). A catalogue of the known responses in plants and animals to microbie invasion would reveal a multitude of types and de- grees of relationships and reactions. The responses, in general, may be divided into two types; physiologic and morphologic. Further, these responses may be either temporary or permanent. Our study is particularly con- cerned with those modifications in physiology and mor- phology which may become permanent in a species. It becomes necessary also to refer to some of the temporary responses associated with disease in order to understand more fully the factors involved in Symbionticism. The reactions that have been indicated in the relative or host symbiont in the previous chapter were seen to vary in different symbiotic relationships. In the protozoa, it has not been possible to recognize any response, either physio- logic or morphologic. Obviously, if a protozoan animal develops an absolute symbiosis with a bacterial organism, 92 AN ANALYSIS OF SYMBIONT REACTIONS 93 and there develops a permanent morphological variation in the host, this would constitute a new species, or, at least, a new variety. The determination of the progenitor of the symbiotic variation might then be purely a matter of chance. A physiological response in the protozoan host would be even more difficult to determine specifically. On account of these obvious difficulties, our analysis must be limited, more or less, to the multicellular organisms, in which morphologic variations can not only be traced in ontoge- netic development, but may also be recognized in a tissue. The simplest type of animal that exhibits a definite mor- phologic response to the presence of a microsymbiont is the Coelenterata. In the Portuguese man-of-war, the cells in the canals of the so-called liver that contain algal sym- bionts are different in appearance from the remaining cells. So, also, in Adamsia diaphana, a hexacoral, the cells of the septa that contain the symbionts are morphologically dis- tinct from the remaining septal cells. It might be said that the morphologic variation in the cells preceded the invasion of the symbionts, and that this implies a physio- logic variation which produced prototactic conditions that attracted the algae. Such an interpretation, however, would seem to place the “effect before the cause.” The special cells are not present before algae make their ap- pearance. A comparison with related, but non-symbiotic species, reveals the fact that the special cells are not pres- ent. Buchner has shown that the algal symbionts are carried in special cells in a large number of animals. These special cells develop in conjunction with the algal sym- bionts, and hence were called “mycetocytes.” Morphologic reactions in the relative symbiont are spe- cifically shown in the turbellarians, Convoluta roscoffensis and C’. paradoxa. In other turbellarian worms, an excre- tory system elaborates and functions, while in the above- named species, the excretory system fails to develop. The 94 SYMBIONTICISM AND THE ORIGIN OF SPECIES absence of this system in these worms is associated with the presence of algal symbionts. Again, it might be argued that this failure in development of an excretory system produced prototactic properties in the worms that are specific for certain algae, and that this circumstance is the crucial factor in the development of the symbiotic rela- tionships. This explanation would not supply a cause for the lack of development. Since excretory systems are present in closely related, but non-symbiotic species, and in the absence of any other evident factor, it appears that the symbiotic algae may be responsible for the absence of the excretory system in Convoluta roscoffensis and C. paradoxa. It is not possible for us to explain the nature of the mechanism whereby the presence of the algae brings about the permanent agenesis of an entire organ system. We can understand how the loss of activity of an organ may result in complete atrophy, but we do not know how such a deficiency can be so impressed upon heredity that the ‘‘defect”’ shall be permanent in succeeding generations. The presence of a foreign microérganism, suggests the possibility that the microsymbiont in some way influences the germplasm of the host. The absence of the symbionts in the eggs of the host, also complicates the situation. ‘True the symbionts are present in the egg capsule which contains the elements of food necessary for the symbiont, but the symbionts do not enter the developing worm until it has reached a certain stage in its life cycle. We must assume that if the microsymbiont impresses a new characteristic, or perhaps, better stated, destroys a potential characteristic in the germ plasm, this must be accomplished while the microsymbionts are present in the general or somatic tis- sues of the host. The loss of the property of food-ingestion in Convoluta roscoffensis, indicates a definite and fixed physiologic re- sponse to the presence of the algal symbionts. This AN ANALYSIS OF SYMBIONT REACTIONS 95 physiologic modification, however, does not appear to be impressed upon the germ plasm, as the young worms take in food from the out-side like any non-symbiotic turbellarian. This response appears to be limited to a definite growth period of the individual. The presence of special cells containing bacterial sym- bionts in the Blattidii (cock-roaches) is another example of morphologic response on the part of a host to the presence of microsymbionts. We know nothing of the functional significance of the “bacteriocytes”’ in these insects. The microsymbionts are transmitted from one generation to another in the germ cell. In the plant kingdom, the formation of root-nodules in conjunction with the invasion of microérganisms represents a specific illustration of a morphologic response in the host. Wright! distinguishes two types of bacteria which produce root nodules on the soy-bean. These two types are morphologically alike, but have different serological and cultural characteristics. The nodules produced by the two types have a different arrangement on the roots of the host plant. In these plants the biologic association of the symbionts has not developed to a point of absolute dependence, although the association apparently is valua- ble to both. The symbiotic relationships present in all the lichens represent one of the most significant examples of symbiosis that we shall attempt to analyze. The group of lichens is distinctive in that every member of it is a symbiotic com- plex. The plant consists of algae symbiotically combined with a fungus. The lichens are particularly significant and instructive since the various species exhibit different degrees of symbiotic relationship. In the simpler forms, reproduction of the two symbionts is independent. It is necessary for the offspring of the two symbionts to find 1 Personal communication. 96 SYMBIONTICISM AND THE ORIGIN OF SPECIES each other, and again join in the production of a daughter lichen. In the next higher group, reproduction of the two symbionts is still independent, but an accessory method of multiplication is represented by a process of budding. The daughter lichens produced by this method contain both of the symbionts from the first. In the higher groups of lichens, a reproductive organ, the ‘‘soridium”’ is formed. Both symbionts supply materials in the formation of the reproductive elements. In these higher lichens the sym- biosis has become absolute, and reproduction is no longer independent. ‘The relationships in these three groups of lichens appear to indicate that there is a gradual develop- ment towards a complete interdependence of the two sym- bionts. In the more lowly lichens, there is a possibility of inde- pendent existence of the two symbionts. In the higher lichens, the two symbionts are incapable of independent existence in nature. There is no question regarding sym- biosis in the lichens, as the symbionts are readily distin- guishable. Here is an example of an entire group of or- ganisms whose origin unquestionably depended upon the establishment of a symbiotic relationship. The symbiotic relationships in which luminiferous bac- teria are the definitive symbionts, have produced morpho- logic responses in the host that are of the greatest signifi- cance in the theory of Symbionticism. In a number of these symbioses, definite organs have been formed in the host, associated with luminescence and harboring the lumi- niferous symbionts. These organs vary in complexity in different animals. In some of the beetles, the organ consists of two kinds of cells—the bacteriocytes, containing the luminferous symbionts, and the “urate cells’ which become associated with them and have a subsidiary func- tion in connection with the organ as a whole (reflector). The cells that harbor the luminiferous symbionts are modi- AN ANALYSIS OF SYMBIONT REACTIONS 97 fied chitin cells, according to the authors who have investi- gated them. Here is a morphologic response in which not only chitin cells become modified by the microsymbiont but other cells (the ‘‘urate cells’) are apparently attracted to the bacteriocytes, take up a definite position, and con- tribute to the formation of the luminous organ. This is a comparatively simple organ, performing a definite func- tion, but its origin is directly traceable to bacterial invasion. The organ is a permanent and characteristic structure of the species. The microsymbiont is transmitted in the egg cell from generation to generation. The organ associated with light production in the ceph- alopods (Rondeletia minor, Sepiola intermedia, Pterygio- theutis maculata) is rather more complex than the lumi- niferous organ in the beetles. In these forms we recognize four distinct tissues entering into the formation of the organ. The bacterial symbionts associated with the first structure to develop—the “‘accessory nidamental glands’ — do not at first possess luminescence in some species. It is only after the organ is completely formed that the lumi- niferous bacteria make their appearance in that part of the organ formed from the “‘accessory nidamental glands.” It is not definitely known if these symbionts are a transforma- tion of the original bacteria associated with the “nidamental glands,” or if they come from some other source after the organ is formed. On the basis of experiments with cultured luminiferous bacteria, it is safe to assume that the original bacteria become modified during development and become luminous. One of the accessory structures in the organ (lens) is definitely known to be associated with bacteria in its formation. The history of the development of the luminiferous organ in the cephalopods illustrates the influence of microérgan- isms in organ production. Various types of cells, appar- ently under the influence of the essential symbionts, become 98 SYMBIONTICISM AND THE ORIGIN OF SPECIES modified and associated in the organ. The behavior of the microsymbiont in this symbiosis is also significant and will be discussed presently. We have followed some of the responses that take place in the relative symbiont, or the host, in symbiosis, we have next to consider the modifications that arise in the definitive symbiont. These are the more difficult to analyze, but they form the “connecting link” in the theory of Symbion- ticism. It is in this connection that the responses shown by algal symbionts furnish important evidence. Keeble and other investigators have described the medi- fications that algae undergo in the symbiotic situation. In the first place, some of the algal species that enter into symbiosis are flagellate in the free-living condition. These flagella are lost when the algae enter the tissues of the host. Some species, after they have taken up the symbiotic position undergo further modifications; the cell wall, nu- cleus, and pyrenoid disappear, so that all that remain of the original algal cell are the chloroplasts. ‘These chloroplasts, apparently, continue to perform their normal functions, for starch granules can be demonstrated by the iodine reaction in the cytoplasm of the host cell. We may then inquire,—what is the nature of the chloro- plast? It has been looked upon as an “organ” in the plant cell. Just what is meant by an intracellular ‘‘organ”’ is difficult to determine from its usage in the literature. In general, it appears that the conception of cytoplasmic “organ” has carried with it the idea of a passive or non- living body. Such a conception is difficult to correlate with what we know of the activities of chloroplasts. We might further ask, Is the chloroplast only a ‘‘container”’ for the chlorophyl, and is the chlorophyl only a sort of “catalyzer?”’ We can extract chlorophyl from plants, mix it with carbon-dioxide and water and place the mix- ture in sunlight, but no starch is produced. ‘There appears AN ANALYSIS OF SYMBIONT REACTIONS 99 to be some sort of an accompanying organization necessary if chlorophyl is to produce starch. This “organization,” apparently, is present in the chloroplast. What, then, is the nature of this “organization?” Is the chloroplast a living body? We do not know, but we have good reasons to suppose that it 7s a living body. This will be discussed in the following chapter. How are bacteria modified in symbiosis? The answer to this question is complicated by a multitude of varia- tions in the responses of different strains of bacteria. Some of these can hardly be more than guessed at in our present state of knowledge. When a bacterium is subjected to a cytoplasmic environment, a number of factors become operative—physical, chemical, thermal, photic and electri- cal. We know that free-living bacteria may be influenced by the same set of factors. It is a comparatively simple matter to determine the morphologic responses of bacteria in artificial culture media. We can determine some of the physiological and chemical modifications of microérganisms under these conditions, but corresponding examinations of symbiotic bacteria cannot readily be accomplished. In many instances, bacterial symbionts exhibit pleomorphism, but free-living bacteria may exhibit the same tendency (Reed, ’24). The responses shown by some pathogenic bacteria illustrate types of response that may be present among microsymbionts. Pleomorphism or morphologic variation, although not limited to microsymbionts, is commonly present in sym- biosis. This has been observed by a number of investiga- tors, and is especially prominent in Bacillus radicicola in the root nodules of legumes. The symbiont at the time of invasion is a minute rod-shaped organism. Later it develops into a much larger organism, and becomes forked at one end. Finally it transforms into a comparatively large, globular body. These transformation stages, ap- parently, do not occur in the free-living state. 100 SYMBIONTICISM AND THE ORIGIN OF SPECIES Another property developed by the Bacillus radicicola in the symbiotic state, is that of “fragility.” This is especially prominent in the final morphogenic state—the large globu- lar body. In an ordinary bacteriological preparation in which the nodule is crushed on a slide, the large bodies are practically never seen, undoubtedly, due to their fragile nature. In histological sections, the large globular form is difficult to demonstrate with the ordinary technique. The organism of Rocky Mountain spotted fever and other Rickettsia bodies exhibit similar fragility. All bacterial symbionts do not necessarily exhibit the same degree of fragility, but on the contrary, many microsymbionts ap- pear to retain their hardier properties. It is a difficult matter to explain this variation in fragility. In general, it is safe to assume that fragility is a conse- quence of symbiosis; the greatest degree of fragility is associated with absolute symbiosis. Other factors, how- ever, must be considered in any attempt to explain fra- gility. It must be recognized that the chemical constitution and the physiologic activity of the symbionts may be different in every symbiotic complex. We must also ad- mit that there may be different ‘degrees’ of symbiosis, as exemplified in the three groups of lichens. When a sym- biosis is as ‘‘loose’’ as it is in the lower lichens, the individ- uals still retain their hardier characteristics, and are readily capable of an independent existence. On the other hand, when the symbiosis is as ‘‘close’’ as it is in the higher lichens, the symbionts have lost their hardier characteris- tics and are no longer able to maintain an independent existence. The ‘‘degree’”’ of symbiosis determines, in a large measure, the independence of the symbionts. This circumstance has a bearing on the difficulty in cultivating microsym- bionts in artificial culture media (see Meyer, ’25). In some forms in which the symbiosis is not so close, the microsym- AN ANALYSIS OF SYMBIONT REACTIONS 101 bionts are readily cultivated. The physiological attri- butes of the microérganisms also must be taken into ac- count. The microdrganisms responsible for a number of diseases can be demonstrated in the tissues and in the blood, but attempts at artificial cultivation of the as- sumed causative organism have only resulted in failure. Obviously, if the proper ingredients were supplied in the culture medium, and other crucial factors were properly adjusted, the microérganism in question should grow freely in this situation. The Rickettsia bodies of Rocky Mountain spotted fever have resisted cultivation in arti- ficial culture media in the experiments of Wolbach, Noguchi, and many other investigators. Pathogenic microédrganisms exhibit properties that throw light on some of the possible responses of microsymbionts. It has been shown by a number of bacteriologists that the “specificity” of certain microérganisms may be altered when grown in certain culture media. It is also a common observation that the viability of some bacterial cultures diminishes with the age of the culture. It has further been established that the morphology of a microérganism may be altered, by changing the environmental factors. Above all, it must be emphasized that our knowledge of the behavior of bacteria is decidedly limited. Variations in response of microsymbionts is exemplified in the luminiferous bacterial symbionts. Harvey (’20, ’24), who has especially studied the physiology of biolumines- cence, states that Zirpolo (’17, ’22) cultivated bacteria from the luminiferous organ of Rondeletia minor (a squid), while Montora (’22) was unable to grow bacteria from a similar organ in another squid, Hetoroteuthis dispar. Attempts to cultivate bacteria from the luminiferous or- gans in a number of other animals have resulted in failure. It is perhaps this circumstance, in part, that led Harvey to say. “I believe the animals in which luminous sym- biotic bacteria can be demonstrated are relatively few.” 102 SYMBIONTICISM AND THE ORIGIN OF SPECIES The experiments described in a previous chapter, demon- strated a variation in the fragility of mitochondria at var- ious periods in the life of the animal. It was shown that they are hardy in the fetus and new born, and gradually develop fragile properties with the age of the animal. It was, also, indicated in these experiments that the mito- chondria of the adult liver tissue do not grow readily in a medium which supports luxuriant growth when the mitochondria are derived from the liver of the fetus and the new-born. These results indicate the danger of arriv- ing at positive conclusions from negative results in bacterial culture experiments. Another observation in connection with the physical nature of mitochondria is significant in connection with the ultimate morphologic response of a microsymbiont. Cowdry and other investigators have described cells in which the mitochondria appeared to have gone into “solu- tion’ in the cytoplasm. The author has also observed and described the same phenomenon in lymphocytes. It was supposed that some injury or accident was responsible for this condition. In the light of the response shown by some algal symbionts in which the cell wall, nucleus and pyrenoid disappeared in symbiosis, it now appears that another interpretation is possible for this mitochondrial “solution” phenomenon. It is possible that the so-called “dissolution” is a normal end-response of microsymbiosis, or may represent a temporary condition due to variations in surface tension either in the host cell or the microsym- biont. In the latter event, it might be possible for the mitochondrial material to separate again and reform into bodies when the surface tension returns to its original condition. Biologists are unanimous in the view that pro- toplasm is a colloidal complex. It differs from an arti- ficial colloidal mixture in so far as it contains what we call life. Does this additional property (life) modify the AN ANALYSIS OF SYMBIONT REACTIONS 103 nature of protoplasm so that it does not respond to the influence of chemico-physical forces? There is no evidence at hand to show that such is the case. On the contrary, it is well known that many responses of protoplasm are due to chemical and physical influences. Obviously, it will require more extended observations and experimental analyses to determine the nature of these reactions in mycrosymbiosis. CHAPTER VIII SYMBIONTICISM AND THE ORIGIN OF SPECIES When a variation develops in a living organism and this deviation becomes hereditary, we recognize a new species or a “new variety,’ depending upon the extent of the variation. In the chapters on ‘‘Microsymbiosis’” and “An Analysis of Symbiont Reactions,’ a number of ex- amples were given in which tissues and organs were shown to originate as the result of the invasion of microdrganisms, or Symbionticism. The variations that were present in these forms were of sufficient magnitude to constitute new species. In the majority of these examples, it was possible to recognize the essential cause of these variations by the particularly prominent characteristics of the microsym- bionts concerned. Various biological conditions and phenomena, coupled with the bacterial nature of mito- chondria and the responses of organisms to microbic inva- sions, leads the author to conclude that Symbionticism ts the fundamental factor in the origin of species. While the above mentioned evidence furnishes the basis for the hy- pothesis, it is pertinent to test the theory further, and attempt to determine how it articulates with other known biological phenomena. If bacteria are the building stones, or the “primordial stuff,’’ from which all higher organisms have been con- structed and modified, then we should be able to find in bacteria the same metabolic products that are found in the specialized cells of the higher forms of life. The re- searches that have been done on bacteria are so extensive that it is beyond the realm of possibility for any one inves- tigator to acquaint himself with the entire literature. The 104 SYMBIONTICISM AND ORIGIN OF SPECIES 105 summaries and data which may be found in text-books and monographie works on bacteriology, however, contain many examples of known bacterial activity which form valuable evidence for our purpose. It has been claimed that there are no enzymatic properties in the human body that are not also represented in bacteria (Jordan, ’24). It may not be possible to completely verify this state- ment. Evidence that supports this however is abundant. In Marshall’s ‘‘Microbiology” (’21) considerable attention is given to the metabolic products of microdrganisms. After a perusal of this book, it is evident that bacteria produce metabolic products that are identical in their properties with those elaborated by plant and animal cells. A brief summary of some of these activities may serve to illustrate this point. The better known enzymes of animals and plants are concerned with food digestion. While we do not know the chemical composition or structure of any of these enzymes, their chemistry is known in terms of the reactions they produce with carbohydrates, fats, and proteins. These enzymes are known to be the products of various glands collectively known as ‘digestive glands” in the higher animals. Some of them are also present in plants. The enzyme concerned with starch decomposition—amylase (ptyalin, diastase)—is a particular product of the salivary glands, and also of the pancreas. Amylase hydrolyzes starch and changes it into maltose, a sugar. Regarding the occurrence of amylase in nature, Marshall states: “Diastase, or amylase is a starch-dissolving enzyme which is one of the most common enzymes in nature. It is found in all green plants, and it forms during the sprouting of starchy seeds. Many moulds and a few bacteria produce this enzyme, while yeasts generally cannot decompose starch for lack of diastase.’’ Cellulose is closely related to starch, and is decomposed into a soluble sugar by an enzyme 106 SYMBIONTICISM AND THE ORIGIN OF SPECIES called ‘‘cellulase’’ or ‘‘cytase.” It is interesting and signifi- cant in this connection that those animals (the herbivora) in which cellulose forms the chief article of diet do not pos- sess any cytase-producing glands, but depend upon certain bacteria present in their intestines to supply the enzyme for cellulose digestion. Lipase is an enzyme which has the ability to split fat into glycerin and fatty acid. Itis produced in the stomach and pancreas of the higher animals and is present in some plants. A few bacteria are known that have the ability to split fat into the same components. Pepsin and trypsin (proteases) are proteolytic enzymes produced in the stomach and intestines of the higher ani- mals. These enzymes have been little studied in the micro- organisms. Marshall states, however, that “there is so far as can be determined, little appreciable difference be- tween the proteolytic enzymes obtained from different organisms, whether low or high in the plant or animal world, consequently many experiences with animal pep- sin and trypsin can be applied to microbial enzymes.”’ The blood-clotting enzyme, thrombase, does not occur in microorganisms, according to Marshall. Rennet is a substance present in the stomach of calves. Rennet is found in many species of microérganisms. Ren- net-forming bacteria are found in milk and dairy products, in soil and other habitats. Pigment formation is an activity that is widely distrib- uted in plants and animals. Many bacteria are known in which pigments are produced. A few strains of bacteria are known that have the prop- erty of decomposing hemoglobin. One of these forms is Streptococcus hemolyticus. Certain functions in the mam- malian body are of a similar nature. It is believed that the spleen has the property of destroying red blood cor- puscles when their hemoglobin content falls to a certain SYMBIONTICISM AND ORIGIN OF SPECIES 107 level (Hemmeter, ’26). The liver is known to have the property of decomposing hemoglobin. The suprarenal gland, apparently, has the property of producing adrenalin or epinephrin. Mathews (’15) states that it has been claimed that certain bacteria have the property of changing tyrosin into adrenalin. From this brief survey it is evident that many of the products that are characteristic of certain specialized cells in higher animals are also the products of bacterial metabo- lism. The basis for a correlation of this circumstance is to be found in the bacterial nature of mitochondria. Sym- bionticism offers a ready explanation for the origin of the “specialized cells’ in the higher plants and animals. The probability that Symbionticism is the factor concerned in the acquisition of these specialized properties, is enhanced by the results of a number of mitochondria investigators. Regaud and Mawas (’09) concluded that the secretions of the parotid and submaxillary glands originate from the mitochondria. Prenant (13) associated pigment forma- tion with mitochondrial activity; Mulon (710) concluded that the secretion of the suprarenal glands originates from the mitochondria; Hoven (710), Horning (’25) and others believe that zymogen of the pancreas originates from mito- chondria. Other cell products and structures that have apparently originated from mitochondria are catalogued in Chapters IT and IV. Modern researches in the plant kingdom have revealed some significant developmental activities. Our interest is especially directed to the chloroplasts and other plastids. Practically all green plants—the Bryophyta, Pteridophyta and Spermatophyta, as well as the lowly algae—possess chloroplasts which in turn contain chlorophyl. The Cyano- phyceae (Schizophyceae), or blue-green algae contain no chloroplasts. In these forms, the chlorophyl and the blue- pigment (not always blue, the characteristic hue of the 108 SYMBIONTICISM AND THE ORIGIN OF SPECIES Red sea is due to Cyanophyceae) are diffused throughout the cytoplasm. The Cyanophyceae do not contain a dis- tinct nucleus. In the central part of the cell, there is a material that has nuclear staining properties and is usually considered a nucleus, but it lacks a nuclear membrane in most cases. The Cyanophyceae together with the bacteria (Schizomycetes) constitute a plant division, the Schtzo- phyta, in the opinion of most botanists. In other words, the blue-green algae are similar and closely related to the bacteria. The origin of the chloroplasts in algae and higher plants has interested botanists for many years. Schimper (’85) first suggested the continuity of chloroplasts in de- velopment. He believed that chloroplasts arise from preéxisting chloroplasts by a process of division. Guillier- mond (’21) and others have shown that chloroplasts origi- nate from mitochondria in ontogeny by a process of trans- formation. The various stages of transformation have been so clearly illustrated by Guilliermond that very Little doubt exists in regards to the accuracy of his observations. His discoveries have been confirmed by a large number of investigators, and this method of plast-formation has been introduced into text books. Not only are chloroplasts formed in this manner, but other kinds of plasts (chromo- plasts, amyloplastids, leucoplasts) are also formed from mitochondria by a process of differentiation. ‘The chloro- phyl in these plastids is not apparent in the mitochondrial stage of development, but appears in later differentiation. In many instances a pyrenoid is associated with the chloro- plast. The shape of the plast is somewhat variable. It is usually circular in outline, but in Euglena viridis (a proto- zoan) it is star-shaped, while in Microglena punctifera (also a protozoan) it is in the form of two elongated rods. It is evident from the data that have just been given, that chloroplasts and other plasts of the higher green plants SYMBIONTICISM AND ORIGIN OF SPECIES 109 bear a close morphologic similarity to the blue-green algae (Cyanophyceae). Physiologically the two structures are also similar. The possible phylogenetic origin of plasts from the blue-green algae appears to be a logical conclusion in line with Symbionticism. How are we to harmonize the mitochondrial origin of plasts in ontogeny with a relation- ship to the blue-green algae? Are the plasts to be con- sidered transformations of symbiotic bacteria? It must be mentioned in this connection, that a large number of bacteriologists claim that no bacteria are known which contain chlorophyl; other bacteriologists, on the other hand, insist that chlorophyl is present in some forms. A number of circumstances and experiences, however, assist us in correlating and harmonizing the plasts with Symbion- ticism. It has been shown in microsymbiosis, and particu- larly in the behavior of mitochondria, that microsymbionts may be altered by ‘their environment. The mitochondrial pleomorphism experiments clearly demonstrated the pleo- morphic responses of these microdrganisms to slight varia- tions in the environment. The absence of chlorophyl in the mitochondrial stage of the chloroplast is not a barrier to the acceptance of a genetic origin from the blue-green algae. It was shown in connection with the development of the luminous organ in some squids, that luminous bac- teria were not present until the organ was completely formed. Extrinsic factors as well as inherent tendencies operate in the development of “specificity”? in microédrgan- isms. It has been shown also by various investigators, and is commonly accepted that light is a fundamental factor in the production of chlorophyl. These observations and experimental results certainly indicate reactions and processes in microérganisms, par- ticularly microsymbionts, that clear the way for a better appreciation of the possible genetic relationship of chloro- plasts and the blue-green algae. It may appear that the 110 SYMBIONTICISM AND THE ORIGIN OF SPECIES immediate mitochondrial origin of chloroplasts is opposed to the acceptance of a genetic relationship to the blue- green algae. On further reflection and analysis, however, such a presumption is hardly valid. The chloroplasts have in the main the characteristics of living bodies. Cell histogenesis in ontogenetic development demonstrates the possibilities of differentiation. It is well known that the germ cell contains potentialities for the elaboration of all the tissues of an individual, whether it be a man or a worm. These “potentialities” are not sufficient within themselves to produce the completed individual. It recently has been emphasized by Child (’24) and others, that environmental factors play an important réle in histogenetic differentia- tion of tissues and organ formation. These environmental factors, perhaps, are chiefly of a chemical nature, although mechanical and physical factors are known to be involved. The metabolic products of one organ or tissue may be the stimulating influence that inaugurates differentiation in other organs or tissues. According to Stockard (721), the active differentiation of a tissue or the formation of an organ may exercise an inhibitory influence on the differen- tiation of a number of other organs and tissues. Conklin (19) has given a clear exposition of the status of histogenetic development in the following words: ‘‘to a certain extent differentiations are already present in the germ cells, but during the course of development their number is greatly increased. This increase in differentia- tion is caused by modification of previously existing differentiations, by transformation rather than by formation de novo, by evolution rather than by creation. New things appear in the course of ontogeny because of new combinations of things already present, just as new chemical substances with new qualities appear as a result of new combinations of elements.” The genetic origin of chloroplasts and other plasts from SYMBIONTICISM AND ORIGIN OF SPECIES 111 the blue-green algae presupposes a morphologic reaction, upon the acquisition of the symbiotic relationship. This is in harmony with the usual reactions observed in micro- symbionts. Furthermore, it harmonizes with the adaptive behavior of living organisms in general. Merejkovsky (720) has recently advanced the hypothesis that the chloroplast is a microsymbiont, genetically related to the blue-green algae, and that all the higher green plants are symbiotic complexes. He has emphasized the morpho- logic and physiologic similarity of chloroplasts to the Cyanophyceae. It appears, from an analysis of Merejkov- sky’s treatise, that he could not harmonize a mitochondrial origin of plastids in plant ontogeny with a genetic origin from the Cyanophyceae. He does not believe that the plastids originate from mitochondria in the ontogenetic development of the plant. In lowly forms lke the algae, it is possible for the plastids to divide and multiply as such; the transmission of microsymbionts in algae from one gen- eration to another is a comparatively simple matter.. In the higher plants reproduction is complicated, and the transmission of an absolute symbiont must be associated with the germ cell or its accessory parts. The potentiali- ties of the entire plant are crowded into the germ cell; it is not surprising therefore to find the symbiont also modified for transmission. While we agree with Merejkovsky’s conception of the symbiotic nature of all the green plants (we would extend his conception to include all plants), we cannot agree with his rejection of the association of mitochondria in ontogenetic chloroplast formation. The careful researches of Guilliermond and others, appear to have definitely established this relationship. Merejkov- sky, perhaps has been misled by the assumed fixed phospho- lipin constitution of mitochondria. The question may arise,—Under what circumstances does Symbionticism take place? It is impossible to give a 112 SYMBIONTICISM AND THE ORIGIN OF SPECIES specific explanation that would fully answer the question. Nevertheless, we can appreciate the complexity of the situation when we review the behavior of known micro- organisms and the factors regulating their activities. The reactions of pathogenic microédrganisms, perhaps, have been investigated more fully than those of any other group; their behavior is more significant, also, because of the rela- tionship they bear to other forms of life. A brief discussion will suffice to indicate some of the ‘‘peculiarities’”’ of micro- bial behavior, as well as the importance of variations in the host with respect to infection. A large number of pathogenic microérganisms have a limited specificity as regards the plant or animal species in which they are capable of producing disease. The avian tubercle bacillus, for example, is capable of producing tuber- culosis in birds, cattle and swine, but apparently not in man. The bovine strain of the tubercle bacillus produces disease in cattle, man and certain laboratory animals, but not in the horse. Some microérganisms have a marked tendency to localize in certain tissues, for example the globoid bodies of anterior poliomyelitis. The diphtheria bacillus is limited in its infections to the mucous mem- branes, especially those of the respiratory tract, and to wounds exposed to the air. The host also plays an important réle in the determina- tion of infection. The tubercle bacillus does not ordinarily produce definite disease in a normal, healthy individual. When vitality is lowered, the organism may become im- planted and develop. The response of the host to an in- fection, or to the presence of a pathogenic organism varies within wide limits. In a particular type of infection, one individual may respond with a violent reaction while another host may manifest very little or no response. An example of marked tolerance is found in those cases spoken of as “carriers.” Typhoid carriers are rather common and may not be conscious of the infection they harbor. SYMBIONTICISM AND ORIGIN OF SPECIES 113 The influence of environmental factors on the response to infection has repeatedly been demonstrated. Pasteur rendered the naturally resistant hen susceptible to anthrax by chilling the bird with cold water. Frogs, which are immune to anthrax at ordinary room temperature, quickly die after an inoculation if placed at a temperature of 25° to 35°C. (Jordan). Obviously, not all symbiotic relationships result in the origin of species. There are a number of microsymbiotic relationships, apparently, which lack a morphological re- sponse. This is especially true in extracellular symbiosis, and probably obtains in some intracellular symbiotic rela- tionships. It is practically impossible to draw a sharp line between parasitic relationships and symbiosis. Fur- thermore, there may be responses in the host under these conditions that are not permanent. It is evident that in those cases of symbiosis in which the response in the host is permanent and produces new species, a peculiar combination of factors, both extrinsic and intrin- sic, in two distinct symbionts have met and are harmonious. It would be futile to attempt to analyze and determine the nature of these factors. An analysis of fertilization may serve to elucidate their significance. The sperm of a species possess a definite affinity for the ovum of the homol- ogous species. In some instances the sperm may pene- trate an ovum of a closely related species and fertilize it. The individual that develops from this fertilization is usually sterile or at least abnormal. Changing the com- position of the sea water surrounding the eggs of certain marine animals, may be sufficient to prevent fertilization by the sperm. Certain temperatures may produce similar results. In other words, if normal reproduction is to take place, it is imperative that certain extrinsic and intrinsic conditions be satisfied. The responses of a host to the presence of a micro- 114 SYMBIONTICISM AND THE ORIGIN OF SPECIES organism in Symbionticism may not be limited to a single tissue or organ. If the particular microsymbiont has the property of producing some chemical substance, this may not only affect the particular group of cells in the produc- tion of a new cell type harboring the symbiont, but the chemical substance may alter the physiology of a number of distant tissues and organs. Such reactions are com- monly found in connection with pathogenic microérganisms. The pathogenic organism may be localized in a particular group of cells producing abnormalities in the tissues of that organ, but the toxins may affect specifically other tissues of the host. The “nidamental gland,” whose origin is as- sociated with symbiotic bacteria, apparently influences muscular tissue in such a way that it transforms or dif- ferentiates into a reflector for the luminiferous organ in the squid. It is possible that many tissues in an animal pre- sent secondary modifications or responses to symbiont influences. It appears to the author that Symbionticism offers a rational explanation for many of the variations in the morphology and physiology of plants and animals. When these variations are of sufficient magnitude and permanence they constitute new species. The fact that mitochondria are universally present in the cells of all organisms higher than the bacteria, that mitochondria are bacterial in nature and that microsymbiosis can determine morphologic and physiologic changes in organs and cells, can lead to no conclusion other than that Symbionticism is a fundamental causative factor in the origin of species. CHAPTER IX SYMBIONTICISM IN RELATION TO HEREDITY AND DEVELOPMENT When Darwin advanced his theory on the origin of species, there was practically nothing known concerning the mechanism of heredity or the factors concerned in development. Darwin felt the need of an hypothesis that would explain the operations of heredity, and advanced his own theory of ‘‘pangenesis”’ to fill this gap. The situa- tion is quite different today; instead of formulating a new theory of heredity to fit our ideas as to the origin of species, it becomes necessary to fit the conception of Symbionticism to a more or less established mechanism of heredity. Since the rediscovery, in 1900, of Mendel’s researches there has grown up an extensive literature on the nature of the factors of heredity. It becomes necessary to limit our discussion to the generalizations and analyses that have been made in reviews on genetics and cytology. We are especially indebted to Wilson’s comprehensive treatise: “The Cell in Development and Heredity” (’25); to Morgan, Sturtevant, Muller, and Bridges (15) for ‘“The Mechanism of Mendelian Heredity;” and to Conklin for an analytic review of the researches in heredity, delivered as a series of lectures under the William Ellery Hale foundation and published in the Scientific Monthly (19-20). Space does not permit an extensive review of the data that have been brought forth in these books and papers. We prefer to discuss some of those features of heredity and development that appear to have a direct bearing upon the theory of Symbionticism. Certain difficulties are encountered in the use of some of 115 116 SYMBIONTICISM AND THE ORIGIN OF SPECIES the terminology of the past on account of the more or less specific conceptions associated with these terms. We are immediately confronted by the phrase “acquired charac- teristics.’ Although this term is sometimes used with a different meaning from that originally proposed, to many it conveys the idea of modifications that occur in an indi- vidual as the result of outside influences of whatever nature. In Symbionticism, it would appear that the ac- quisition of a microdrganism and the morphologic re- sponse of the host constituted an ‘‘acquired characteristic.” The theory of Symbionticism implies that such an acquired character is “impressed” on heredity, and is transmitted from generation to generation. It is apparent, however, that we must differentiate between various kinds of “ac- quired characteristics.”’ We recognize acquired charac- ters that are the result of extrinsic forces, such as tempera- ture, light, moisture, chemical and physical agents, etc. Other acquired characters are physiologic in nature and may be produced by ‘use and disuse.” We might also include the “acquired characteristics” that are artificially produced, such as dehorning of cattle, cutting of plumage, castration, etc. We agree with the majority of students of heredity, that a mechanism whereby acquired charac- teristics of this kind might be transmitted in heredity has never been demonstrated nor logically explained. The “acquired characteristics” associated with Symbionticism are of an entirely different nature and relationship to the organism. It may be necessary to modify some of the ideas that are held in connection with heredity, but these modifications, perhaps, are not so momentous as they might at first appear. It is also proper to point out that some of the conceptions held in heredity are still in the hypothetical stage. Some facts, apparently, have been established in regard to hered- ity and development and something is known as to the SYMBIONTICISM IN RELATION TO HEREDITY 117 nature and behavior of mitochondria and microsymbionts. While a few minor points may be at variance, the theory of Symbionticism appears, in general, to harmonize with the modern conception of heredity. The facts that have been established in connection with mitochondria are few, but to the point. It has been shown by a number of investigators that, in the division of the cell, the mitochondria are more or less equally divided and distributed to the daughter cells (see plate I, fig. 3). Mito- chondria are present in the germ cells. In the ovum they usually retain their characteristic appearance while in the sperm they may coalesce to form the nebenkern and spiral filament. Mitochondria increase by simple fission. The evidence for the bacterial nature of mitochondria has been submitted by the author, and reviewed in a previous chap- ter of this book. Up to the present time, this work has neither been confirmed nor discredited. The question arises: What réle do the mitochondria play in the mechanism of heredity? The remarkable researches that have been made by Morgan, Davenport, Castle, Calkins, Guyer, Bridges, Shull, Sturtevant, and many others in this country, deVries, Janssens, Johannsen, Bateson, Doncaster, and others in Europe, have fully established the localization of the heredity transmitting factors in the chromosomes of the nucleus. The history of these researches appear to prove beyond a doubt that the chromosomes are the bearers of heredity. Morgan and his associates, in particular, have been able to corre- late certain characteristics with certain chromosomes. The discovery of the odd or X-chromosome by McGlung (02) made it possible to correlate definite characters with this X-chromosome. Factors that are located in chromo- somes of this nature have been called sex-linked by Morgan. By studying variations in the animal or plant, and corre- lating these with the behavior of chromosomes, it has 118 SYMBIONTICISM AND THE ORIGIN OF SPECIES been found that ‘‘elements” of one chromosome may cross over to another. These “elements” have come to be generally known by the name ‘‘genes.”’ Various modifications in chromosomes have been ob- served by different investigators. It is only the more ex- treme variations, however, that can be detected. It is generally admitted that these modifications are abnormal in nature; they result in abnormal distribution of chromo- somes in development and are of no importance in evolution. Conklin says: Abnormalities in the distribution of chromosomes of a cell persist in all its daughter cells and are not usually corrected. On the other hand, abnormalities in the distribution of cytoplas- mic substances to the cleavage cells frequently occur and usually the cell returns to a normal condition by a process of regulation. But when once the normal number of chromosomes has been changed there is no regular means of restoring that number. Momentary changes in the temperature, density or chemical composition of the medium may thus produce permanent changes in the distribution of chromosomes and of germplasm. Such changes in the distribution and number of chromosomes are usually of so gross a character that the development is not only very abnormal but also the resulting organism is incapable of continued life and reproduction. Consequently such changes do not usually give rise to new races or species; only in the most favorable cases, where at least the full set of haploid chromosomes is present does the new form survive and reproduce. The central thought that has developed in connection with the researches in genetics is focused on ‘‘genes.”” The investigations that have led up to this position are too numerous to review here. ‘To those who may doubt the validity of this concept, we can only refer to the extensive reviews and discussions which have been published—such as Conklin’s, Morgan’s, Bateson’s, Wilson’s and others. Certainly, the experimental and observational evidence SYMBIONTICISM IN RELATION TO HEREDITY 119 points to a smaller unit than the chromosome, whatever the nature of this unit may be. The genes have been thought of in terms of atoms or molecules. The work ac- complished in genetics in which genes have been accepted as the transmitting unit, has enabled biologists to appreciate the nature of the mechanism concerned in hereditary varia- tions. But we must raise the question: Do hereditary variations lead to evolutionary variations? The variations that have been experimentally produced by deVries and others, apparently, are hereditary variations, explainable on the basis of the behavior of genes. But have these mutations represented true species? Many modern biologists are not convinced that they are stable species. Such mutations may breed true for a time, but it appears that these variations are not of the character nor of the rank of evolutionary variations. We must attempt to look further into the hypothetical nature of genes, and attempt to correlate ‘“‘genes” with evolutionary variations. It appears from a perusal of the literature that ‘‘genes’”’ are to be considered as some sort of molecular entities, of the same nature in all forms of life, but varying in their arrangement and grouping in the chromosomes of different species. This conception, apparently, carries with it the idea that new genes are never added to the chromosomes, but that the genes in an amoeba are the same as those in man; the essential difference being the arrangement and grouping in the chromosomes. It is believed also that the genes may be modified by intrinsic environmental factors. Morgan, perhaps, had this conception of genes in mind when he said, ‘‘Evolution consists largely in introducing new factors that influence characters already present.” This conception of ‘‘preformed” or limited genes has led to more or less bizarre hypotheses on the mechanism of organic evolution. 120 SYMBIONTICISM AND THE ORIGIN OF SPECIES The ‘‘presence or absence” hypothesis assumed that dominant characters are due to the presence of a factor and recessive characters to its absence, and since regressive mutations, in which some dominant character becomes recessive, are much more numerous than progressive ones, it was suggested by Shull, Bateson, and Davenport that evolution might be due to the loss or disintegration of factors or genes. ‘This conception results,” said Shull (’07), ‘an an interesting paradox, namely the produc- tion of a new character by the loss of an old unit,” and he sug- gests that at least the later stages of evolution may be a process of analysis due to the disappearance of one unit after another. Bateson (714) also proposed the same thing in his well-known inquiry ‘“‘whether the course of evolution can at all reasonably be represented as an unpacking of an original complex, which contained within itself the whole range of diversity which living things present;’’ and in the same category is the speculation by Davenport that ‘‘the foundations of the organic world were laid when a tremendously complex molecule capable of splitting up into a vast number of simpler molecules, was evolved.” [Quoted from Conklin.] These conceptions, perhaps, have not been generally ac- cepted, although the essential idea of “preformed” or limited genes appears to be retained by most students in genetics. It is necessary for us to question the logical probability of one feature of these conceptions of genes. Obviously, it is impossible morphologically to examine a gene, but we are justified in entering into a speculative analysis of these factors, since all the analyses that have gone before have been speculative. This does not necessarily imply that they have been without a basis. But, just as the most generally-accepted view of the nature of mitochondria was based on an assumed or misleading conception of the chemical constitution of these bodies, so also, it appears that an assumed or misleading conception of the nature of genes has led to a “blind pocket” in heredity and evolu- SYMBIONTICISM IN RELATION TO HEREDITY 121 tion studies. What evidence is there to assume that new genes are not acquired in evolution? The basis for this assumption apparently, has rested on the conception that the ‘‘mechanism of heredity is the mechanism of evolution.” Our conceptions in any field of intellectual and scientific endeavor are based upon the analysis of conditions so far as they have been made. When more phenomena are dis- covered, new points of view enter into our speculative analyses, and it may become necessary to alter our pre- vious conceptions. It is evident that the modern concep- tions of genes were justified on the evidence that had accumulated. There was no evidence at hand to indicate that ‘‘new genes” were acquired in phylogenetic develop- ment. A mechanism whereby new genes might be acquired had not been indicated by cytological researches. Only one alternative remained, and that was to assume that life originated as a “‘tremendously complex molecule, capable of splitting up into a vast number of simpler molecules,” or in other words, that the amoeba contains the same genes that are present in man. On the basis of the new point of view that is associated with Symbionticism, we are forced to the conclusion that new genes must be acquired in organic evolution. This conception does not alter our ideas as to the behavior of genes in heredity, but it intro- duces a “new” factor in the conception of organic evolu- tion. This new point of view, furthermore, displaces hypotheses that are antagonistic to the general and broader principle embodied in the theory of organic evolution, namely, that evolution consists of ever-increasing accre- tions resulting in greater complexity of structure as well as of function. It is logical to assume that there have been ever-increasing additions to organisms during organic evolution. These “additions’’ must have come from the outside, and represent true accretions which are responsible for the origin of species. 122 SYMBIONTICISM AND THE ORIGIN OF SPECIES The established mechanism of heredity permits the development of variations in organisms, but these varia- tions, apparently do not lead to the production of new species. ‘These variations may be of a retrogressive nature, resulting in the loss of parts or characters, but apparently no fundamentally new character is produced by a rearrange- ment or loss of genes. It apparently is necessary that “new” genes be added to chromosomes in order to produce a fundamentally new character. How, then, does Symbionticism affect the accepted mechanism of heredity and correlate it with our ideas of organic evolution? The conception that mitochondria may be involved in the transmission of hereditary charac- ters was enunciated by Meves in 1908.