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.     

Nuclear transplant studies of the determination of mating type in germ nuclei of Paramecium tetraurelia

The micronucleus from vegetative cells of one mating type (O or E) in Paramecium tetraurelia was transplanted by micropipet into amicronucleate cells of opposite mating type (E or O). When autogamy was induced in the recipient cells, they developed new macronuclei and micronuclei derived from the transplanted micronucleus and usually expressed the same mating type as the recipients. The results indicate that micronuclei in the asexual phase may be undetermined for mating type. Recipient E cells in which the macronucleus had been previously removed were transplanted with a whole macronucleus from an O cell. Their mating type was soon transformed E to O before the occurrence of autogamy, and remained O after autogamy. This demonstrates that the transplanted macronucleus determined the O cytoplasmic state to determine the developing zygotic macronucleus for mating type O. It is unlikely that the micronucleus is determined for mating type in O or E cell during the asexual cycle.     

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.     

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.     

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.     

3种披碱草属牧草交配系统研究

本文通过测定3种披碱草属牧草的花粉-胚珠比（P/O值）、杂交指数（OCI）,结合不同授粉方式下这3种牧草的结实率,探讨这3种披碱草属牧草的交配系统, 为披碱草属牧草杂交育种、丰产栽培等提供理论依据。结果表明,老芒麦、麦薲草和披碱草花粉-胚珠比（P/O值）均处于31.9～396.0,交配系统属于兼性自交;杂交指数OCI值为2,交配系统也属于兼性自交;结实率统计表明,以自交为主,异交可育。因此,3种披碱草属牧草的交配系统属于兼性自交类型。     

Circadian rhythmicity within single cells of Paramecium bursaria

Cell populations of Paramecium bursaria show mating reactivity in the light period, but not in the dark period, when exposed to a light-dark cycle (LD 12:12). After they are transferred to constant-light (LL) conditions (1,000 lux), they continue to show a circadian rhythm of mating reactivity. The rhythm gradually dampens in LL so that mating reactivity in populations becomes arrhythmic in LL within 2 weeks. We wanted to know whether the arrhythmicity of this population was due to the absence of circadian rhythmicity within each individual cell, or merely due to asynchrony of a population of individually rhythmic cells. Therefore, single cells were isolated randomly from an arrhythmic population that had been in LL for a long time. Then the mating reactivity of these single cells was individually tested every 3 hr for 2 days. Each single cell showed a circadian mating rhythm in LL. This shows that the disappearance of the mating rhythm in cell populations under LL is not caused by disappearance of circadian rhythmicity within individual cells, but is due to desynchronization among cells in a population. When an arrhythmic population in LL is darkened for 9 hr, the mating reactivity rhythm of the cell population reappears. This occurs by resynchronization of the rhythms among individual cells, as can be shown by exposing single cells to pulses of 9 hr of darkness. This dark treatment causes phase shifts of single-cell rhythms, and a phase response curve is obtained for this stimulus. This phase-shifting behavior explains the efficacy of 9-hr dark pulses in restoring the population's rhythm.     

Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

A circadian rhythm of mating type reversals in Paramecium multimicronucleatum, syngen 2, and its genetic control

Two new alleles, C and c, involved in mating type expression were demonstrated. A dominant allele, C(cycler), must be present for the expression of the rhythm involving a sequential alternation of the two complementary mating types (III and IV). Cultures can be entrained with light-dark cycles. The phase of each clone can be characterized by its III to IV and IV to III transitions in relation to the zero hour of a given light-dark cycle. Phase is a stable phenotypic trait during asexual reproduction, but following sexual reproduction it does not display Mendelian segregation. Instead phase is determined through nuclear differentiation, i.e., the trait is controlled by differently determined macronuclear alagen (caryonidal inheritance) which normally segregate at the second cell division after conjugation. The phase of a clone within its genetic limits is a function of the photofractions and the light intensities used in the entraining treatment. By examining a number of clones a variety of phase angles between the mating type cycle and the entraining light-dark cycle are found. Dividing cells which are sexually unreactive and therefore do not express the rhythm can be entrained and following entrainment, phase is inherited through repeated cell replications at a rate greater than one fission a day in continuous darkness or continuous dim light. This result unique to this system indicates that the cellular processes underlying the phase and period of this circadian rhythm persist (unexpressed: sexual reactivity requires slight starvation) through repeated cell replications even when the division cycle is considerably shorter than the expressed circadian period. The rhythm has a circadian period in continuous darkness or light (tested for six days) of less than 24 hours. The reversal of mating type ceases in continuous light at higher intensities. Cells homozygous for the recessive allele, c(acyclic), do not reverse mating type but are either mating type III or IV, again as a consequence of nuclear differentiation. Since individual cells with the dominant allele express both mating types, differentiation for mating type can not involve the absence in the macronucleus of mating type determining factors.     

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.     

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.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

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.     

Florivory increases selfing: an experimental study in the wild strawberry, Fragaria virginiana

Florivores are antagonists that damage flowers, and have direct negative effects on flowering and pollination of the attacked plants. While florivory has mainly been studied for its consequences on seed production or siring success, little is known about its impact on mating systems. Damage to flowers can alter pollinator attraction to the plant and may therefore modify patterns of pollen transfer. However, the consequences of damage for mating systems can take two forms: a decrease in flower number reduces opportunities for intra-inflorescence pollen deposition (geitonogamy), which, in turn, may lead to a decrease in selfing; whereas a decrease in floral display may also reduce overall visitation and thus increase the chances of self-pollination via facilitated or autonomous autogamy. We investigated the effects of damage by a bud-clipping weevil ( Anthonomus signatus ) in Fragaria virginiana in an experimental setting mimicking natural conditions. We found that increased damage led to an increase in selfing, a result consistent with the increased autogamy pathway. We discuss the implications of this finding and evaluate the generality of florivore-mediated mating system expression.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

Microinjection of plasmid DNA encoding the A surface antigen of Paramecium tetraurelia restores the ability to regenerate a wild-type macronucleus.

Strain d48 of Paramecium tetraurelia contains the A i-antigen gene in the micronucleus, but the gene is lost when micronuclear products develop into the macronucleus. It has recently been shown that when injected into d48, macronucleoplasm from the wild type transforms d48 cells to wild type. It is shown here that wild-type cytoplasm can also bring about transformation, with a marked stage-specific sensitivity for both donor and recipient. It was also found that a plasmid containing the cloned A gene could transform d48 to wild type. Injection of nucleoplasm from animals in the vegetative stage of the cell cycle into the cytoplasm of recipients at various stages of autogamy caused high-frequency transformation of cells able to express the A serotype both before and after the next autogamy. Injection of nucleoplasm into vegetative macronuclei produced over 70% transformants able to express the A serotype after the next autogamy. The ability of nucleoplasm to transform was acquired at the second cell cycle after autogamy and was maintained throughout the vegetative stage. When cytoplasm was obtained from donors during autogamy and injected into the cytoplasm of recipients 1 to 2 h after the sensitive period, quite high frequencies of stable revertants were found when tested both before and after the next autogamy. Cells that were injected into the macronucleus with the cloned A plasmid expressed the A serotype after five fissions in over 20% of the lines and maintained this ability through successive fissions; all transformants except one stably expressed the A serotype even after the next autogamy.     

EVOLUTION OF THE NUMBER OF SEXES

In sexually reproducing isogamous organisms, gametes (or diploid cells in ciliates) are classified into two or more groups called sexes, and mating occurs only between cells of different sexes. We have studied the evolutionary stability of the number of sexes maintained in a population by examining population-genetic models. For models in which the diploid genome determines the sex of conjugal cells, a one-locus system with three alleles of pecking-order dominance is assumed. Unlike traditional bisexual models, the genetic dynamics then depend on a rule, called mating kinetics, which determines the proportion of matings between each pair of sexes for given proportions of cells of the three sexes. The evolutionary consequences greatly depend on the mating kinetics assumed. Of the four mating kinetics examined, two give a large advantage to rare sexes whose cells quickly find heterosexual partners, which implies an evolutionary increase in the number of sexes. In contrast, the other two mating kinetics, in which gametes wait for suitable mates without being eliminated from the gamete pool during this waiting period, produce neutrally stable dynamics with curves or a surface of equilibria. Then random drift or differential fitness among sexes would result in the loss of sex alleles until only two remain in the population. This suggests a turnover of sexes; a new sex invades and replaces resident sexes after temporary coexistence. Similar results are obtained in models with haploid sex-determination and with autogamy. These two processes, however, may help to maintain many sexes indirectly by preventing the accumulation of recessive lethal mutations on sex chromosomes. The relationship of these models to models of self-sterility factors in plants and sex factors in honeybees is discussed. To summarize, the number of sexes should increase when conjugal cells must find mates during a limited period of time, otherwise a two-sex system should evolve. We conclude that there may be more isogamous species with three or more sexes than are currently known.     

A Study of Odd Mating-Type Determination Factor by Transfer of Macronuclear Karyoplasm in PARAMECIUM TETRAURELIA

The unique feature of the "B system" of mating-type determination found in Paramecium tetraurelia is the existence of a cytoplasmic difference between odd (O) and even (E) cells created and maintained by the action of their macronuclei. Thus far, the presence of a determining factor that controls the differentiation of the developing zygotic macronucleus for O mating type has not been verified. Results of crosses between cells of differing clonal age and complementary mating type suggest that, for one to two fissions after autogamy, O cells produce some factor that determines the gametic nucleus (micronucleus) as mating type O. Direct evidence for the production of O-determining factor by the young O macronucleus was obtained by transplanting young O macronuclear karyoplasm (a part of the macronucleus) into E cells: 32-35% of E exautogamous clones transformed to O; transformation of E exautogamous clones to O reached as high as 72% by transfer of young O macronuclear karyoplasm from a conjugant, 3-4 hr after mixing. This indicates that O determinants produced by the O macronucleus can also act during the sensitive period of development of the new macronucleus. These O-determining factors may be produced or activated at the sexual stage and then decrease in activity in subsequent fissions after new macronuclear reorganization.     

The genetic basis for mating-induced sex differences in starvation resistance in Drosophila melanogaster

Multiple genetic and environmental factors interact to influence starvation resistance, which is an important determinant of fitness in many organisms, including Drosophila melanogaster. Recent studies have revealed that mating can alter starvation resistance in female D. melanogaster, but little is known about the behavioral and physiological mechanisms underlying such mating-mediated changes in starvation resistance. In the present study, we first investigated whether the effect of mating on starvation resistance is sex-specific in D. melanogaster. As indicated by a significant sex × mating status interaction, mating increased starvation resistance in females but not in males. In female D. melanogaster, post-mating increase in starvation resistance was mainly attributed to increases in food intake and in the level of lipid storage relative to lean body weight. We then performed quantitative genetic analysis to estimate the proportion of the total phenotypic variance attributable to genetic differences (i.e., heritability) for starvation resistance in mated male and female D. melanogaster. The narrow-sense heritability (h²) of starvation resistance was 0.235 and 0.155 for males and females, respectively. Mated females were more resistant to starvation than males in all genotypes, but the degree of such sexual dimorphism varied substantially among genotypes, as indicated by a significant sex × genotype interaction for starvation resistance. Cross-sex genetic correlation was greater than 0 but less than l for starvation resistance, implying that the genetic architecture of this trait was partially shared between the two sexes. For both sexes, starvation resistance was positively correlated with longevity and lipid storage at genetic level. The present study suggests that sex differences in starvation resistance depend on mating status and have a genetic basis in D. melanogaster.     

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.