Cytological investigation of the mechanism of parthenogenesis in Drosophila mercatorum

Thelytokous parthenogenesis (female progeny only) in animals is believed to arise initially in unfertilized eggs produced by bisexual females via the fusion of two haploid nuclei following meiosis, to produce diploid female progeny. The transition from sexual to parthenogenetic mechanisms of reproduction requires that the egg replace the paternal contributions of a haploid genetic complement and the basal body, which is thought to be essential for centrosome formation. The transitional facultative parthenogenetic stage is usually associated with a high rate of failed or abortive development, but the molecular and mechanistic reasons for this failure remain unclear. We show that a facultative parthenogenetic strain of Drosophila mercatorum produces a high percentage of unfertilized eggs competent to restore diploidy and form centrosomes de novo following meiosis. The female meiotic products replicate and divide by an acentrosomal mechanism in most oocytes and cytoplasmic centrosomes form in 35% of the oocytes. However, after pronuclear replication the cytoplasmic centrosomes must "capture" two haploid nuclei in order to restore diploidy. In practice, this process frequently fails due to centrosome-mediated capture events of single or more than two haploid nuclei, as well as multiple nuclear capture events in a single embryo when excess free centrosomes are not inactivated following formation of the first zygotic nucleus. Additionally, as development proceeds, many of the centrosomes that initiate syncytial development do not remain functional, possibly due to centrosome maturation defects, and later stages of syncytial development fail. The combined effect of the high error rate associated with nuclear capture and the failure of centrosome maturation during later developmental prevents successful parthenogenesis in most of the eggs that initiate development. This shows that the high rate of failed development associated with the transition from sexual to parthenogenetic reproduction is limited by the low probability of the formation of a diploid zygotic nucleus with the correct complement of centrosomes in D. mercatorum.     

Reciprocal inheritance of centrosomes in the parthenogenetic hymenopteran Nasonia vitripennis

Background: In the majority of animals, the centrosome-the microtubule-organizing center of the cell-is assembled from components of both the sperm and the egg. How the males of the insect order Hymenoptera acquire centrosomes is a mystery, as they originate from virgin birth.Results: To address this issue, we observed centrosome, spindle and nuclear behavior in real time during early development in the parthenogenetic hymenopteran Nasonia vitripennis. Female meiosis was identical in unfertilized eggs. Centrosomes were assembled before the first mitotic division but were inherited differently in unfertilized and fertilized eggs. In both, large numbers of asters appeared at the cortex of the egg after completion of meiosis. In unfertilized eggs, the asters migrated inwards and two of them became stably associated with the female pronucleus and the remaining cytoplasmic asters rapidly disappeared. In fertilized eggs, the Nasonia sperm brought in paternally derived centrosomes, similar to Drosophila melanogaster. At pronuclear fusion, the diploid zygotic nucleus was associated only with paternally derived centrosomes. None of the cytoplasmic asters associated with the zygotic nucleus and, as in unfertilized eggs, they rapidly degenerated.Conclusions: Selection and migration of the female pronucleus is independent of the sperm and its aster. Unfertilized male eggs inherit maternal centrosomes whereas fertilized female eggs inherit paternal centrosomes. This is the first system described in which centrosomes are reciprocally inherited. The results suggest the existence of a previously undescribed mechanism for regulating centrosome number in the early embryo.     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

Centrosome inheritance in insects: fertilization and parthenogenesis.

Centrosome biogenesis is unclear, although much structural and biochemical research has been performed in several experimental systems. An alternative model to study the assembly of functional centrosomes could be the process of zygotic centrosome formation at the beginning of embryonic development. Although it seems obvious that the sperm cell provides the centrosome at fertilization, some pieces of evidence are not in line with this point of view and give controversial results. Such an analysis could provide useful information if applied to a large variety of organisms. Since insects are a highly diverse group of organisms they provide a variety of models in which to study the process of centrosome reconstitution during fertilization. Moreover, many insect species reproduce by parthenogenesis, a special mode of reproduction in which embryonic development occurs without male contribution. Studies of unfertilized parthenogenetic eggs may therefore teach us much about the process of centrosome assembly in the absence of preexisting centrioles.     

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. I. the Modes of Diploidization in the Gynogenesis Induced by a Male-Sterile Mutant, ms(3)K81

Sperm that are produced by males homozygous for ms(3)K81 , a male sterile mutant of Drosophila melanogaster, are defective in syngamy but are capable of activating eggs to develop gynogenetically. The activated eggs usually produce haploid embryos, but a small fraction (10 ^-4–10^-5) of them give rise to diploid impaternate adults. To know the cytological mechanisms by which these impaternates restore diploidy, the genotypes of impaternate progeny obtained from females doubly heterozygous for visible markers were examined. The results show that, as generally found among parthenogenetic Drosophila, diploidy is restored after completing meiosis either by pronuclear fusion or by gamete duplication (doubling of a haploid cleavage nucleus). The fusion of two nonsister nuclei following meiosis II (central fusion) was indicated to be a predominant mode of diploidization in this species. Two meiotic mutants, mei-9 and mei-S332, which are known to greatly increase meiotic nondisjunction, did not cause an increased incidence of impaternates. This seems to exclude the possibility that some impaternates might have been derived from diploid egg nuclei produced through nondisjunction.     

Loss of function of MULTICOPY SUPPRESSOR OF IRA 1 produces nonviable parthenogenetic embryos in Arabidopsis

In sexually reproducing species, fertilization brings together in the zygote the genomes of the female and male gametes. In several animal species, female gametes are able to initiate embryogenesis in the absence of fertilization, a process referred to as parthenogenesis. Parthenogenesis has been engineered in mice by tampering with expression of loci under epigenetic controls [1]. In plants, embryo development in the absence of fertilization has been reported in cases in which meiosis is bypassed leading to apomictic development, and parthenogenetic development from a reduced egg cell has been only reported in rare accidental cases [2]. We report that single mutations in the gene MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) are able to initiate parthenogenetic development of the embryo in Arabidopsis thaliana from eggs cells produced by meiosis. The WD40 repeat protein MSI1 is part of the evolutionarily conserved Polycomb group (PcG) chromatin-remodeling complexes [3] and is homologous to the Retinoblastoma binding proteins P55 in Drosophila and RbAp48 in mammals [4]. Nonviable haploid parthenogenetic msi1 embryos express molecular markers and polarity similar to diploid wild-type (wt) embryos produced by fertilization, indicating a maternal contribution to early patterning of the Arabidopsis embryo.     

Aster self-organization at meiosis: a conserved mechanism in insect parthenogenesis?

Unfertilized eggs usually lack maternal centrosomes and cannot develop without sperm contribution. However, several insect species lay eggs that develop to adulthood as unfertilized in the absence of a preexisting centrosome. We report that the oocyte of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum is able to self-organize microtubule-based asters, which in turn interact with the female chromatin to form the first mitotic spindle. This mode of reproduction provides a good system to investigate how the oocyte can assemble new centrosomes and how their number can be exactly monitored. We propose that the cooperative interaction of motor proteins and randomly nucleated surface microtubules could lead to the formation of aster-like structures in the absence of pre-existing centrosomes. Recruitment of material along the microtubules might contribute to the accumulation of pericentriolar material and centriole precursors at the focus of the asters, thus leading to the formation of true centrosomes. The appearance of microtubule asters at the surface of activated oocytes could represent a possible common mechanism for centrosome formation during insect parthenogenesis.     

Diploidy restoration in Wolbachia-infected Muscidifurax uniraptor (Hymenoptera: Pteromalidae)

Thelytokous reproduction, where females produce diploid female offspring without fertilization, can be found in many insects. In some Hymenoptera species, thelytoky is induced by Wolbachia, a group of cytoplasmically inherited bacteria. We compare and contrast early embryonic development in the thelytokous parthenogenetic species Muscidifurax uniraptor with the development of unfertilized eggs of the closely related arrhenotokous species, Muscidifurax raptorellus. In the Wolbachia-infected parasitic wasp M. uniraptor, meiosis and the first mitotic division occur normally. Diploidy restoration is achieved following the completion of the first mitosis. This pattern differs in the timing of diploidy restoration from previously described cases of Wolbachia-associated thelytoky. Results presented here suggest that different cytogenetic mechanisms of diploidy restoration may occur in different species with Wolbachia-induced thelytoky.     

Spindle Assembly and Mitosis without Centrosomes in Parthenogenetic Sciara Embryos

In Sciara, unfertilized embryos initiate parthenogenetic development without centrosomes. By comparing these embryos with normal fertilized embryos, spindle assembly and other microtubule-based events can be examined in the presence and absence of centrosomes. In both cases, functional mitotic spindles are formed that successfully proceed through anaphase and telophase, forming two daughter nuclei separated by a midbody. The spindles assembled without centrosomes are anastral, and it is likely that their microtubules are nucleated at or near the chromosomes. These spindles undergo anaphase B and successfully segregate sister chromosomes. However, without centrosomes the distance between the daughter nuclei in the next interphase is greatly reduced. This suggests that centrosomes are required to maintain nuclear spacing during the telophase to interphase transition. As in Drosophila, the initial embryonic divisions of Sciara are synchronous and syncytial. The nuclei in fertilized centrosome-bearing embryos maintain an even distribution as they divide and migrate to the cortex. In contrast, as division proceeds in embryos lacking centrosomes, nuclei collide and form large irregularly shaped nuclear clusters. These nuclei are not evenly distributed and never successfully migrate to the cortex. This phenotype is probably a direct result of a failure to form astral microtubules in parthenogenetic embryos lacking centrosomes. These results indicate that the primary function of centrosomes is to provide astral microtubules for proper nuclear spacing and migration during the syncytial divisions. Fertilized Sciara embryos produce a large population of centrosomes not associated with nuclei. These free centrosomes do not form spindles or migrate to the cortex and replicate at a significantly reduced rate. This suggests that the centrosome must maintain a proper association with the nucleus for migration and normal replication to occur.     

The Chromosomes of Turkey Embryos during Early Stages of Parthenogenetic Development

In the early stages of parthenogenetic development in turkey eggs, many blastoderms are mosaics of haploid, diploid and polyploid cells. The genome composition of these blastoderms can be identified by C-banding. They may be generally described as either A-Z/2A-ZZ/nA-nZ or A-W/2A-WW/nA-nW and are found in a nearly 1:1 ratio. The blastoderms showing the W body (W⁺) become lethal within two days of incubation. The haploid cell proportion decreases rapidly during the early stage of development, and, as haploid cells decrease, the proportion of polyploid cells appears to increase. At six days of incubation, various kinds of parthenogenetic development can be observed. Their genome compositions are either diploid (2A-ZZ) or mosaic (A-Z/2A-ZZ). These findings suggest that diploid parthenogenesis occurs by either suppression of meiosis II or chromosome doubling some time after the first cleavage division. The frequent occurrence of mosaic blastoderms indicates that the majority, if not all, of the parthenogenetic embryos initiate their development in haploid ova.     

Whole-genome scan in thelytokous-laying workers of the Cape honeybee (Apis mellifera capensis): central fusion, reduced recombination rates and centromere mapping using half-tetrad analysis

While workers of almost all subspecies of honeybee are able to lay only haploid male eggs, Apis mellifera capensis workers are able to produce diploid female eggs by thelytokous parthenogenesis. Cytological analyses have shown that during parthenogenesis, egg diploidy is restored by fusion of the two central meiotic products. This peculiarity of the Cape bee preserves two products of a single meiosis in the daughters and can be used to map centromere positions using half-tetrad analysis. In this study, we use the thelytokous progenies of A. m. capensis workers and a sample of individuals from a naturally occurring A. m. capensis thelytokous clone to map centromere position for most of the linkage groups of the honeybee. We also show that the recombination rate is reduced by >10-fold during the meiosis of A. m. capensis workers. This reduction is restricted to thelytokous parthenogenesis of capensis workers and is not observed in the meiosis of queen within the same subspecies or in arrhenotokous workers of another subspecies. The reduced rate of recombination seems to be associated with negative crossover interference. These results are discussed in relation to evolution of thelytokous parthenogenesis and maintenance of heterozygosity and female sex after thelytoky.     

Parthenogenesis in Xenopus eggs requires centrosomal integrity

Xenopus eggs are laid arrested at second metaphase of meiosis lacking a functional centrosome. Upon fertilization, the sperm provides the active centrosome that is required for cleavage to occur. The injection of purified centrosomes mimics fertilization and leads to tadpole formation (parthenogenesis). In this work we show that the parthenogenetic activity of centrosomes is inactivated by urea concentrations higher than 2 M. The loss of activity is correlated with a progressive destruction of the centriolar cylinder and extraction of proteins. This shows that centrosomes are relatively sensitive to urea since complete protein unfolding and solubilization of proteins normally occurs at urea concentrations as high as 8-10 M. When present, the parthenogenetic activity is always associated with a pelletable fraction showing that it cannot be solubilized by urea. The parthenogenetic activity is progressively inactivated by salt concentrations higher than 2 M (NaCl or KCl). However, only a few proteins are extracted by these treatments and the centrosome ultrastructure is not affected. This shows that both parthenogenetic activity and centrosomal structure are resistant to relatively high ionic strength. Indeed, most protein structures held by electrostatic forces are dissociated by 2 M salt. The loss of parthenogenetic activity produced at higher salt concentrations, while the structure of the centrosome is unaffected, is an apparent paradox. We interpret this result as meaning that the native state of centrosomes is held together by forces that favor functional denaturation by high ionic strength. The respective effects of urea and salts on centrosomal structure and activity suggest that the centrosome is mainly held together by hydrogen and hydrophobic bonds. The in vitro microtubule nucleating activity of centrosomes can be inactivated at salt or urea concentrations that do not affect the parthenogenetic activity. Since egg cleavage requires the formation of microtubule asters, we conclude that the extracted or denatured microtubule nucleating activity of centrosomes can be complemented by components present in the egg cytoplasm. Both parthenogenetic and microtubule nucleating activities are abolished by protease treatments but resist nuclease action. Since we find no RNA in centrosomes treated by RNase, they probably do not contain a protected RNA. Taken together, these results are consistent with the idea that the whole or part of the centrosome structure acts as a seed to start the centrosome duplication cycle in Xenopus eggs.     

No patrigenes required for femaleness in the haplodiploid wasp Nasonia vitripennis

The parasitoid wasp Nasonia vitripennis is an emerging model organism for developmental and behavioral genetics. It reproduces by haplodiploidy; males typically develop parthenogenetically from haploid eggs and females from fertilized diploid eggs. A polyploid mutant strain is available in which females are triploid and lay haploid and diploid eggs that normally develop into males when unfertilized. In contrast to previous reports, approximately 2% of triploid females were found to occasionally produce daughters as well as gynandromorphs from diploid unfertilized eggs. Daughter production increased with age and differed among familial lineages. This is the first report of parthenogenetic female development in Nasonia. The results show that a paternally provided genome is not required for femaleness and call for modifications of existing models of sex determination in Nasonia.     

Drosophila parthenogenesis: a tool to decipher centrosomal vs acentrosomal spindle assembly pathways

Development of unfertilized eggs in the parthenogenetic strain K23-O-im of Drosophila mercatorum requires the stochastic interactions of self-assembled centrosomes with the female chromatin. In a portion of the unfertilized eggs that do not assemble centrosomes, microtubules organize a bipolar anastral mitotic spindle around the chromatin like the one formed during the first female meiosis, suggesting that similar pathways may be operative. In the cytoplasm of eggs in which centrosomes do form, monastral and biastral spindles are found. Analysis by laser scanning confocal microscopy suggests that these spindles are derived from the stochastic interaction of astral microtubules directly with kinetochore regions or indirectly with kinetochore microtubules. Our findings are consistent with the idea that mitotic spindle assembly requires both acentrosomal and centrosomal pathways, strengthening the hypothesis that astral microtubules can dictate the organization of the spindle by capturing kinetochore microtubules.     

The Population Genetics of Parthenogenetic Strains of DROSOPHILA MERCATORUM. II. the Capacity for Parthenogenesis in a Natural, Bisexual Population

Drosophila mercatorum is a bisexual species, but certain strains are capable of parthenogenetic reproduction in the laboratory. We investigated the parthenogenetic capacity of the virgin daughters of females captured from a natural, bisexual population in Hawaii. An isozyme survey indicated the natural population is polymorphic at about 50% of its loci, and its individuals heterozygous at 18% of their loci. The predominant mode of parthogenesis in D. mercatorum causes homozygosity for all loci in a single generation. Despite this radical change in genetic state, 23% of the virgin female lines produced adult parthenogenetic progeny, and 16% produced parthenogenetic progeny themselves capable of parthenogenetic reproduction. The parthenogenetic rats as measured by the number of parthenogenetic progeny themselves capable of parthenogenesis divided by the number of eggs laid is arougn 10(-5) for the virgin female lines. We argue that one of the major reasons for this low rate is that very few of the impaternate zygotes have a genotype that can survive and reproduce under the genetic conditions imposed by parthenogenetic reproduction. This intense selective bottleneck can be passed in a single generation if enough unfertilized eggs are laid, and once passed is accompanied by a large (perhaps a thousandfold) increase in the rate of parthenogenesis and by modifications in many phenotypic traits such as morphology and behavior.     

Drosophila parthenogenesis: a model for de novo centrosome assembly

The Drosophila egg contains all the components required to properly execute the early mitotic divisions but is unable to assemble a functional centrosome without a sperm-provided basal body. We show that 65% of unfertilized eggs obtained from a laboratory strain of Drosophila mercatorum can spontaneously assemble a number of cytoplasmic asters after activation, most of them duplicating in a cell cycle-dependent manner. Such asters are formed by a polarized array of microtubules that have their Asp-associated minus-ends converging at a main focus, where centrioles and typical centrosomal antigens are found. Aster assembly is spatially restricted to the anterior region of the oocyte. When fertilized, the parthenogenetic egg forms the poles of the gonomeric spindle by using the sperm-provided basal body, despite the presence within the same cytoplasm of maternal centrosomes. Thirty-five percent of parthenogenetic eggs and all unfertilized and fertilized eggs from the sibling bisexually reproducing D. mercatorum strain do not contain cytoplasmic asters. Thus, the Drosophila eggs have the potential for de novo formation of functional centrosomes independent of preexisting centrioles, but some control mechanisms preventing their spontaneous assembly must exist. We speculate that the release of the block preventing centrosome self-assembly could be a landmark for ensuring parthenogenetic reproduction.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Inheritance of gynandromorphism in the parasitic wasp Nasonia vitripennis

The parasitic wasp Nasonia vitripennis has haplo-diploid sex determination. Males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. Females and males can be easily distinguished by their morphology. A strain that produces individuals with both male and female features (gynandromorphs) is studied. We provide data on female/male patterning within and between individuals, on environmental effects influencing the occurrence of gynandromorphism, and on its pattern of inheritance. A clear anterior/posterior pattern of feminization is evident in gynandromorphic individuals that developed from unfertilized haploid eggs. The proportion of gynandromorphic individuals can be increased by exposing the mothers to high temperature and also by exposing embryos at early stages of development. Selection for increased gynandromorph frequency was successful. Backcross and introgression experiments showed that a combination of a nuclear and a heritable cytoplasmic component causes gynandromorphism. Analyses of reciprocal F(2) and F(3) progeny indicate a maternal effect locus (gyn1) that maps to chromosome IV. Coupled with previous studies, our results are consistent with a N. vitripennis sex determination involving a maternal/zygotic balance system and/or maternal imprinting. Genetics and temperature effects suggest a temperature-sensitive mutation of a maternally produced masculinizing product that acts during a critical period in early embryogenesis.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. II. Characterization of a Gynogenetically Reproducing Strain

A strain of Drosophila melanogaster, named gyn-F9, can reproduce by gynogenesis. On mating with a male sterile mutant, ms( 3)K81, gyn-F9 females produced impaternate progeny at a rate of about 15 flies per female, which was almost 2000 times as frequent as that of the control. When the females were mated with normally fertile males, the number of offspring varied extremely from parent to parent, with average fertility being much lower than that of normal females. Nearly one-third of these bisexual progeny were either triploid females or intersexes. Among the rest of the progeny, some were diploid impaternates having developed without syngamy. The gynogenetic property of gyn-F9 is primarily governed by a few genes, most likely two recessive genes, one each located on the second and third chromosomes. The impaternates were found to restore their diploidy by the fusion of two nonsister nuclei out of the four egg pronuclei which result from the second meiotic division (central fusion). Although nondisjunction occurs frequently in the meiosis of gyn-F9, this is unlikely to bring about an appreciable number of diploid gametes developing into impaternates. Possible mechanisms of the evolutionary origin of parthenogenesis are discussed in relation to these findings.