Part I, continued:

The Sexual Process in Bacteria

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

   The question of a sexual process in bacteriaand actinomycetes remains undetermined. Does it occur in these organisms? This questionhas been vividly discussed in the literature of recent years. Former views on bacteriaas primitive organisms gradually changed under the pressure of accumulated factualmaterial. A search for complicated cycles of development in bacteria as well as forcopulation processes was started. In the period of enthusiasm for theories of a complicatedcycle, various forms of sexual process in various specimens of bacteria were described.Many authors assumed that all bacteria reproduce by copulation, which is in generalpeculiar to all other higher and lower organisms. This process takes place in variousspecimens of bacteria, although in a primitive manner, externally manifested in variousways. 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 deservinggreat attention was described by Schaudin (1902). This investigator observed fusionof 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, onlysometimes , 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 ofone and the same cell. The whole process is described by the author as follows: afterisolation of the coarse-grained bacillus in a drop of the intestinal juice of thebeetle, glistening granules, surrounded by a clear halo are visible in the centerof the cell after 30 minutes. Exactly as in division the granules are distributedin a row and after 20-40 minutes they form a transverse septum, which does not differin living or in fixed and stained cells from the septum observed upon ordinary celldivision. The bacillus remains in this phase approximately 1-2 hours, after whichthe transverse septum becomes pale and thinner, the granules distributed in a rowalong the septum disappear and the cell of the bacillus looks as it did before theformation of the septum (Figure 21). Before the dissolution of the septum multiplegranules of chromatin appear in the plasma, they are distributed in a long line andform a peculiar chromatin thread. This thread disintegrates into granules upon sporeformation, the majority of granules moving to the poles of the cell and forming largebodies 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, thechromatin 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 sporesare formed.

   The described way of autogamous copulation closelyresembles 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 filamentousblue-green algae of the genus Oscillaria, but in contradistinction to them,it does not possess a pigment. Each organism represents a filament of greater orshorter length, divided by transverse septa into short cells.

   During the course of its development Oscillospiraforms spores. Before sporulation two and sometimes three adjacent cells of one filamentfuse; the septa separating these cells dissolve and disappear. and the protoplastsof cells fuse into one body, which, by an appropriate reorganization, transformsinto a spore. Upon fusion of the protoplasts, the central bodies and chromatin granulesdistributed 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 dividedprotoplast in the yeasts--Saccharomyces paradoxus. As is known, in this organismthe protoplast of the cell divides into 3-4 parts before sporulation. First the divisionof the nucleus takes place, and is followed by that of the plasma. From each partof the protoplast a spore is formed. Mature spores released from the maternal cellgerminate into vegetative cells (Bachinskaya, 1914).

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

   After the fragmentation of the protoplast intoseparate parts, spore formation begins, and small bits of protoplasm become roundedand dense with the cell walls outlines. Afterward maturation of spores stops andthe process proceeds in the opposite direction. The contours of the outlined cellwall disappear and the protoplasm becomes less dense, and undergoes vacuolation.Separate not organized parts appear; very often the residual cell walls may be seenamong 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 processesof 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 agoas a reversed development of the organism (Nadson and Krasil'nikov. 1920). Afterwardwe came to another conclusion, and related thin phenomenon to autogamous copulation,somehow similar to that which takes place in Bac. bütchlii or in protozoalorganisms.

   The process of autogamy may evidently take placebetween various parts of the cell, which did not undergo preliminary division. Theexpression of extreme autogamy was noted by many authors. Data on observations offusion of chromatin granules into one body before sporulation and sometimes beforedivision have already been cited above. We observed such a fusion of cellular partsin certain large bacilli, which were isolated from the intestine of a guinea pig.The chromatin granules distributed throughout the protoplast combine into one largebody. After some time the body either divides into two and is followed by the celldivision, or it becomes the center of spore formation. In some microbes the fusionof chromatin is accompanied by formation of long threadlike fibers, which are straightor spirally bent from one pole of the cell to the other. Such formations were observedby us (1928), in a peculiar microbe--Metabacterium octosporus. This microbelives 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. Theprotoplasm in young cells is homogeneous, dense, and stains a dark color with basicdyes. As the cell grows, granules of chromatin and metachromatin appear in the plasma.

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

   Copulation of the hologamic type in various specimensof bacteria was described by many authors. In essence, this manner of copulationis as follows: after a series of consecutive divisions, two isolated cells combinein various ways and exchange their contents or fuse into one organism. The fusionof protoplasts proceeds through copulation canals and small bridges. Formation ofbridges between two cells was noted by many investigators. Rindfleisch (1872) describeda 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 mostwidespread. He described the formation of small bridges in the purple sulfur bacteria--Chromatiumokenii, Ch. weissii, Ch. violaceus and in the Spirillum photometricum.According to his observations sometimes three or more cells are consecutively interconnectedby canals, which in his opinion are copulation canals. Potthoff even observed fusionof 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 ofcell fusion in Azotobacter and some other bacteria. According to his observationsthe cells combine by bridges, through which the fusion of protoplasts occurs. A similartype of copulation was described by Lieske (1926) in bacteria of the coli group whichwere isolated from a tobacco extract. Cunningham (1931) found cells combined by meansof a canal in 20 strains of Bac. saccharobutiricus.

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

   Smith (1944) described fusion of cells in Pseudobacterfunduliformis; Lindegren and Mellon (1932) in the tubercle bacillus (Mycob.tuberculosis, var. avium). Klieneberger-Nobel (1949) observed the fusion of nuclearbodies 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 developmentof cells in Bact. Proteus (OX-19) by means of microphotography. They noticedformation of strongly swollen spherical cells. These spheres are combined with oneto three rodlike cells. As the growth of the spherical cell proceeds, the rods becomeshorter, paler and disappear completely; their substance appears to have enteredinto the spherical elements. On this basis the authors concluded that these spheresrepresented zygote cells which undergo disintegration with the formation of eitherfine-grained elements or are transformed into rodlike elements by consecutive divisions.Under favorable conditions the granules may be transformed into regenerative elementsand 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 Stempenand Hutchinson we can not agree with their interpretation. In our opinion the sphericalcells observed by them represent not zygotes but involution forms. The forms describedby 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 alsonot convincing. They mostly represent a result of deductions based on the study offixed and stained preparations. Direct microscopic observations on development andfusion of cells were not carried out by them.

   Our study of a large number of bacteria did notenable us to establish any external signs of cell fusion, which resembled a sexualprocess. In some species peculiar small bridges between the cells are observed. However,a detailed investigation of such formations showed that they represent the resultof an unfinished constriction of cells. As was indicated above, when cells of Azotobactermultiply by constriction, long plasmodesms are often formed, which resemble copulationcanals (Figure 25). These plasmodesms sometimes move to a lateral surface of thecell, and even possess a small swelling with a tiny granule inside it, in the centralpart, which resembles a sexual process even more. We did not observe any indicationsof 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 ofcells in Chromatium described by Potthoff (1924). We had the opportunity tofollow in detail the formation of bridges in Chromatium okenii and Chromatiumwarmingii in a hanging drop. As in Azotobacter, the cells of these bacteriaunder special conditions , multiply by constriction and form long canals (Figure25 B). The canals are often displaced to a lateral surface. This displacement, asindicated 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 regardthe extracellular fusion of the protoplast of two and more cells after the disintegrationof their cell walls as a sexual process. This fusion of the substance of the disintegratedcells was named by Löhnis "symplasm". It is observed on the agingof cells, and under unfavorable growth conditions. The cells, often strongly swollen,disintegrate, their cell wall disrupts and undergoes lysis and the cell contentsleak out and fuse with the contents of other cells. A protoplasmic extracellularmass or symplasm is obtained. According to Löhnis processes characteristic ofcopulation proceed, there. After some time, new organisms having the nature of azygote are formed in the symplasm.

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

   Several investigators describe combinations ofcells of bacteria which strongly resemble the conjugation of protozoal organisms.Rodlike cells unite at their ends into groups, several cells in each group, and formpeculiar 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 groupsof cells which are joined by their ends may often be seen. The cause of these formationsand their meaning are not clear. Stapp and others (1931, 1949, 1955) subjected thesecells to careful study and came to the conclusion that the formation of cells intostarlike groups represents a sexual process during which a combination and fusionof hereditary material proceeds. They follow the formation of these groups, the fusionof the cells and further events, step by step. It was established that the cellsindeed combine by their ends into firm groups. The nuclear substance, chromatin granulesand nucleoids move toward the site of fusion, the cell wall dissolves and these granulesand nucleoids of the combined cells fuse into one large body. The latter remainsin 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 divisionof nucleoids is followed by the division of the cell. Two. three, four, five andmore 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 observedby us in some strains of root-nodule bacteria of the pea, bean, vetch, and otherlegumes. 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 oftenseen. By a careful cytochemical analysis and observations of their development ina hanging drop it was established that the cells are joined quite firmly by the endsand 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 cellwas 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 becomeinvisible and then the cells begin to grow and multiply. The plasma of such reorganizedcells is optically homogeneous; granular inclusions and nucleoids appear later.

   As a rule. the cells in starlike groups aftertransfer to a fresh nutrient substrate do not develop immediately. A long time passesbefore they begin growth and multiplication. Until that moment they remain in thestate of reorganization of the protoplasts. We assume that the picture of cell combinationinto starlike groups described in root-nodule bacteria is connected with some metabolicprocess. The cells mutually exchange the products of their metabolism, one cell assimilatessome substances released by the other.

   Recently papers were published in which the sexualprocess in bacteria is regarded as an exchange of substances between one cell andanother upon direct contact without any special copulation structure and withoutdisintegration of the cell wall. It is suggested that specific cell substances--carriersof hereditary properties--penetrate through the cell walls of the continguous cellsand 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 constitutea small percentage and are formed in special cases of metabolic disturbances, underthe action of some environmental factors [such as] "ultraviolet light",and chemical agents; they also occur in old cultures. They lack the ability to performcertain functions, which are restored upon contact with other bacteria carrying theopposite features (Braun. 1953 ; Lederberg and Tatum. 1954. and others).

   This method of mutual fertilization was describedin 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 mixingof cells of various strains of Bact. coli after their subsequent plating onan agar medium, one obtains new strains with properties of the parent cells (seechapter on variation).

   Some investigators assume that bacteria performa 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 transformsinto a zygospore. From the latter a vegetative cell develops under favorable conditions.

   Enderlein (1925) made the course of a developmentalcycle 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--gonitesor gonidia, subjected to meiosis. The gonites develop either into spermites or intooites. The former are small, rodlike, straight or slightly bent, very motile witha flagellum on one extremity; the latter are larger. of spherical form. nonmotile.An oite fertilized by a spermite transforms into a mycete, initiating normal vegetativecells. The sexual cells--oite and spermite represent haploid forms, as a result offertilization, a diploid--the mycete--form is created.

   Enderlein has no factual material to confirmhis hypothesis. Since he was not a microbiologist he mechanically transferred datafrom zoology into the microbial world.

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

   Almquist (1925) describing the sexual processbetween differentiated sexual cells in bacteria, assumes that this process may proceednot only between cells of one species but also between those of different species.producing hybrid offspring.

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

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

   In actinomycetes the question of a sexual processremains 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 directcontact.

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

   Fusion of cells may be observed under directconstant observation in a hanging drop in many species of actinomycetes. The processproceeds as follows: upon germination of spores, small appendages shaped like smalltubes are formed (outgrowths); these tubes come into close contact at the ends, atthe site of contact the cell walls dissolve and a canal is formed. Through this canalthe contents of the two germinating spores combine and fuse into one protoplast .The combined outgrowths produce a common sprout which extends into a long filamentand 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 differentspecies of actinomycetes (1938a), Later (1950) this process of fusion of germinatingspores was noted in many species of actinomycetes. By its external manifestationthe 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, nucleoidsand chromatin granules occurs upon fusion of the growth tubes. If in yeasts a similaract 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 theprotoplast of conjugated cells.

   The joining of cells by means of anastomosesis observed in actinomycetes considerably more often than the fusion of spores. Thismay 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 smallbridges in the form of a canal connecting two more or less distant filaments. Thiscanal may be quite long when it connects two filaments located at a great distancefrom one another. The canal may occur between branches located side by side and closelyrelated, originatng from one hypha, as well as between distant branches originatingfrom various hyphae of the same mycelium. (Figure 27 B).

   Anastomoses have no septa, and because of thisthey 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 filamentinto 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 actinomycetesa process takes place which may be qualified as an autogamous copulation.

   Formation of anastomoses occurs not only betweenfilaments of one and the same culture but also between filaments of various strainsof one and the same species: we observed anastomoses between various strains of Actinomycesstreptomycini, producers of streptomycin, isolated from various soils of differentareas of the Soviet Union.

   This form of fusion of mycelial filaments isnot observed between cultures belonging to different species or even to differentvarieties. We carried out observations under the microscope. in a drop of a semiliquidnutrient medium. Spores of two actinomycetal species which were inoculated at differentsites of the drops germinated and gave rise to hyphae, which grew out and after sometime came close together. At the site where the filaments touched, the formationof anastomoses could be observed.

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

   Probably not all branches form anastomoses. Manyof them, which came in contact with neighboring hyphae did not attach themselvesand did not form anastomoses. It to possible that anastomoses are formed by brancheswhich 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 heterogeneoussystem, where separate filaments and branches have different sexual qualities.

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

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

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

   The hypothesis of rejuvenation puts forward theproblem: do cells become old upon prolonged asexual multiplication? Bütchlii(1880, 1887) and then Maupas (1888, 1889) tried to solve this problem experimentallyon 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 indicatedhypothesis. Numerous observations made by contemporary investigators on bacteriaand yeasts also show the erroneousness of this point of view.

   The explanation of the essence of the fertilizationprocess, given by the chromosomal theory of heredity, is built entirely on the assumptionthat gametes contain particles of a special hereditary substance. According to thistheory, factors responsible for the hereditary transmission of properties are locatedin the chromosomes of cells. They are connected with the chromatin substance of thelatter and, according to modern ideas, with desoxyribonucleic acid, which constitutesthe fundamental component of the nuclear substance.

   Upon fusion of the copulating cells an entirelymechanical combination of cytoplasm and nuclei occurs. The substances of chromosomesare not dissolved in the protoplast, they do not disappear, and they preserve theirpeculiarities as well as their characteristic features of heredity. The chromosomesof the female and male cells preserve their individuality in the chemical as wellas in the biological sense.

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

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

   An entirely new interpretation of the questionof fertilization to given by the academician Lysenko. He is of the opinion that theessence of the sexual process consists of the combination of the chromosomal setsof the gametes but not of exchange of substances between these gametes. Accordingto the author, fertilization represents a peculiar process where the copulating cellsmutually assimilate substances. The protoplast of one cell is assimilated by theprotoplast of the second.

   According to Lysenko, the essential differencein fertilization from all other biological assimilation processes consists of thefollowing: "In any physiological process one part is the assimilating one andthe other the assimilated one. . . . . The substances which are assimilated are usedas building material for the assimilating component. In the sexual process, whentwo seemingly equivalent cells combine, there is a mutual assimilation. Each buildsitself from the substance of the other, according to its own pattern. Finally, neitherof these cells remains, but instead of the two a third new cell is produced"(Lysenko, 1948. p 383).

   The peculiarity of this exchange or assimilationconsists in that there is not one but two cells which assimilate. During the processof 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--initiatesa new organism combining the paternal and maternal traits. The living substance ofthe zygote is of dual quality, it contains elements of both the paternal and maternalorganisms, these elements are there, not in the form of special corpuscles or genes,concentrated in chromosomes. but exist throughout the whole living organism. Thisheterogeneous quality of the newly formed zygotic cells insures the formation ofan organism with new properties inherited from the parent couple. In this way thecontinuity of inheritable traits and the evolution of the species to secured. Accordingto Lysenko's conclusion, the biological importance of the process of fertilizationconsists in an increase of viability of the organisms. As a result of fertilization,organisms with a dual heredity, maternal and paternal, are obtained. "Dual heredityconditions a high vitality (in the direct sense of the word) of organisms and greatadaptability to variable conditions of life" (Lysenko, 1948, p 381).

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

   The above-mentioned principles of the biologicalimportance of the fertilization process. elaborated by academician Lysenko on thebasis of Darwin's and Michurin's theories. reflect one of the laws of adaptabilityand evolution of the living population.

   The usefulness of the sexual process was stressedby Darwin and subsequently by many other investigators. Maupas (1888-89), summarizingthe results of numerous observations and experiments, came to the conclusion thatconjugation in Infusoria constitutes a process which is indispensable to the renewalof viability. According to his opinion, for every species of Protozoa there existsonly a certain number of asexual generations, the cells multiply vegetatively toa certain limit, then age, degenerate and inevitably die. The sexual process leadsto restoration of the vital activity of cells. It rejuvenates the Infusoria and initiatesa new series of sexual generations.

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

   In recent years the concept of the biologicalusefulness of the sexual process was well confirmed by the work of Cleveland. Thisauthor studied the phenomenon of fusion of gametes in the flagellates Polymastigidaand Hypermastigida in detail. In one of them--Trichonymphs--the gametes are morphologicallyidentical. Upon copulation the male cell penetrates into the female cell and dissolvesthere, or more precisely, the protoplasts of both gametes mix completely and forma new organism. In Oxymonas the fused gametes remain in the form of a double organismfor a few days and then the fusion of important organoids follows. Not only nucleiand chromosomes, but also resistant formations of axostyles which do not have anyrelation to the protoplast fuse. In the zygote a double, larger axostyle is formed(according to Dogel', 1951).

   The decisive importance of metabolism in thesexual 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 asuccessful fusion of gametes and formation of healthy progeny, at least a small differencein 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 formsi. e.. by the fusion of sexual cells in which the nucleus, nucleolus and other importantinclusions of the cell always take part.

   Externally the sexual process expressed itselfin different ways in various specimens of microorganisms. Besides the true oögamyi.e. , fertilization occurring between highly differentiated sexual cells as in someyeasts and fungi, copulation often takes place between individuals externally similarto one another. Gametes do not often differ from the vegetative cells. There aremicrobial forms, in which the sexual process occurs not between cells but betweenparts of one and the same cell, i.e., autogamously.

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

   As was shown above, there is in the literaturefactual material which constitutes a basis for the assumption that in bacteria andactinomycetes processes occur between cells or parts of cells which are consistentwith the notion of fertilization.

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

   According to contemporary concepts, any sexuallydifferentiated individual (female or male) and any sexually differentiated gameteconcurrently contains all the rudiments necessary for development of the oppositesex. As a result of the increased development of one of the rudiments, and the suppresseddevelopment of the other, the male or female tendency of the cell is expressed. Thesexual tendency becomes expressed under the influence of various kinds of environmentaland internal factors.

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

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

   Milliard (1898) and Schafner (1927) obtained100% formation of female flowers in hemp, by regulating the duration of daylight(according to Sabinin, 1940). There are many other examples of similar changes ofthe sexual tendency in higher plants.

   Kuhn (1941), Zhukovskii and Medvedev (1948) andothers, assume that the formation of specific substances, sex-"determinants"are determined by light stimuli, short-wave rays of the solar spectrum. It to assumedthat sexual tendencies are connected with the photochemical reactions of specificsubstances. 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 indicatingthat the sexual tendency is determined not by specific individual compounds but bythe whole living substance of the cell.

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

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

   Among the known species of Chlamydomonas,there are dioecious organisms, hermaphrodites, and organisms where the sex cannotbe clearly distinguished. Among the latter, the Hissen group (from the town Hissen)of Chalmydomonas pseudoparadoxa, is of special interest. In this group thereis no copulation between the cells. but after treatment with filtrates obtained fromcultures of another dioecious form of Chlamydomonas, the sexual process betweenthe gametes proceeds in a normal way. Thereby one of the "Hissen" clonesis activated only by filtrates of one type of gamete, and other clones--only by filtratesof another type of gamete of the dioecious group (Moewus 1933, 1935, 1950: Lewin,1954, Smith, 1951; Hartmann, 1923, 1943, 1955). Moewus performed crossings betweendioecious groups, and also between the clones of Chlamydomonas paradoxa andChlamydomonas pseudoparadoxa which are identical in respect to sex. He establishedthat 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 dataconfirm earlier observations described by Hartmann (1923, 1943), which lead to himconclusion on the relativity of the sexual tendency and served as the foundationof his general theory of sexuality. In 1925 Hartmann showed that the existing unisexualmale and female groups of algae Chlamydomonas paupera were of different potencyor valence, hence, cells of one and the same sexual potency may copulate as gametesof different sexes. Hartmann, and subsequently other investigators, observed a relativityof sexual tendency in the dioecious green algae Spyrogyra quinina. In thesealgae the separate filaments consist either of only female cells, or only of malecells.

   Under special conditions, cells of one and thesame filament sometimes react as female cells, and other times as male cells. Thisis well demonstrated when three or more adjacent filaments take part in the sexualprocess. Cells of the middle filament copulate with those of one of the neighboringfilaments 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 Moewuson the alga--Chlamydomonas eugametos. In experiments with this alga not onlyfacts pertinent to relative sexual tendency in gametes of one and the same sexualitywere established but also facts relative to its physiological and biochemical differences.It was shown, that sex changes depended on growth conditions, the composition ofthe nutrient medium, light sources and other factors.

   It was previously known that dioecious gameteshave distinct physiological properties, the two sexes secrete different substancesinto the medium. The presence of these substances stimulates the gametes to copulate.The formation and presence of these two differentiated substances in the medium wereinvestigated 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 gametesand the determination of sex, as well as during fertilization itself, a complexityof variously named substances took part. In three of the most minutely investigatedspecies of algae Chlamydomonas eugametos, Chl. dreadenais and Chl. brauniithe following substances were found.

   1. A motility substance, stimulating thegametes to motility. It is formed under the influence of light and then secretedinto the medium. If a filtrate of Chlamydomonas is added to a culture withnonmottle gametes. the latter acquire flagella and become motile. In the absenceof light and filtrate the algae are nonmotile and do not copulate. The motility substanceaffects 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 substancewas 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 fromsaffron, proved, on investigation, to have the same effect on cells of algae of Chlamydomonasas filtrates of the substance obtained from them. The sensitivity of algae cellsto the latter to very high. Even at a dilution of 1:250 billions, crocin as wellas the substance of filtrates activates the cells of Chlamydomonas eugametosin the dark. After 4 -5 minutes the gametes become motile and ready for the sexualprocess.

   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 afiltrate 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 occurin the gamones: one affects female gametes, the other affects male gametes. Gamonesaffecting female gametes are formed by them, and gamones affecting gametes of theopposite sex are formed by male gametes under the influence of the violet and blueparts of the solar spectrum. Therefore, the male cells need a more prolonged radiationthan 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 gamoneappears later (after 74-76 min). The female gamone was named gynogamone, the maleandrogamone.

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

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

   Studying various groups and variants of Chlamydomonaseugametos, Moewus found that they have different sexual valency, i. e., havedifferent ability to start the copulation process. Expressing this valency by fournumbers one may state, that the weakest valency (male 1 and female 1), isobserved in Chl. eugametos forma synoica; it is expressed somewhat strongerin Chl. eugametos forma simplex (male 2 and female 2), Thevalency 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 wereobtained; in the first group gametes with a valency of 1 did not copulate or copulatedin single cases; the experimental cells of the second group with a valency 2 producedabout 20 copulating pairs; in the third group 100 and more pairs were obtained outof the total number of organisms tested.

   The biological effect of gamones consists inthat, 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 byan admixture of bacteria. Forster and Wiese (1954) found agglutination of cells insterile cultures of algae in complete absence of bacteria. These authors establishedthat the process of sex determination in algae to neither conditioned by carotenoidnor crocin but by other specific substances of protein nature--glycoproteins. Hartmannis also of this opinion (1955).

   As was indicated above, at an appropriate degreeof development of only female or only male tendency, the gamete cells may react witheach other as organisms of different sexes. The more the degree or valency of sexualpotency is expressed, and the stronger the difference in the potency of the reactingcells, the greater the intensity in which copulation proceeds. Moewus cited six combinationsfor copulation of gametes of the same sex. Combinations male 4 x female 1 givethe highest percentage of copulating pairs, the combinations male 4 x female 3--least.Gametes male 4 and female4 do not copulate at all.It was shown that, the greater the difference in the content of cis- and trans-substancein copulating gametes, the more vigorous is their fusion. If the difference in thecontent of these substances is small (not exceeding 20%), copulation does not occur.

   In 1923 Hartmann stressed the quantitative characterof 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 anexpression 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 determiningsex in algae were found by Iollos in 1926. Afterward the existence of these substanceswas 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 femaleones--a female tendency. A specific pH of the medium should be preserved, for thefirst alkaline, for the second acid.

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

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

   The nature of mutual relations between chemicalsubstances taking part in copulation processes of algae, according to Moewus (1950),are as follows: they are all chemically close to the bitter, fragrant substance ofsaffron, largely distributed in the plant kingdom and derived from protocrocin. Protocrocinoccurs 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 protocrocinunder the influence of a special enzyme. Consequently, picrocrocin also appears.Crocin in sexually oriented cells, transforms into cis- and trans-crocetin dimethylester, 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 cellularferments trans-forms into safranal, i.e., into termone, and the termone transformsthe bisexual cells into cells of different sexes--either males or females.

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

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

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

   Of special interest are substances, formed bycells of Paramecium and appearing in the protoplast in the form of small granulesor bodies, called "kappa". These particles, too small to be seen by eye,are determined by indirect methods. Upon division of cells they also multiply andpass 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 thislevel if the rate of cell division in not too high. Upon accelerated multiplicationof cells the size of the "kappa particles decreases, their number becomes smallerand they may disappear completely, In this way one may obtain a line entirely deprivedof these particles. However, if one single "kappa" particle remains inthe cell, an increase of their number to the limits mentioned above occurs, if themultiplication of individuals becomes slower.

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

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

   The described process of conjugation of parameciais apparently affected by some chemical substances formed in the dead cells. By itsaction it resembles the gamones of algae.

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

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

   Sexual potency at a relative sexuality is alsonoted 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 auxotrophicvariants possess different degrees of sexual tendency. As was indicated earlier,these bacteria are able to exchange some essential vital substances upon a directcontact of cells. This mutual exchange or mutual assimilation of cell metabolic productsoccurs 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 femalesexual tendency, manifested to various degrees. In other words, bacterial cells possessdifferent sexual valencies. According to this, the process of cell fusion expressesitself in various ways. The greatest productivity is obtained upon mixing a cultureof maximal male potency with a culture of maximal female potency. Defining the degreeof potency by the four-number system, Lederberg showed that productivity upon mixingF+4 with F was greater thanthat 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 combinationsof 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 assumethat the sexual function in various specimens of lower organisms proceeds quite differently.In some forms the sexual process occurs between sharply differentiated male and femalegametes, 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 andthe same cell between separate parts of the protoplast with a different sexual potencyor with different potencies of the same sexuality.

   The sexual tendency and sexual potency are notconstant 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 maybecome a male or female cell depending upon various growth conditions.

   Consequently, sexual tendency is of a relativenature. Each cell has two elements--a male and a female one. The presence and manifestationof these elements during the physiological and morphological development of the organismsdetermines the latter's sexuality.

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

   Hartmann and his collaborators, developing thetheory of relative sexuality, indicate that from the very beginning organisms onearth were of a bisexual nature and contained both male and female elements. Theseelements evoke a sexual tension in the cells, stimulating them to unite and to leveloff or stop this tension. Sexual tension has a decisive role in the whole chain offertilization processes. It is created as a result of an irregular development ofone or the other sexual element. Sexual tension may occur not only between separatecells but also between parts of one and the same cell. In bacteria and other specimensof microorganisms which are on a lower evolutionary level, sexual tension betweenparts of the protoplast is apparently of essential importance in the rejuvenationof the organisms. The sexual tension created in the cells also determines the natureof autogamy in microbes. It should be assumed that in nonsporeforming bacteria autogamydoes not proceed as in sporeformers, that in actinomycetes it takes place in a differentmanner than in fungi.

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