Studies on Conjugation in Paramecium polycaryum

SYNOPSIS. The process of autogamy in unassociated individuals of Paramecium polycaryum was reported by the author in 1954. In May, 1955, conjugation was first seen in this species in cultures collected by me at Annamalainagar, South India, thus removing it from the list of non-conjugating species. This appears to be the first instance in which the process of autogamy was detected prior to observation of conjugation in the same species. Autogamy occurs in singles of the Indian race and appears to be similar, cytologically, to that of American races. The details of the micronuclear behavior in conjugation parallel those of autogamy in singles. In fact, the conjugation process seems to be one of double autogamy (cytogamy), rather than of reciprocal gametic interchange. Paroral cones, often of fair size, are formed but breakdown of the cones to permit micronuclear passage has not been observed. In conjugation there are the usual three pregamic divisions; the first shows four characteristic crescents. The resulting nuclei may all participate in the second division. Fertilization occurs in the paroral cone area. Frequently, separation of the conjugants takes place immediately after the first division of the synkaryon. The old macronucleus undergoes very little change prior to the last postzygotic micronuclear division in the ex-conjugant, when it goes into a skein condition. Four macronuclear and four micronuclear anlagen are formed in the ex-conjugants at the completion of reorganization. On occasion giant individuals of P. polycaryum were observed to have ingested numbers of Tetrahymena pyriformis. The presence of an unidentified rod-like organism in the cytoplasm of the paramecia (non-conjugating) was detected in one collection from Bangalore, India.     

Life Cycle of Autogamous Strains of Euplotes minuta*

SYNOPSIS. The life cycles of 5 autogamous strains of Euplotes minuta are reported. Interautogamic intervals (measured as number of fissions) are quite variable among clones belonging to the same strain, while their variability is much reduced (15 fissions) among sublines of the same clone. By selecting the clone with the shortest immature period to start successive autogamous generations, it has been found that all clones undergo autogamy almost synchronously and have a very short period of immaturity at the 5th autogamous generation. Both conjugation and autogamy, however, are consistently followed by immature periods in all autogamous strains examined. Mating capacity as well as competence for autogamy are reached almost simultaneously in all clones of the strains studied with the exception of about 1/3 of the A-31 clones in which autogamy occurs significantly earlier than conjugation. The results are discussed from the genetic point of view and in relation to the sexual mechanisms operating in nature within different populations of the species.     

Autogamy in Paramecium cell cycle stage-specific commitment to meiosis

Autogamy is a process of meiosis and fertilization which takes place in unpaired Paramecium cells, and which is triggered by starvation. This study examines the consequences of nutritional down-shift at various points within the cell cycle on the occurrence of autogamy. It shows that cells become committed to autogamy in a two-step process. An initial point of commitment to autogamy occurs about 100 min prior to the median time of cell division (cell cycle duration, 330 min). Cells which have become committed to autogamy initiate meiosis following the next fission, others complete another vegetative cell cycle before undergoing meiosis. Treatments that perturb the cell cycle and displace the point of commitment to division also displace the point of initial commitment to autogamy to the same extent.The initial commitment to autogamy can be reversed by refeeding. The second, final, point of commitment to autogamy occurs about 30 min after the fission, immediately prior to initiation of meiosis, and coincides with the beginning of meiosis. If cells are refed at this point, or at later stages, autogamy continues.Autogamy is not well synchronized either in naturally starved cultures or in those subjected to abrupt nutritional down-shift. This is a consequence of the cell cycle stage dependence of entry into autogamy. Autogamy occurs synchronously in samples of dividers selected from asynchronous cultures 2 or more hours after nutritional down-shift. The timing of the events of conjugation and autogamy coincide when the pre-autogamous fission is aligned temporally with the initial contact of mating cells.     

Commitment to autogamy in Paramecium blocks mating reactivity: implications for regulation of the sexual pathway and the breeding system

Commitment to autogamy blocks mating reactivity in Paramecium. Cells which had previously developed mating reactivity, lost reactivity 30-90 min prior to the preautogamous fission. Mating reactivity develops at a standard level of starvation when cells are allowed to exhaust their food supply naturally. In abruptly starved cultures, mating reactivity appears 3.3 h after downshift. Autogamy is also triggered by starvation. The level of starvation required for initiation of autogamy decreases progressively as cells age. When the autogamy starvation threshold drops to such a low level that all cells become committed to autogamy before any of them develop mating reactivity, reactivity does not occur under natural starvation conditions and the period of maturity for conjugation has come to an end. There is no absolute immature period for autogamy.     

Autogamy is induced in Paramecium bursaria by methyl cellulose

When isolated single cells of Paramecium bursaria were treated with 1.25% methyl cellulose, most of the single cells showed nuclear changes which were substantially similar to those seen in conjugation, suggesting that the cells undergo autogamy. Additional cytological observations indicated that the cells underwent normal nuclear processes during autogamy. The ability to induce autogamy will facilitate genetic studies in P. bursaria.     

Factors Determining the Frequency of the Killer Trait within Populations of the Paramecium aurelia Complex

The factors maintaining the cytoplasmically inherited killer trait in populations of Paramecium tetraurelia and Paramecium biaurelia were examined using, in part, computer simulation. Frequency of the K and k alleles, infection and loss of the endosymbionts, recombination during conjugation and autogamy, cytoplasmic exchange and natural selection were incorporated in a model. Infection during cytoplasmic exchange at conjugation and natural selection were factors that would increase the proportion of killers in a population. Conversely, k alleles reduced the proportion of killers in a population, acting through conjugation and autogamy. Field studies indicate that the odd mating type is prevalent in P. tetraurelia isolated from nature. Conjugation and therefore transmission by cytoplasmic transfer would be rare. Competition studies indicate a strong selective disadvantage for sensitives at concentrations found in nature. Natural selection must therefore be the factor maintaining the killer trait in P. tetraurelia.     

Chemical Induction of Autogamy in Paramecium multimicronucleatum,Syngen 21

The combination of KCl + acriflavine + Ca²⁺-poor condition, known as conjugation-inducing-chemicals, was found to be autogamy-inducing-chemicals as well. Suspension of a single cell of Paramecium multimicronucleatum, syngen 2 in this medium for three hours or more resulted in autogamy that was evidenced by cytological similarity to conjugation.     

Inheritance of Autogamy and the Killer Trait in Euplotes minuta

SYNOPSIS. Eight interfertile strains of Euplotes minuta collected from two localities in the Mediterranean Sea are shown to comprise a single breeding system of multiple mating types. One strain regularly passes through autogamy and, in crosses to non-autogamous strains, preliminary evidence for the genic control of autogamy was obtained. The cytogenetic events of conjugation and autogamy are briefly described. Three strains are "killers." Killer cells liberate a substance into the surrounding medium which has a lethal effect on cells of other strains called "sensitives." This substance, "euplotein," is not produced by sensitives. The inheritance of the killer trait is apparently under the control of a cytoplasmic factor but present attempts to identify the factor microscopically were without success. The discussion stresses the potential usefulness of this species for studies of protozoan genetics and development.     

Ultrastructural Features of the Oral Region of Amicronucleate Paramecium tetraurelia in Autogamy1

ABSTRACT Amicronucleate Paramecium tetraurelia fails to develop an oral apparatus but resorbs the pre-existing one in the sexual cycle. Previous cytological studies have revealed the presence of a shallow, oral depression with a few scattered basal bodies after autogamy or conjugation. The present ultrastructural study of the ectoplasm beneath the oral depression revealed only the presence of a disorganized filamentous reticulum and small blocks of cytopharyngeal ribbons. In addition, beneath the oral depression, a large number of discoidal vesicles were found, similar to but larger in diameter than those normally associated with the cytopharynx of vegetative cells. The oral structures found in amicronucleates in autogamy might be remnants of the pre-existing oral apparatus, but the possibility that they were newly developed was not excluded. The morphogenetic interest of these structures was discussed. It is evident that amicronucleates in autogamy do not develop an extensive oral ultrastructural organization.     

Identification of DNA Segments Capable of Rescuing a Non-Mendelian Mutant in Paramecium

The non-Mendelian mutant d48 of Paramecium tetraurelia contains micronuclear wild type A genes, but at autogamy and conjugation proper processing fails and new macronuclei lack A genes. When cloned A genes are injected into the macronucleus of d48, proper processing is restored at the next autogamy; d48 is rescued, becoming permanently wild type. In the present study we have injected portions of the A gene into d48. We find that the ability to rescue extends over a large portion of the gene, with highest activity near a series of 221-bp repeat units in the middle of the gene. Regions outside the A gene are inactive.     

Morphological Drift Accompanying Nascent Population Differentiation in the Ciliate Euplotes vannus

A rare phenomenon can occur in ciliated protists of the genus Euplotes, which can undergo genetic recombination by the normal outbreeding process of conjugation following mild starvation. Occasionally, the dominant mutation for the autogamy trait arises. Individuals possessing the trait show obligate self-fertilization upon mild starvation. This yields, after normal asexual division, a population of individuals that are reproductively isolated from the parental outbreeding strain. A morphometric analysis of sympatric autogamous and non-autogamous populations of Euplotes vannus from Somalia demonstrates that there has been morphological drift in gross body proportions in the autogamous populations. However, the positional patterns of the locomotory organelles on the ventral surface remain unchanged. The changes in body proportions in the autogamous populations are relevant to the mechanics of the conjugation process, which involves fusion of the oral regions of paired cells belonging to complementary mating types.     

Persistence of Oral Structures in the Sexual Process of Amicronucleate Paramecium tetraurelia

ABSTRACT In the sexual process, amicronucleate Paramecium tetraurelia , unlike micronucleates, fail to produce an oral apparatus, but resorb the pre-existing one. Exceptions were found in some amicronucleate cell lines in which about 1% of the cells possessed oral structures, including pieces of oral membranelles, sometimes complete with buccal cavity, after autogamy or conjugation. By following oral development in the sexual process in some detail, the present study supports the view that these oral structures are derived from the pre-existing oral apparatus and not newly developed from the oral primordium. The possible involvement of the micronucleus and the pre-existing oral apparatus in oral resorption is discussed. The possession of a functional oral apparatus after the sexual process may open up a new evolutionary avenue to the amicronucleates.     

Nuclear Behavior and Morphogenetic Changes in Fission and Conjugation of Aspidisca costata (Dujardin)

Nuclear behavior and accompanying cytoplasmic changes have been traced in conjugation and binary fission in Aspidisca costata, a fresh water hypotrich. Cases of autogamy in singles also have been found. In conjugation the animals pair with left ventral sides apposed, at an angle of ∼ 100°, connected by a broad bridge. After 2 maturation divisions 4 potential gametic nuclei are formed. Thereafter 1 of 2 alternative plans is followed. The essential difference between them is the occurrence in Scheme B of degeneration of 2 products of the 2nd prezygotic division, thus reducing to 2 the 4 potential gametic nuclei–all viable in Scheme A. Single or double synkarya are thus generated, respectively, in each conjugant, certain of them necessarily by autogamy. After 1 or 2 postzygotic divisions, 4 final nuclei are formed: 1 macronuclear anlage, 2 micronuclei, and a degenerate nucleus. The exconjugants at 1st are astomatous, although a new complete set of cirri (exclusive of the anterior “tooth”) arises during conjugation. A later reorganization in the exconjugants must restore the buccal apparatus and probably also replaces the cirri and tooth. The morphogenetic changes in fission have been observed. All the cirri are replaced, in 2 sets, at each cycle. These originate in a single locus, a pouch anterior to the contractile vacuole. A new adoral zone of membranelles (AZM) arises for the opisthe, as a new tooth. The old AZM and the old tooth are retained by the proter. It is suggested that the tooth is the homolog of the 1/I cirrus of other hypotrichs.     

Mating Types in Glaucoma scintillans

SYNOPSIS. Two sexually isolated varieties or syngens of Glaucoma scintillans have been found in Japan. Each of these includes 8 (or 9) mating types, any one of which can conjugate with the other 7 (or 8). Under appropriate conditions, a high percentage of the animals in a mixture of 2 mating types of the same syngen underwent conjugation. Conjugating pairs appeared to be formed only between animals of different mating types. In a few combinations, inter-syngenic conjugation (usually in less than 5% of the animals) was seen. Neither selfing nor autogamy was seen. Clones from conjugants pass thru a long period of immaturity and a period of adolescence before attaining full maturity. Limited observations indicated that inheritance of mating types is synclonal. Nuclear reorganization processes during conjugation are similar to those reported for Tetrahymena pyriformis.     

Mating Types and Mating Type Inheritance in Euplotes minuta Yocom (Ciliata, Hypotrichida)

SYNOPSIS. Thirty-one stocks of a marine ciliate, Euplotes minuta Yocom, collected from different localities, can be grouped in seven mating types. True pairs are formed only in mixtures of stocks belonging to different mating types. No selfing pairs or intraclonal conjugation have ever been observed. Synclonal inheritance of mating types is the rule, although about 10% of pairs show clonal inheritance. The latter can be explained by assuming syncaryon formation through cytogamy or through caryogamy of pronuclei derived from different products of meiosis. Mating type determination is due to 7 alleles at the single locus mt . There is complete dominance among the 7 alleles which can be orderly seriated, as shown in Table 3, according to their dominance relationship. The 5 stocks, and only these 5, of mating type VII have the autogamy trait. The mortality rate in F₁ and F₂ is very low–a maximum of 10%; however, the F₂'S obtained by autogamy from F₁ progenies in which mating type VII is involved have a very high mortality rate. The two facts (high mortality rate in F₂ and strict association of autogamy trait with mating type VII in natural populations) have been considered as evidences of a probable isolation mechanism existing between mating type VII and the other 6 mating types. Thus, the 7 mating types have been assigned to the same syngen only tentatively.     

Variability of autogamy-maturation pattern in genetically identical populations of Paramecium tetraurelia

Autogamy in Paramecium tetraurelia is a form of sexual reproduction in a single cell that results in homozygosity in every genetic locus. Autogamy becomes inducible by natural starvation several fissions after the previous autogamy, and percent autogamy increases gradually with clonal age to reach 100%. We here report the degree of variability of the autogamy-maturation pattern, and how it is inherited through autogamous generations. We assessed the autogamy-maturation pattern by monitoring percent autogamy at the ages of 9, 18 and 27 fissions in the wild-type stock 51. To determine how the autogamy-maturation pattern is inherited, clones that showed the lowest and the highest percent autogamy at age 18 in a given autogamous generation (Gn) were examined for their percent autogamy in the next autogamous generation (Gn+1). This procedure was repeated through successive autogamous generations. We found that percent autogamy at ages 9 and 27 was rather stable (low and high, respectively), while it was extremely variable at age 18 ranging from 3% to 100%. We also found that percent autogamy at age 18 in the progeny clones was variable irrespective of percent autogamy at age 18 in the parental clones; there was no regular rule such as producing progeny with higher (or lower) percent autogamy from parents with lower (or higher) percent autogamy.     

Paramecium biaurelia--relation of strains

Inter- and intra-strain crosses were made in Paramecium biaurelia of the P. aurelia species complex for studying the relation of strains within the species. Altogether ten strains originating from Scotland, Spain, Romania, Czech Republic, Ukraine, Italy, Germany, Russia, and Poland (two strains) were studied. A high percentage of surviving clones in both generations, F1 (obtained by conjugation) and F2 (obtained by autogamy), was observed in strain crosses, indicating a strong relation between the strains, and absence of genetic barriers between them in P. biaurelia.     

Microsurgical analysis of the clonal age and the cell-cycle stage required for the onset of autogamy in Paramecium tetraurelia

When autogamy was induced in competent cells of Paramecium tetraurelia by depriving them of food, the onset of autogamy was preceded by a critical fission which occurred in the starvation medium. When the cells were fed again immediately after the fission, they did not undergo autogamy. However, they did undergo autogamy when they were fed later than 1 hr after the critical fission. The irreversible differentiation for autogamy seems to be at about 1 hr after the critical fission. This procedure thus provides the opportunity to induce autogamy synchronously. The result of macronuclear transplantation demonstrated that autogamy was under the control of macronucleus. Moreover, the clonal age required for autogamy was found to be shortened by repetitive elimination of a part of the macronucleus. The result can be explained by the hypothesis that clonal age is measured in rounds of chromosome replication or DNA synthesis rather than cell divisions.     

Molecular biology of the genes for immobilization antigens in Paramecium

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition to known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51C and 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.