Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all-female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes.     

Evidence for recombination of mitochondrial DNA in triploid crucian carp

In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish.     

The sex-chromosome constitution and early postimplantation development of diandric triploid mouse embryos

Diandric triploid mouse embryos were produced by standard micromanipulatory techniques, using eggs isolated from female mice with a normal chromosome constitution that had been mated to homozygous Rb(1.3)1Bnr males (which carry a large metacentric "marker" chromosome, viz., a Robertsonian translocation involving chromosomes 1 and 3). The tripronucleate embryos were transferred to the oviducts of pseudopregnant mice, which were subsequently autopsied at about midday on the 10th day of gestation. Although a relatively small number of the isolated conceptuses consisted of morphologically abnormal egg-cylinder-like structures or empty gestational sacs, most were at clearly distinguishable embryonic stages, from the primitive streak stage to embryos with about 20 pairs of somites present. These embryos all appeared to be morphologically normal but were substantially smaller than normal (diploid) fertilized embryos analyzed at similar stages of development. A total of 63 diandric triploid conceptuses were recovered and analyzed cytogenetically. They were G-banded to determine their sex-chromosome constitution and confirm their diandric triploid status. No obvious difference was observed in the developmental potential of the 58,XXX class of diandric triploids, compared to that of the 58,XXY class. The ratio of 58,XXX to 58,XXY embryos was close to the expected ratio of 1:2, assuming that unfertilized eggs have an equal chance of becoming fertilized by an X- or a Y-bearing spermatozoon and that the additional (i.e., "donor") male pronucleus also has an equal chance of having either an X or a Y sex chromosome present. However, the development of the 58,XYY class appeared to be restricted, even at the stage of gestation analyzed, in that no embryos with this genetic constitution were observed that had progressed beyond the early somite stage. The present findings are discussed in relation to the cytogenetic findings in human triploid conceptuses, the majority of which are spontaneously aborted during the first half of pregnancy. In man, the 69,XYY class (equivalent to the 58,XYY class in our study) is only rarely encountered, and it has been assumed that these triploid embryos are probably lost at a very early stage of gestation.     

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.     

Does triploidization produce functional sterility of triploid males of tench <Emphasis Type="Italic">Tinca tinca</Emphasis> (L.)?

Triploidy interferes with gametogenesis in all fish species tested so far. In fish it results in complete female sterility however, males are still able to develop testis. The reason why sterility levels in triploid fishes differ among species and between sexes is unclear. In the present study the reproductive capacity of triploid males of tench was studied. Flow cytometry revealed sperm cells of triploids to be largely aneuploid with high mosaic DNA, oscillating from haploid DNA to diploid DNA content. Analysis of variance showed an insignificant influence of ploidy level on the percentage of motile spermatozoa, as well as on spermatozoa velocity. Experimental crosses between normal diploid female and triploid males resulted in the appearance of triploid progeny, which exhibited genotypes composed of microsatellite alleles inherited from the founder female and additional allele derived from the donor male. We can conclude that the triploid males analysed in the present study were capable to fertilize eggs derived from diploid females.     

The cytogenetics of a triploid Hordeum bulbosum and of some of its hybrid and trisomic derivatives

Summary The progeny from a cross between diploid H. vulgare and triploid H. bulbosum were mostly triploid (VBB) hybrids, the other progeny were haploid (V) barley (H. vulgare). From a cross between diploid and triploid H. bulbosum, four of the seven possible trisomic lines were isolated. The Giemsa banded karyotype of H. bulbosum was produced, and two of the lines were identified as trisomic for chromosomes 6 and 7. The cytology and transmission rates of the trisomics were examined.     

Erratum to Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Sexual maturation in female triploid plaice, Pleuronectes platessa, and plaice × flounder, Platichthys flesus, hybrids

The gonads of immature female triploid plaice ( Pleuronectes plafcssa ) and plaice × flounder ( Platichthys flesus ) hybrids produced from cold-shocked eggs were examined. They were less than half the size of those in diploids of similar age and contained only small numbers of developing oocytes. These were similar both in size and cytological appearance to those in the controls. The bulk of the ovarian tissue was composed of nests of small undifferentiated cells resembling the oogonia in the ovaries of diploid fish. Mature, 5 year old triploid hybrids which had not produced eggs over the previous two spawning seasons were killed in January when the diploid controls were nearing spawning condition. The ovaries of these fish were smaller than those of the controls and contained degenerating oocytes at maturation stage V. The advantage of sterile female triploid fish for fish-cultivation is discussed.     

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.     

Diploid males and their triploid offspring in the paper wasp Polistes dominulus

Although the hymenopteran sex-determining mechanism generally results in haploid males and diploid females, diploid males can be produced via homozygosity at the sex-determining locus. Diploid males have low fitness because they are effectively sterile or produce presumably sterile triploid offspring. Previously, triploid females were observed in three species of North American Polistes paper wasps, and this was interpreted as indirect evidence of diploid males. Here we report what is, to our knowledge, the first direct evidence: four of five early male-producing Polistes dominulus nests from three populations contained diploid males. Because haploid males were also found, however, the adaptive value of early males cannot be ignored. Using genetic and morphological data from triploid females, we also present evidence that both diploid males and triploid females remain undetected throughout the colony cycle. Consequently, diploid male production may result in a delayed fitness cost for two generations. This phenomenon is particularly relevant for introduced populations with few alleles at the sex-determining locus, but cannot be ignored in native populations without supporting genetic data. Future research using paper wasp populations to test theories of social evolution should explicitly consider the potential impacts of diploid males.     

Unusual triploid males in a microchromosome-carrying clone of the Amazon molly, Poecilia formosa

The Amazon molly, Poecilia formosa, is an all-female fish of hybrid origin which reproduces by gynogenesis, i.e. it depends on sperm of males of closely related species to trigger parthenogenetic development of the embryo. Therefore the offspring is clonal and identical to the mother. In rare cases the exclusion mechanism fails and paternal introgression occurs. This may result either in triploid offspring - if the whole haploid chromosome set of the sperm fuses with the diploid egg nucleus - or in siblings with microchromosomes - if only subgenomic amounts of paternal DNA are included. In one of our diploid, microchromosome-carrying laboratory stocks we observed eight triploid individuals which all developed into males. We investigated the mitotic and meiotic chromosomes, the synaptonemal complex (SC), and sperm production of these males, and compared them to males of the gonochoristic parental species (P. latipinna and P. mexicana) and their hybrids. This comparison revealed that P. formosa males are functional males with reduced effective fertility. They show a deviation from the typical 23 bivalents in the synaptonemal complexes as well as in diakinesis due to the triploid state. They produced offspring but only with gynogenetic Amazon molly females. This shows that the probably aneuploid sperm from P. formosa males can trigger parthenogenetic development of unreduced eggs.     

Diploid sperm produced by artificially sex-reversed clone loaches

Clone loaches reproduce unisexually in a wild population of Hokkaido Island, Japan. These clone loaches produce genetically identical unreduced eggs which develop to diploid individuals without any genetic contribution of sperm donors. In the present study, sex reversal of clone loaches was attempted and the reproductive potential of resultant clone males was examined. Clone loaches administered 0.5 ppm of 17-alpha methyltestosterone (MT) for 30 days from 1 month after hatching differentiated into physiological males. These sex-reversed clone males produced fertile spermatozoa with a diploid DNA content. Diploid spermatozoa had significantly larger heads than normal haploid sperm, but had a normal shape showing a head, mid-piece, and tail. The motility of diploid spermatozoa was low after ambient water was added. Concentration of diploid spermatozoa per unit of sperm was lower than that of control haploid spermatozoa. Microsatellite genotyping revealed that triploid progeny from the cross between a normal diploid female and a sex-reversed clone male had two alleles specific to the diploid clone male and one allele of the mother loach. These results indicated that the sex-reversed clone males produced fertile diploid spermatozoa genetically identical to the clone lineage.     

Disruption of normal meiosis in artificial inter-populational hybrid females of <Emphasis Type="Italic">Misgurnus</Emphasis> loach

Artificial cross between two genetically different populations of Japanese Misgurnus loach was made to examine the reproductive capacity of the artificial inter-populational hybrid females. Ploidy status and microsatellite genotypes of the eggs laid by these hybrids were inferred from those determined in progenies developed by normal fertilization with haploid loach sperm, induced gynogenesis with UV-irradiated goldfish sperm and/or hybridization with intact goldfish sperm. Some hybrid females laid unreduced diploid eggs genetically identical to the mother. However, these diploid eggs could not develop by spontaneous gynogenesis, but grow to triploid by incorporation of a sperm nucleus. Other hybrid females laid haploid eggs together with diploid eggs and/or various aneuploid and polyploid eggs. Thus, a disruption of normal meiosis occurred in inter-populational hybrid females. The results suggested that the two populations should be so distant as to give rise to atypical formation of unreduced and other unusual eggs in their hybrids.