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Part I, continued:

   One of the most important expressions of life of organisms is the sexual process. In higher organisms this process is morphologically well expressed and has been investigated in detail. It is also well expressed and has been investigated in lower organisms--algae, fungi, and protozoa.

   The question of a sexual process in bacteria and actinomycetes remains undetermined. Does it occur in these organisms? This question has been vividly discussed in the literature of recent years. Former views on bacteria as primitive organisms gradually changed under the pressure of accumulated factual material. A search for complicated cycles of development in bacteria as well as for copulation processes was started. In the period of enthusiasm for theories of a complicated cycle, various forms of sexual process in various specimens of bacteria were described. Many authors assumed that all bacteria reproduce by copulation, which is in general peculiar to all other higher and lower organisms. This process takes place in various specimens of bacteria, although in a primitive manner, externally manifested in various ways. It may proceed, according to the opinions of some authors, in the form of autogamy, hologamy, or oögamy (see Krasil'nikov, 1932 b, c).

Figure 21. Phenomenon of autogamy in Bac. bütchlii

   A primitive type of copulation in bacteria deserving great attention was described by Schaudin (1902). This investigator observed fusion of a divided cell in the relatively large bacillus which he discovered--Bac. bütchlii. This microbe lives in the intestine of the black beetle, however, apparently, only sometimes , since many investigators could not find it there.

   According to Schaudin's data, copulation in Bac. bütchlii does not occur between separate organisms, but between parts of one and the same cell. The whole process is described by the author as follows: after isolation of the coarse-grained bacillus in a drop of the intestinal juice of the beetle, glistening granules, surrounded by a clear halo are visible in the center of the cell after 30 minutes. Exactly as in division the granules are distributed in a row and after 20-40 minutes they form a transverse septum, which does not differ in living or in fixed and stained cells from the septum observed upon ordinary cell division. The bacillus remains in this phase approximately 1-2 hours, after which the transverse septum becomes pale and thinner, the granules distributed in a row along the septum disappear and the cell of the bacillus looks as it did before the formation of the septum (Figure 21). Before the dissolution of the septum multiple granules of chromatin appear in the plasma, they are distributed in a long line and form a peculiar chromatin thread. This thread disintegrates into granules upon spore formation, the majority of granules moving to the poles of the cell and forming large bodies there. The plasma is concentrated around these bodies and a prospore is formed, and later, a mature spore. There the cell that underwent division fuses again, the chromatin of the two cellular parts fuses into one body and then divides in two. This is followed by division of the protoplast into two parts, from which spores are formed.

   The described way of autogamous copulation closely resembles a similar process in some algae--in different species of Spyrogyra condensata, Sp. sprellana and other forms.

   An analogous picture was observed by us (1928) in the bacteria Oscillospira which live in the intestine of the guinea pig. In its internal and external structure this organism is similar to the filamentous blue-green algae of the genus Oscillaria, but in contradistinction to them, it does not possess a pigment. Each organism represents a filament of greater or shorter length, divided by transverse septa into short cells.

   During the course of its development Oscillospira forms spores. Before sporulation two and sometimes three adjacent cells of one filament fuse; the septa separating these cells dissolve and disappear. and the protoplasts of cells fuse into one body, which, by an appropriate reorganization, transforms into a spore. Upon fusion of the protoplasts, the central bodies and chromatin granules distributed into them fuse into one large body (Figure 22).

Figure 22. Copulation of cells in bacteria Oscillospira guilliermondii before spore formation:

a--fusion of three adjacent cells; b--fusion of two cells. The arrow shows consecutive stages of development.

   We observed the fusion of parts of a divided protoplast in the yeasts--Saccharomyces paradoxus. As is known, in this organism the protoplast of the cell divides into 3-4 parts before sporulation. First the division of the nucleus takes place, and is followed by that of the plasma. From each part of the protoplast a spore is formed. Mature spores released from the maternal cell germinate into vegetative cells (Bachinskaya, 1914).

   Often before the spores become permanent vegetative organisms, they combine into pairs and copulate. Copulation of spores may proceed inside the capsule. Often 2-4 spores copulate. This kind of copulation is observed in many yeasts (see Krasil'nikov. 1935) and is regarded as a usual form. However in the yeast organism mentioned, aside from this form, an abortive copulation which does not proceed to completion in noted.

   After the fragmentation of the protoplast into separate parts, spore formation begins, and small bits of protoplasm become rounded and dense with the cell walls outlines. Afterward maturation of spores stops and the process proceeds in the opposite direction. The contours of the outlined cell wall disappear and the protoplasm becomes less dense, and undergoes vacuolation. Separate not organized parts appear; very often the residual cell walls may be seen among them (Figure 23). Then a fusion of the parts and the nuclei occurs. As a result, the cell capsule takes on the initial appearance of a vegetative cell, and soon processes of growth and multiplication start again.

Figure 23. Autogamous copulation in the yeasts Saccharomyces paradoxus Batschin. Consecutive phases of sporulation:

-b--protoplast divides into 3 -4 parts; c--onset of wall formation around the prospore; d--the wall disappears; e--content of prospores fuses; f--cell is transformed anew into vegetative cell; g--budding starts.

   This, process was regarded by us some time ago as a reversed development of the organism (Nadson and Krasil'nikov. 1920). Afterward we came to another conclusion, and related thin phenomenon to autogamous copulation, somehow similar to that which takes place in Bac. bütchlii or in protozoal organisms.

   The process of autogamy may evidently take place between various parts of the cell, which did not undergo preliminary division. The expression of extreme autogamy was noted by many authors. Data on observations of fusion of chromatin granules into one body before sporulation and sometimes before division have already been cited above. We observed such a fusion of cellular parts in certain large bacilli, which were isolated from the intestine of a guinea pig. The chromatin granules distributed throughout the protoplast combine into one large body. After some time the body either divides into two and is followed by the cell division, or it becomes the center of spore formation. In some microbes the fusion of chromatin is accompanied by formation of long threadlike fibers, which are straight or spirally bent from one pole of the cell to the other. Such formations were observed by us (1928), in a peculiar microbe--Metabacterium octosporus. This microbe lives in the intestine of the guinea pig; it is of a large size, 10-20 x 3-5 µ, nonmotile, and forms from one to eight spores. The cells multiply by division. The protoplasm in young cells is homogeneous, dense, and stains a dark color with basic dyes. As the cell grows, granules of chromatin and metachromatin appear in the plasma.

   Before spore formation, chromatin aggregates into a spirally bent thread which is distributed along the longer axis in the central part of the cell. Afterward this thread divides into parts, usually into four to eight, sometimes into two to three Each part becomes a center of organization and spore formation.

   Copulation of the hologamic type in various specimens of bacteria was described by many authors. In essence, this manner of copulation is as follows: after a series of consecutive divisions, two isolated cells combine in various ways and exchange their contents or fuse into one organism. The fusion of protoplasts proceeds through copulation canals and small bridges. Formation of bridges between two cells was noted by many investigators. Rindfleisch (1872) described a similar combination in bacteria and considered it as a process of copulation. Klebs (1896) and Albrecht (1881) observed the fusion of cells by means of canals in spirochaeta, Fuhrman (1906)--in coccoid bacteria, Forster (1892)--in purple bacteria, etc.

   The observations of Potthoff (1924) are most widespread. He described the formation of small bridges in the purple sulfur bacteria--Chromatium okenii, Ch. weissii, Ch. violaceus and in the Spirillum photometricum. According to his observations sometimes three or more cells are consecutively interconnected by canals, which in his opinion are copulation canals. Potthoff even observed fusion of chromatin granules (Figure 24.

Figure 24. Joining of cells by means of "canals" in Chromatium okenii (after Potthoff, 1924)

   Löhnis (1921) described such a process of cell fusion in Azotobacter and some other bacteria. According to his observations the cells combine by bridges, through which the fusion of protoplasts occurs. A similar type of copulation was described by Lieske (1926) in bacteria of the coli group which were isolated from a tobacco extract. Cunningham (1931) found cells combined by means of a canal in 20 strains of Bac. saccharobutiricus.

   Mellon (1925) described spherical elements in a culture of Bact. coli. In his opinion they represented zygospores formed upon fusion of two rodlike cells. Stoughton (1932) noted similar formations in Bact. malvacearum. He regarded a part of the spherical bodies as zygotes, and part as budding germs.

   Smith (1944) described fusion of cells in Pseudobacter funduliformis; Lindegren and Mellon (1932) in the tubercle bacillus (Mycob. tuberculosis, var. avium). Klieneberger-Nobel (1949) observed the fusion of nuclear bodies in bacteria before the formation of L-forms. According to her observations, rodlike cells are fragmented into small parts, each possessing one chromatin body. These small parts then combine. As a result, the unusual. so-called L-forms are formed.

   Stempen and Hutchinson (1951) observed development of cells in Bact. Proteus (OX-19) by means of microphotography. They noticed formation of strongly swollen spherical cells. These spheres are combined with one to three rodlike cells. As the growth of the spherical cell proceeds, the rods become shorter, paler and disappear completely; their substance appears to have entered into the spherical elements. On this basis the authors concluded that these spheres represented zygote cells which undergo disintegration with the formation of either fine-grained elements or are transformed into rodlike elements by consecutive divisions. Under favorable conditions the granules may be transformed into regenerative elements and develop into vegetative cells (Hutchinson and Stempen 1954).

   Analogous formations were observed by Dienes (1930, 1943) in Bact. proteus, Bact. moniliformans and in Pseudobacterium (Bacteriides) (from Dubos, 1948).

   Examining the microphotographs obtained by Stempen and Hutchinson we can not agree with their interpretation. In our opinion the spherical cells observed by them represent not zygotes but involution forms. The forms described by them were observed by us in Azotobacter, Mycobacteria and other microorganisms. They are often combined with rodlike elements.

   The cited data on the fusion of cells are also not convincing. They mostly represent a result of deductions based on the study of fixed and stained preparations. Direct microscopic observations on development and fusion of cells were not carried out by them.

   Our study of a large number of bacteria did not enable us to establish any external signs of cell fusion, which resembled a sexual process. In some species peculiar small bridges between the cells are observed. However, a detailed investigation of such formations showed that they represent the result of an unfinished constriction of cells. As was indicated above, when cells of Azotobacter multiply by constriction, long plasmodesms are often formed, which resemble copulation canals (Figure 25). These plasmodesms sometimes move to a lateral surface of the cell, and even possess a small swelling with a tiny granule inside it, in the central part, which resembles a sexual process even more. We did not observe any indications of fusion of unseparated cells.

Figure 25. Joining of cells by plasmodesms (incomplete division):

(A) Chromatium warmingii and (B) Azotobacter chroococcum: a. b, c, d, e, f. g, i, l, m--consecutive stages of cell division.

   The same may be said in relation to fusion of cells in Chromatium described by Potthoff (1924). We had the opportunity to follow in detail the formation of bridges in Chromatium okenii and Chromatium warmingii in a hanging drop. As in Azotobacter, the cells of these bacteria under special conditions , multiply by constriction and form long canals (Figure 25 B). The canals are often displaced to a lateral surface. This displacement, as indicated above, takes place because the cell grown at the end, in the growing point, and thus shifts the site of attachment of the canal sideward (Krasil'nikov 1932b, c).

   Löhnis and some other investigators regard the extracellular fusion of the protoplast of two and more cells after the disintegration of their cell walls as a sexual process. This fusion of the substance of the disintegrated cells was named by Löhnis "symplasm". It is observed on the aging of cells, and under unfavorable growth conditions. The cells, often strongly swollen, disintegrate, their cell wall disrupts and undergoes lysis and the cell contents leak out and fuse with the contents of other cells. A protoplasmic extracellular mass or symplasm is obtained. According to Löhnis processes characteristic of copulation proceed, there. After some time, new organisms having the nature of a zygote are formed in the symplasm.

   According to our observations, the described symplasm sometimes does indeed contain germs or regenerative bodies. However they are usually formed in the cell before its disintegration and do not constitute a fusion product of two or more protoplasts. Symplasm represents a post-mortem formation of cells, a mixture of protoplasm of already dead or moribund cells. It to possible that in single cases. regenerative units are formed In such a mixture of still living protoplasts (Krasil'nikov 1932 d 1954 b).

   Several investigators describe combinations of cells of bacteria which strongly resemble the conjugation of protozoal organisms. Rodlike cells unite at their ends into groups, several cells in each group, and form peculiar groups. Inside the cells a shift of the nuclear inclusions takes place. A similar fusion of cells is observed in Pseudomonas radiobacter, Ps. tumefaciens, Bac. stellatus and other forms.

   In cultures of these bacteria, starlike groups of cells which are joined by their ends may often be seen. The cause of these formations and their meaning are not clear. Stapp and others (1931, 1949, 1955) subjected these cells to careful study and came to the conclusion that the formation of cells into starlike groups represents a sexual process during which a combination and fusion of hereditary material proceeds. They follow the formation of these groups, the fusion of the cells and further events, step by step. It was established that the cells indeed combine by their ends into firm groups. The nuclear substance, chromatin granules and nucleoids move toward the site of fusion, the cell wall dissolves and these granules and nucleoids of the combined cells fuse into one large body. The latter remains in one of the cells, which starts to develop and gives rise to a new generation. The body gradually becomes looser. decreases in size and begins division. The division of nucleoids is followed by the division of the cell. Two. three, four, five and more cells may take part in the process of union described (Figure 26).

Figure 26. Union of cells into starlike complexes (after Stapp, 1956): a--Ps. Radiobacter; b--Ps. tumefaciens; c--Bac. stellatus.

   A detailed picture of cell fusion was observed by us in some strains of root-nodule bacteria of the pea, bean, vetch, and other legumes. When these bacteria were cultivated on synthetic media (CPI. Capelu or "chkapeka" in Russian) or on extracts of leguminous plants, starlike groups of cells were often seen. By a careful cytochemical analysis and observations of their development in a hanging drop it was established that the cells are joined quite firmly by the ends and are not disrupted under pressure. This connection is not mechanical, but organic, although a dissolution of the cell wall at the sites of the attachment of the cell was not observed by us. At the end of each cell one chromatin granule (nucleoid) is distinctly seen. After some time these nucleoids undergo dissolution or become invisible and then the cells begin to grow and multiply. The plasma of such reorganized cells is optically homogeneous; granular inclusions and nucleoids appear later.

   As a rule. the cells in starlike groups after transfer to a fresh nutrient substrate do not develop immediately. A long time passes before they begin growth and multiplication. Until that moment they remain in the state of reorganization of the protoplasts. We assume that the picture of cell combination into starlike groups described in root-nodule bacteria is connected with some metabolic process. The cells mutually exchange the products of their metabolism, one cell assimilates some substances released by the other.

   Recently papers were published in which the sexual process in bacteria is regarded as an exchange of substances between one cell and another upon direct contact without any special copulation structure and without disintegration of the cell wall. It is suggested that specific cell substances--carriers of hereditary properties--penetrate through the cell walls of the continguous cells and in this way the process of fertilization is achieved. In the author' a opinion, in each culture of bacteria there are unisexual and bisexual cells. In the latter, fertilization takes place by the autogamy described above. The unisexual cells constitute a small percentage and are formed in special cases of metabolic disturbances, under the action of some environmental factors [such as] "ultraviolet light", and chemical agents; they also occur in old cultures. They lack the ability to perform certain functions, which are restored upon contact with other bacteria carrying the opposite features (Braun. 1953 ; Lederberg and Tatum. 1954. and others).

   This method of mutual fertilization was described in several strains of Bact. coli variant K = 12 by Tatum and Lederberg (1947, 1954); Hayes (1953), Catcheside (1951), and others. They indicate that on the mixing of cells of various strains of Bact. coli after their subsequent plating on an agar medium, one obtains new strains with properties of the parent cells (see chapter on variation).

   Some investigators assume that bacteria perform a sexual process of the organic type. According to observation of Ferran (1885), cells of Vibrio cholerae form male gametes--antheridia, and female gametes--archegonia. The archegonium, according to him, is fertilized by the antheridium and transforms into a zygospore. From the latter a vegetative cell develops under favorable conditions.

   Enderlein (1925) made the course of a developmental cycle of bacteria in general and of the sexual process in particular very complicated. According to his views, at a determined stage of development cells form special bodies--gonites or gonidia, subjected to meiosis. The gonites develop either into spermites or into oites. The former are small, rodlike, straight or slightly bent, very motile with a flagellum on one extremity; the latter are larger. of spherical form. nonmotile. An oite fertilized by a spermite transforms into a mycete, initiating normal vegetative cells. The sexual cells--oite and spermite represent haploid forms, as a result of fertilization, a diploid--the mycete--form is created.

   Enderlein has no factual material to confirm his hypothesis. Since he was not a microbiologist he mechanically transferred data from zoology into the microbial world.

   In spite of lack of objective evidence, this hypothesis had many followers among microbiologists. There were and still are followers who try to find a basis for it. Data from bacterial life are given by them in order to confirm Enderlein's views (Broadhurst, Mariyama Pease, 1931, Almquist, 1925; Mellon, 1925; De Lamater, 1951, and others).

   Almquist (1925) describing the sexual process between differentiated sexual cells in bacteria, assumes that this process may proceed not only between cells of one species but also between those of different species. producing hybrid offspring.

   The latter were obtained by him upon mixing cultures of Bac. typhi and Bac. dysenteriae. The hybrids differed from the initial cultures and at the same time had features common to both.

   Almquist and other followers of Enderlein's views bring data which is not very convincing as evidence. The so-called antheridia and oögonia or sporogonia observed by them have nothing in common with the differentiated sexual cells of bacteria. In cultures of the latter particularly, as is known to microbiologists, in old cultures, there are always greatly enlarged organisms, and very small forms, which, if one cares to do so, may be regarded as female and male sexual cells. However, none of these authors showed the essential importance of these cells in the process of fusion under direct and constant observation in a hanging drop. All statements are based on analogies with known facts of sexual processes in other organisms.

   In actinomycetes the question of a sexual process remains to be elucidated. There are only a few casually stated opinions. For instance, Kober (1929) expressed the view that in actinomycetes fusion of cells occurs by direct contact.

   According to our observations, the fusion of cells in actinomycetes occurs in two ways: a) by a combination of outgrowths of the spores, b) by combination of mycelial filaments by means of anastomoses. Both are found under usual growth conditions (Krasil'nikov, 1938 a).

   Fusion of cells may be observed under direct constant observation in a hanging drop in many species of actinomycetes. The process proceeds as follows: upon germination of spores, small appendages shaped like small tubes are formed (outgrowths); these tubes come into close contact at the ends, at the site of contact the cell walls dissolve and a canal is formed. Through this canal the contents of the two germinating spores combine and fuse into one protoplast . The combined outgrowths produce a common sprout which extends into a long filament and then grows, becoming a mycelium (Figure 27).

Figure 27. Fusion of cells in actinomycetes:

A--Joining of germinating spores in Act. chromogenes. B--union of mycelial filaments by means of anastomoses: a--joining of filaments genetically distant from one another; b--joining of branches of daughter and maternal hyphae genetically related; c--joining of genetically related sister branches.

   This fusion of spores was observed by us in different species of actinomycetes (1938a), Later (1950) this process of fusion of germinating spores was noted in many species of actinomycetes. By its external manifestation the described fusion of spores did not differ from similar fusions in many yeasts (Krasil'nikov. 1935; Kudryavtsev, 1954; Guilliermond, 1920; 1941, Gäumann, 1949).

   As in other yeasts, fusion of chromatin, nucleoids and chromatin granules occurs upon fusion of the growth tubes. If in yeasts a similar act is regarded as a sexual process, there is no basis for not accepting it in Actinomycetes, though we have not yet sufficiently investigated the cytochemical changes in the protoplast of conjugated cells.

   The joining of cells by means of anastomoses is observed in actinomycetes considerably more often than the fusion of spores. This may be seen in any culture on various nutrient media, liquid or agar. Externally, anastomoses in actinomycetes do not differ from those in fungi. As in the latter, hyphae are formed between the mycelial filaments of actinomycetes. These are small bridges in the form of a canal connecting two more or less distant filaments. This canal may be quite long when it connects two filaments located at a great distance from one another. The canal may occur between branches located side by side and closely related, originatng from one hypha, as well as between distant branches originating from various hyphae of the same mycelium. (Figure 27 B).

   Anastomoses have no septa, and because of this they do indeed represent canals, through which the protoplasts of filaments fuse. This may be seen directly under the microscope in a preparation of a living culture. Separate granules, which are in constant Brownian movement, pass from the filament into the canal, from there these granular inclusions move into the second filament. An exchange of protoplasmatic substance occurs between the hyphae and fusion of substances, such as chromatin.

   Consequently, between the hyphae of actinomycetes a process takes place which may be qualified as an autogamous copulation.

   Formation of anastomoses occurs not only between filaments of one and the same culture but also between filaments of various strains of one and the same species: we observed anastomoses between various strains of Actinomyces streptomycini, producers of streptomycin, isolated from various soils of different areas of the Soviet Union.

   This form of fusion of mycelial filaments is not observed between cultures belonging to different species or even to different varieties. We carried out observations under the microscope. in a drop of a semiliquid nutrient medium. Spores of two actinomycetal species which were inoculated at different sites of the drops germinated and gave rise to hyphae, which grew out and after some time came close together. At the site where the filaments touched, the formation of anastomoses could be observed.

   The process of formation of anastomoses proceeds in the following manner: a side branch of the mycelial filament grows in the direction of a neighboring filament, touches it, and attaches itself by its end. After some time, the walls of the filaments dissolve at the site of attachment and a canal is formed, through which fusion of protoplasts occurs.

   Probably not all branches form anastomoses. Many of them, which came in contact with neighboring hyphae did not attach themselves and did not form anastomoses. It to possible that anastomoses are formed by branches which have some sexual property, and are different from those of usual mycelial hyphae. If it is so, then the whole culture of mycelium should be regarded as a heterogeneous system, where separate filaments and branches have different sexual qualities.

   In general, it should be noted that, while solving the problem of a sexual process in bacteria and actinomycetes, it is necessary to clarify the meaning of the terms "sexual process" and how it is regarded in biology in general and in lower organisms in particular.

   There are various theories regarding the biology of the sexual process. They may be reduced to two basic ones: the theory of plasma mixing and the theory of rejuvenation.

   The theory of the mixing of plasma was first presented by Weismann in the 1880's. Fusion of two cells and mixing of germ plasm or amphimixis, was primarily considered by Weismann, in relation to heredity and formation of species. Such a point of view in still widespread among biologists at present. Developing his views, Weismann stated that the germ plasm was immortal and is passed from generation to generation in an unmodified state. This view of a "potential immortality" of the germ plasm was widely developed in his studies on protozoa. He stated that unicellular organisms were endowed with the ability to proliferate indefinitely. With each division, the two cells formed anew are equivalent. This point of view in refuted at present.

   The hypothesis of rejuvenation puts forward the problem: do cells become old upon prolonged asexual multiplication? Bütchlii (1880, 1887) and then Maupas (1888, 1889) tried to solve this problem experimentally on infusoria. Many lengthy observations were carried out in this direction by Metal'nikov, Hertwig (1902) and others. The investigations led the authors to reject the indicated hypothesis. Numerous observations made by contemporary investigators on bacteria and yeasts also show the erroneousness of this point of view.

   The explanation of the essence of the fertilization process, given by the chromosomal theory of heredity, is built entirely on the assumption that gametes contain particles of a special hereditary substance. According to this theory, factors responsible for the hereditary transmission of properties are located in the chromosomes of cells. They are connected with the chromatin substance of the latter and, according to modern ideas, with desoxyribonucleic acid, which constitutes the fundamental component of the nuclear substance.

   Upon fusion of the copulating cells an entirely mechanical combination of cytoplasm and nuclei occurs. The substances of chromosomes are not dissolved in the protoplast, they do not disappear, and they preserve their peculiarities as well as their characteristic features of heredity. The chromosomes of the female and male cells preserve their individuality in the chemical as well as in the biological sense.

   This invariable part of the nuclear substance assures the continuity of inheritance. The material carriers of heredity, the genes, which are concentrated in it, perform the transmission of properties and traits from parents to progeny.

   These concepts on the essence of the sexual process are refuted as groundless by modern biology and particularly by Soviet specialists, followers of the school of Michurin.

   An entirely new interpretation of the question of fertilization to given by the academician Lysenko. He is of the opinion that the essence of the sexual process consists of the combination of the chromosomal sets of the gametes but not of exchange of substances between these gametes. According to the author, fertilization represents a peculiar process where the copulating cells mutually assimilate substances. The protoplast of one cell is assimilated by the protoplast of the second.

   According to Lysenko, the essential difference in fertilization from all other biological assimilation processes consists of the following: "In any physiological process one part is the assimilating one and the other the assimilated one. . . . . The substances which are assimilated are used as building material for the assimilating component. In the sexual process, when two seemingly equivalent cells combine, there is a mutual assimilation. Each builds itself from the substance of the other, according to its own pattern. Finally, neither of these cells remains, but instead of the two a third new cell is produced" (Lysenko, 1948. p 383).

   The peculiarity of this exchange or assimilation consists in that there is not one but two cells which assimilate. During the process of fertilization the two combined cells--the maternal and the paternal--are equivalent; upon fusion neither preserves its previous individuality. A new cell is obtained, dissimilar from both the paternal and maternal ones. This new cell--zygote--initiates a new organism combining the paternal and maternal traits. The living substance of the zygote is of dual quality, it contains elements of both the paternal and maternal organisms, these elements are there, not in the form of special corpuscles or genes, concentrated in chromosomes. but exist throughout the whole living organism. This heterogeneous quality of the newly formed zygotic cells insures the formation of an organism with new properties inherited from the parent couple. In this way the continuity of inheritable traits and the evolution of the species to secured. According to Lysenko's conclusion, the biological importance of the process of fertilization consists in an increase of viability of the organisms. As a result of fertilization, organisms with a dual heredity, maternal and paternal, are obtained. "Dual heredity conditions a high vitality (in the direct sense of the word) of organisms and great adaptability to variable conditions of life" (Lysenko, 1948, p 381).

   In the majority of cases prolonged self-fertilization in animals and plants, as well as coupling of closely related animals, leads to the extinguishment of life. As a rule, normal viable organisms occur only in cases when plants and animals which differ at least slightly one from the other are coupled. Normal internal life contradictions, and, consequently, also life impulses are created mainly by means of crossing and breeding.

   The above-mentioned principles of the biological importance of the fertilization process. elaborated by academician Lysenko on the basis of Darwin's and Michurin's theories. reflect one of the laws of adaptability and evolution of the living population.

   The usefulness of the sexual process was stressed by Darwin and subsequently by many other investigators. Maupas (1888-89), summarizing the results of numerous observations and experiments, came to the conclusion that conjugation in Infusoria constitutes a process which is indispensable to the renewal of viability. According to his opinion, for every species of Protozoa there exists only a certain number of asexual generations, the cells multiply vegetatively to a certain limit, then age, degenerate and inevitably die. The sexual process leads to restoration of the vital activity of cells. It rejuvenates the Infusoria and initiates a new series of sexual generations.

   Calkins came to these conclusions after ten years of research on conjugations in the infusorium Uroleptus mobilis. He considers the "wearing out " of plasma and organoids as the cause of aging and degenerative senility of cells. Conjugation is indispensable for the restoration of viability. In this process reconstruction of the whole living substance occurs (according to Dogel', 1951).

   In recent years the concept of the biological usefulness of the sexual process was well confirmed by the work of Cleveland. This author studied the phenomenon of fusion of gametes in the flagellates Polymastigida and Hypermastigida in detail. In one of them--Trichonymphs--the gametes are morphologically identical. Upon copulation the male cell penetrates into the female cell and dissolves there, or more precisely, the protoplasts of both gametes mix completely and form a new organism. In Oxymonas the fused gametes remain in the form of a double organism for a few days and then the fusion of important organoids follows. Not only nuclei and chromosomes, but also resistant formations of axostyles which do not have any relation to the protoplast fuse. In the zygote a double, larger axostyle is formed (according to Dogel', 1951).

   The decisive importance of metabolism in the sexual process is confirmed in numerous works of Hartmann, Moewus and other investigators; their subjects of study were algae and protozoa. As will be shown further, for a successful fusion of gametes and formation of healthy progeny, at least a small difference in the chemical composition of their living substance is necessary.

   The process of fertilization in lower organisms, protozoa, algae and fungi, often proceeds in the same way as that of higher forms i. e.. by the fusion of sexual cells in which the nucleus, nucleolus and other important inclusions of the cell always take part.

   Externally the sexual process expressed itself in different ways in various specimens of microorganisms. Besides the true oögamy i.e. , fertilization occurring between highly differentiated sexual cells as in some yeasts and fungi, copulation often takes place between individuals externally similar to one another. Gametes do not often differ from the vegetative cells. There are microbial forms, in which the sexual process occurs not between cells but between parts of one and the same cell, i.e., autogamously.

   In some yeast organisms, upon combination of cells, only fusion of the plasma takes place, the nuclei do not fuse, and in some yeasts a combination of copulation of protrusions is noted, but there is no fusion of cells (Guilliermond, 1920, Krasil'nikov, 1935).

   As was shown above, there is in the literature factual material which constitutes a basis for the assumption that in bacteria and actinomycetes processes occur between cells or parts of cells which are consistent with the notion of fertilization.

   Proceeding from the conjecture that bacteria. (at least some of them) represent not a primitive, ancestral cell but a quite complex organism whose functions of growth, development and life activity are stabilized, in an evolutionary way, and that many of them are evidently degenerative forms of more organized creatures, one must think, that bacterial cells also have sexual rudiments in a potential form which, in a more developed form, are characteristic of higher organisms.

   According to contemporary concepts, any sexually differentiated individual (female or male) and any sexually differentiated gamete concurrently contains all the rudiments necessary for development of the opposite sex. As a result of the increased development of one of the rudiments, and the suppressed development of the other, the male or female tendency of the cell is expressed. The sexual tendency becomes expressed under the influence of various kinds of environmental and internal factors.

   Even in higher organisms the expression of sexual tendency to often conditioned by environmental influences. For instances in corn the sexual tendency changes upon change of nutrition. If in the early period of growth the plant does not get sufficient nitrogen, female flowers develop mostly; on the other hand, if there is a lack of potassium. more male flowers develop.

   In cucumbers and melons, when there is a deficiency of nitrogen during the embryonic period of development when reproductive organs are initiated, formation of female flowers is mainly observed. The market gardeners of Klin have long employed the smoking of cucumber plantations in order to obtain female flowers.

   Milliard (1898) and Schafner (1927) obtained 100% formation of female flowers in hemp, by regulating the duration of daylight (according to Sabinin, 1940). There are many other examples of similar changes of the sexual tendency in higher plants.

   Kuhn (1941), Zhukovskii and Medvedev (1948) and others, assume that the formation of specific substances, sex-"determinants" are determined by light stimuli, short-wave rays of the solar spectrum. It to assumed that sexual tendencies are connected with the photochemical reactions of specific substances. According to some authors, in the expression of sexual tendency. pigments, especially carotenoids play an important role (Lebedev, 1953).

   In our opinion Sabinin in right in indicating that the sexual tendency is determined not by specific individual compounds but by the whole living substance of the cell.

   In lower plants--algae, the sexual tendency is evidently subjected to more variability than in higher plants.

   At present, there is voluminous data in the literature on the hermaphroditism of species and variants, as well as on experimentally obtained variants in protozoan organisms. Changes of sexual tendency have been studied in many specimens of protozoa and especially in flagellar algae--Chlamydomonas, Polytoma, Stephanosphaera and in some species of Ectocarpus, Dasycladus, Tetraspora and others.

   Among the known species of Chlamydomonas, there are dioecious organisms, hermaphrodites, and organisms where the sex cannot be clearly distinguished. Among the latter, the Hissen group (from the town Hissen) of Chalmydomonas pseudoparadoxa, is of special interest. In this group there is no copulation between the cells. but after treatment with filtrates obtained from cultures of another dioecious form of Chlamydomonas, the sexual process between the gametes proceeds in a normal way. Thereby one of the "Hissen" clones is activated only by filtrates of one type of gamete, and other clones--only by filtrates of another type of gamete of the dioecious group (Moewus 1933, 1935, 1950: Lewin, 1954, Smith, 1951; Hartmann, 1923, 1943, 1955). Moewus performed crossings between dioecious groups, and also between the clones of Chlamydomonas paradoxa and Chlamydomonas pseudoparadoxa which are identical in respect to sex. He established that female clones copulate with other females. and male clones with other males, gametes of one species of algae copulate with gametes of another species. These data confirm earlier observations described by Hartmann (1923, 1943), which lead to him conclusion on the relativity of the sexual tendency and served as the foundation of his general theory of sexuality. In 1925 Hartmann showed that the existing unisexual male and female groups of algae Chlamydomonas paupera were of different potency or valence, hence, cells of one and the same sexual potency may copulate as gametes of different sexes. Hartmann, and subsequently other investigators, observed a relativity of sexual tendency in the dioecious green algae Spyrogyra quinina. In these algae the separate filaments consist either of only female cells, or only of male cells.

   Under special conditions, cells of one and the same filament sometimes react as female cells, and other times as male cells. This is well demonstrated when three or more adjacent filaments take part in the sexual process. Cells of the middle filament copulate with those of one of the neighboring filaments as gametes of male traits (Figure 28).

Figure 28. Schematic representation of triple copulation in the dioecious alga Spirogyra quinina: Middle filament B functions as a male relative to filament A and a female relative to filament C (after Hartmann, 1943)

   Detailed investigations were performed by Moewus on the alga--Chlamydomonas eugametos. In experiments with this alga not only facts pertinent to relative sexual tendency in gametes of one and the same sexuality were established but also facts relative to its physiological and biochemical differences. It was shown, that sex changes depended on growth conditions, the composition of the nutrient medium, light sources and other factors.

   It was previously known that dioecious gametes have distinct physiological properties, the two sexes secrete different substances into the medium. The presence of these substances stimulates the gametes to copulate. The formation and presence of these two differentiated substances in the medium were investigated in detail by Kuhn, Moewus and others and by collaborators of Hartmann, Forster and Wiese (1954) and others.

   It was shown that in the formation of gametes and the determination of sex, as well as during fertilization itself, a complexity of variously named substances took part. In three of the most minutely investigated species of algae Chlamydomonas eugametos, Chl. dreadenais and Chl. braunii the following substances were found.

   1. A motility substance, stimulating the gametes to motility. It is formed under the influence of light and then secreted into the medium. If a filtrate of Chlamydomonas is added to a culture with nonmottle gametes. the latter acquire flagella and become motile. In the absence of light and filtrate the algae are nonmotile and do not copulate. The motility substance affects cells of its own species more strongly than those of foreign species. Consequently, it is specific to some degree.

   The chemical nature of the motility substance was investigated on dense concentrates of culture filtrates of Chlamydomonas, 16 ml of a bright yellow concentrate was obtained from 300 liters after evaporation. The presence of a carotenoid very close to crocin was established. Crocin (C₄₄H₆₄0₂₄) obtained from saffron, proved, on investigation, to have the same effect on cells of algae of Chlamydomonas as filtrates of the substance obtained from them. The sensitivity of algae cells to the latter to very high. Even at a dilution of 1:250 billions, crocin as well as the substance of filtrates activates the cells of Chlamydomonas eugametos in the dark. After 4 -5 minutes the gametes become motile and ready for the sexual process.

   2. Fertilization substance or gamones. Investigations showed that in order to evoke copulation of alga cells of Chl. eugametos, the presence of the "motility" substance alone is not enough. Still other elements, the so-called gamones are needed. Copulation occurs when a filtrate of culture of Chlamydomonas, kept under light, is added to the medium. It was shown that in the filtrate two different substances connected with sex occur in the gamones: one affects female gametes, the other affects male gametes. Gamones affecting female gametes are formed by them, and gamones affecting gametes of the opposite sex are formed by male gametes under the influence of the violet and blue parts of the solar spectrum. Therefore, the male cells need a more prolonged radiation than the female cells. When the same cells are subjected to the effect of light, (after 24-26 min) the female gamone appears first in the filtrate and the male gamone appears later (after 74-76 min). The female gamone was named gynogamone, the male androgamone.

   Gynogamones and androgamones consist of a mixture of two chemically established substances: trans-dimethyl crocetin and cis-dimethyl crocetin. At first the latter to formed under conditions of the culture, and then under the effect of violet and blue light it transforms into trans-dimethyl crocetin.

   The quantitative relation of trans-and cis-dimethyl crocetin determines the sexual tendency of the gametes of algae. The composition of gynogamones of Chlamydomonas eugametos form a simplex, corresponds to three parts of cis- and one part of trans-dimethyl crocetin ester. In other groups and variants the relation of these two gamones is different and may change within the limits of 10%.

   Studying various groups and variants of Chlamydomonas eugametos, Moewus found that they have different sexual valency, i. e., have different ability to start the copulation process. Expressing this valency by four numbers one may state, that the weakest valency (male ¹ and female ¹), is observed in Chl. eugametos forma synoica; it is expressed somewhat stronger in Chl. eugametos forma simplex (male ² and female ²), The valency is higher in Chl. eugametos forma typica (male ³ and female ³) Finally. it is manifested most strongly in the anisogamic type Chl. braunii (male ⁴ and female ⁴). In experiments on crossing these variants, various combinations were obtained; in the first group gametes with a valency of 1 did not copulate or copulated in single cases; the experimental cells of the second group with a valency 2 produced about 20 copulating pairs; in the third group 100 and more pairs were obtained out of the total number of organisms tested.

   The biological effect of gamones consists in that, in the presence of these substances, the gametes are attracted to each other, stick together in groups and copulate. They also cause agglutination of gametes. Moewus assumed that the adherance of algae cells which he observed was caused by an admixture of bacteria. Forster and Wiese (1954) found agglutination of cells in sterile cultures of algae in complete absence of bacteria. These authors established that the process of sex determination in algae to neither conditioned by carotenoid nor crocin but by other specific substances of protein nature--glycoproteins. Hartmann is also of this opinion (1955).

   As was indicated above, at an appropriate degree of development of only female or only male tendency, the gamete cells may react with each other as organisms of different sexes. The more the degree or valency of sexual potency is expressed, and the stronger the difference in the potency of the reacting cells, the greater the intensity in which copulation proceeds. Moewus cited six combinations for copulation of gametes of the same sex. Combinations male ⁴ x female ¹ give the highest percentage of copulating pairs, the combinations male ⁴ x female ³--least. Gametes male ⁴ and female ⁴ do not copulate at all. It was shown that, the greater the difference in the content of cis- and trans-substance in copulating gametes, the more vigorous is their fusion. If the difference in the content of these substances is small (not exceeding 20%), copulation does not occur.

   In 1923 Hartmann stressed the quantitative character of alterations of gamones on the basis of his theory of relativity of sexual tendency. He did not regard male and female cells as absolutely male and female, but as an expression of quantitative relationships of gamones (Figure 29).

Figure 29. Scheme explaining experiments on relative sexuality (after Hartmann, 1931):

white color--male substance in gametes; black--female substance; left (1, 2, 3)--various kinds of male gametes, right (1, 2. 3)--females gametes. Arrows show positive reactions: triple arrows--strong reactions; double--average reactions; single--weak reactions. Gametes with arrows directed to them behave as female gametes with respect to gametes from which the arrows start.

   3. Termones or substances determining sex in algae were found by Iollos in 1926. Afterward the existence of these substances was also revealed by Moewus. Upon the addition of filtrate of dioecious species (Dasycladus) or flagellates (Chlamdomonas) of algae to gametes of monoecious algae Chl. eugametos forma synoica, the whole population acquires a monosexual character, namely, from filtrates of male gametes--a male tendency, and from filtrates of female ones--a female tendency. A specific pH of the medium should be preserved, for the first alkaline, for the second acid.

   According to Moewus (1950), the indicated effect of filtrates is not conditioned by gamones, but by special substances--termones (the female--quinotermone and the male--androtermone). These substances have not been isolated and investigated. It is assumed that they are related to the bitter substance of saffron; gynotermone to picrocrocin, androtermone, to safranal.

   The effect of termones in manifested only on hermaphrodite forms, the dicecious gametes do not react.

   The nature of mutual relations between chemical substances taking part in copulation processes of algae, according to Moewus (1950), are as follows: they are all chemically close to the bitter, fragrant substance of saffron, largely distributed in the plant kingdom and derived from protocrocin. Protocrocin occurs in all cells growing in the dark. Two substances--cis-crocin and trans-crocin, and also crocin, are formed from it. The latter is formed by splitting of protocrocin under the influence of a special enzyme. Consequently, picrocrocin also appears. Crocin in sexually oriented cells, transforms into cis- and trans-crocetin dimethyl ester, i.e., in one type of gamete--into androgamone, in the others into gynogamone. The latter brings the cells to copulate. Picrocrocin under the influence of cellular ferments trans-forms into safranal, i.e., into termone, and the termone transforms the bisexual cells into cells of different sexes--either males or females.

   The relativity of sexual tendency is noted in some protozoan organisms. In the infusorian Paramecium aurelia various groups and types have been revealed whose cells copulate in certain combinations. Lines of Infusoria are described, living in various geographical parts, in which several types of cell coupling have been noted. On these grounds the mentioned organisms were subdivided into independent species (Nanney, 1954).

   Substances were also found in Infusoria which activate the sexual process. Kimball (1939) established that filtrates from cultures of Euplotes patella of one type of coupling evokes conjugation in organisms of a second type of coupling. Thereby types of one line induce cell copulation in all other types, except its own. Types of another category evoke conjugation only in certain types of distant relation. This type of induction resembles the effect of andro- and gynogamones of algae mentioned above.

   Special substances of the type of antibiotics formed by separate types or line's of Infusoria display an effect on processes of copulation in Infusoria. In a series of investigations it was shown that some line a of Infusoria formed the so-called paramecins which suppress life processes in other lines. Some paramecins evoke a strong swelling and vacuolization of cells, others attack the motility organs, and still others affect metabolism. All of them display a great influence on copulation processes in various ways.

   Of special interest are substances, formed by cells of Paramecium and appearing in the protoplast in the form of small granules or bodies, called "kappa". These particles, too small to be seen by eye, are determined by indirect methods. Upon division of cells they also multiply and pass from the parent individual to the daughter cell through a great number of generations. Their number in the cell may reach considerable values (250-450) and remain on this level if the rate of cell division in not too high. Upon accelerated multiplication of cells the size of the "kappa particles decreases, their number becomes smaller and they may disappear completely, In this way one may obtain a line entirely deprived of these particles. However, if one single "kappa" particle remains in the cell, an increase of their number to the limits mentioned above occurs, if the multiplication of individuals becomes slower.

   The process of paramecin formation and lack of cell sensitivity to it is closely connected with the formation of "kappa" particles. The process of formation of the antibiotic slows down with a decrease of the number of the "kappa" particles in the cell. Lack of sensitivity to foreign paramecin is preserved till at least one "kappa" particle remains in the cell. The cells become sensitive to paramecin after the disappearance of these particles. The "kappa" particles are inactivated at 36°. Maximal formation is observed at 27°; at 10° this process slows down, and at 30° it stops. Numerous attempts have been made to explain the nature of this mysterious substance. Some investigators (Altenburg, 1946) regard the "kappa" particles as symbionts, others as viruses (Lindegren. 1945). However the majority of authors reject these views and do not find an explanation for this phenomenon (according to Dogel'l, 1951).

   The process of conjugation and autogamy in Infusoria may be induced by the addition of killed cells to living ones. Metz (1947) and others added about 1,500 to 3,000 cells killed by formalin to each 800-1,200 living cells of a culture of Paramecium aurelia. In this mixture agglutination of living with dead cells occurred. After 60-90 minutes the living cells part from the dead and begin coupling and conjugating. This coupling is assumed by the authors to occur during agglutination when the cells are in the aggregates (Metz, 1954; Tyler, 1948).

   The described process of conjugation of paramecia is apparently affected by some chemical substances formed in the dead cells. By its action it resembles the gamones of algae.

   The relativity of sexual tendency in protozoa is well manifested in experiments with various clones. By suitable cultivation one may obtain male or female gametes at will. Lerkhe grew a large number of clones on a medium of normal composition and on media deficient in nitrogen and phosphorus. Cells, after cultivation on the poor medium. behaved an female gametes; they were larger and acquired a red pigment. Cells, grown on the rich nutrient medium had a male tendency, were smaller and possessed a green pigment.

   The red gametes do not copulate with the red ones, nor the green gametes with the green ones. When mixed they begin the formation of pairs quite rapidly, the green gametes surround the red ones and conjugate with them as individuals with a sharply manifested sexual difference (according to Dogel', 1951).

   Sexual potency at a relative sexuality is also noted in bacteria by some authors. According to the opinions of Lederberg, Hayes, Clark and others, cells of the coli bacillus Bac. coli K-12 of auxotrophic variants possess different degrees of sexual tendency. As was indicated earlier, these bacteria are able to exchange some essential vital substances upon a direct contact of cells. This mutual exchange or mutual assimilation of cell metabolic products occurs only with certain combinations of organisms and is regarded as a sexual process. As in algae, variants of coli bacilli according to the authors, have a male and female sexual tendency, manifested to various degrees. In other words, bacterial cells possess different sexual valencies. According to this, the process of cell fusion expresses itself in various ways. The greatest productivity is obtained upon mixing a culture of maximal male potency with a culture of maximal female potency. Defining the degree of potency by the four-number system, Lederberg showed that productivity upon mixing F+₄ with F was greater than that obtained after mixing of cultures F+₃ with F. The productivity of mixed cultures of F+₂ with F is still lower. Productivity decreases gradually in combinations of cultures F+₄ x F+₁ > F+₄ x F+₂ > F+₄ x F+₃ > F+₄ x F+₄.

   As may be seen from the above, one can assume that the sexual function in various specimens of lower organisms proceeds quite differently. In some forms the sexual process occurs between sharply differentiated male and female gametes, in others this process proceeds between organisms which are sexually homogenous; gametes of the same sex, male or female but with different sexual potency, copulate. There are organisms in which the copulation process is accomplished inside one and the same cell between separate parts of the protoplast with a different sexual potency or with different potencies of the same sexuality.

   The sexual tendency and sexual potency are not constant features of the organism. Both may be altered depending on nutritional conditions, and also on environmental factors. One and the same cell during its development may become a male or female cell depending upon various growth conditions.

   Consequently, sexual tendency is of a relative nature. Each cell has two elements--a male and a female one. The presence and manifestation of these elements during the physiological and morphological development of the organisms determines the latter's sexuality.

   At the present stage of our knowledge, although we may experimentally succeed in affecting the direction of the sexual tendency of the organism, many basic questions in the field of sexuality remain unsolved. It should be noted that the developing theory of Hartmann on general bipolar bisexuality as a basis of the whole sexual phenomenon was preceded by statements of Bütchli and later by Schaudin. Bütchli regarded the sexual process as an act leading to the rejuvenation of the organism. Schaudin assumed that each cell is hermaphroditic or bisexual to a certain degree, and possesses elements of both the male and female sex, As a result of predominance of one or the other element, the cell becomes either male or female. Thus, the author explained cases of autogamous copulation in protozoa, algae, and bacteria.

   Hartmann and his collaborators, developing the theory of relative sexuality, indicate that from the very beginning organisms on earth were of a bisexual nature and contained both male and female elements. These elements evoke a sexual tension in the cells, stimulating them to unite and to level off or stop this tension. Sexual tension has a decisive role in the whole chain of fertilization processes. It is created as a result of an irregular development of one or the other sexual element. Sexual tension may occur not only between separate cells but also between parts of one and the same cell. In bacteria and other specimens of microorganisms which are on a lower evolutionary level, sexual tension between parts of the protoplast is apparently of essential importance in the rejuvenation of the organisms. The sexual tension created in the cells also determines the nature of autogamy in microbes. It should be assumed that in nonsporeforming bacteria autogamy does not proceed as in sporeformers, that in actinomycetes it takes place in a different manner than in fungi.

   In various species of sporeforming bacteria this process is morphologically manifested in a different way. The sexual process in Bac. bütchlii which was described by Schaudin (1902) is not found in other sporeforming bacteria. In them fusion of various cellular elements proceeds without formation of the longitudinal line of inclusions. However the product of fusion--the spore--constitutes in both cases a formation of the same order and may be regarded as a zygote. Formation of similar zygotic cells or spores in specimens of filamentons bacteria, Oscillospira guilliermondii, occurs after fusion of two and sometimes of three and more contiguous cells, In these organisms the sexual differentiation is on a higher level, as it involves, not parts of the protoplast, but whole cells. A more complex process of copulation occurs in organisms which are higher in evolutionary development.