Nucleotide Sequence and Chromosome Mapping of 5S Ribosomal DNA from the Dojo Loach, Misgurnus anguillicaudatus.

There are 2 genetically divergent groups in the dojo loach Misgurnus anguillicaudatus: A and B. Although most wild-type diploids reproduce sexually, clonal diploids (clonal loach) reproduce gynogenetically in certain areas. Clonal loaches produce unreduced isogenic eggs by premeiotic endomitosis, and such diploid eggs develop gynogenetically following activation by the sperm of sympatric wild-type diploids. These clonal loaches have presumably arisen from past hybridization events between 2 different ancestors. The genomic differences between these 2 groups have not been completely elucidated. Thus, new genetic and cytogenetic markers are required to distinguish between these 2 groups. Here, we compared the 5S rDNA region to develop markers for the identification of different dojo loach groups. The nontranscribed sequence (NTS) of the 5S rDNA was highly polymorphic and group-specific. NTSs were found in clades of 2 different groups in clonal loaches. In contrast, we did not find any group-specific sequences in the coding region of the 5S rRNA gene. Sequences were located near the centromere of the short arm of the largest submetacentric chromosomes in groups A and B and clonal loaches. Thus, the 5S rDNA of the dojo loach is conserved at the chromosomal location. Whereas, the sequences of the NTS regions evolved group-specifically in the dojo loach, with the sequences of both groups being conserved in clonal loaches.

FISH Identifies Chromosome Differentiation Between Contemporary Genomes of Wild Types and the Ancestral Genome of Unisexual Clones of Dojo Loach, Misgurnus anguillicaudatus.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

Developmental potential of somatic and germ cells of hybrids between Carassius auratus females and Hemigrammocypris rasborella males.

The cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.

Aberrant Meiotic Configurations Cause Sterility in Clone-Origin Triploid and Inter-Group Hybrid Males of the Dojo Loach, Misgurnus anguillicaudatus.

Gonochoristic wild-type dojo loaches (Misgurnus anguillicaudatus) are diploid (2n = 50) and reproduce bisexually. However, sympatric clonal diploids generate unreduced diploid isogenic eggs that develop gynogenetically. Clone-origin triploidy arises following the incorporation of a haploid wild-type sperm nucleus into the diploid egg. Triploid females produce fertile haploid eggs by meiotic hybridogenesis, while triploid males are sterile. Clonal loaches arose from past hybridization event(s) between genetically diverse groups, A and B. Artificial hybrid females between the 2 groups produce unreduced and/or aneuploid eggs, but the hybrid males are sterile. In this study using FISH, we analyzed chromosome pairing in meiotic cells of clone-origin triploid and inter-group hybrid males to clarify the cytogenetic mechanisms underlying the male-specific sterility. We used a repetitive sequence probe to identify group B-derived chromosomes and a 5.8S + 28S rDNA probe to identify pairs of homologous chromosomes. We found that asynapsis and irregular synapsis occur in triploid and hybrid males containing 2 different genomes and that this may cause the formation of sterile germ cells. These results will help us to understand hybrid sterility from the viewpoint of synapsis behavior.

Clonal reproduction assured by sister chromosome pairing in dojo loach, a teleost fish.

Wild-type dojo loach (Misgurnus anguillicaudatus) commonly reproduces bisexually as a gonochoristic diploid (2n = 50), but gynogenetically reproducing clonal diploid lines (2n = 50) exist in certain districts in Japan. Clones have been considered to develop from past hybridization event(s) between two genetically diverse groups, A and B, within the species. Fluorescence in situ hybridization analyses using the repetitive sequence "ManDra" as a probe clearly distinguished 25 chromosomes derived from group B out of a total of 50 diploid chromosomes of the clone, providing strong molecular cytogenetic evidence of its hybrid origin. In meiosis, diploid wild-type showed 25 bivalents, while diploid clones revealed 50 bivalents, indicating the presence of 100 chromosomes. In meiotic chromosome spreads in sex-reversed clonal males, ManDra signals were detected in 25 out of 50 bivalents, and only one out of two bivalents possessing major ribosomal RNA coding regions exhibited two positive ManDra signals. In clonal females, ManDra signals were detected in approximately 25 out of 50 bivalents. Thus, unreduced gametes should be generated by the pairing between sister chromosomes doubled from each ancestral chromosome from the different groups by premeiotic endomitosis. Sister chromosome pairing should assure production of unreduced isogenic clonal gametes due to the absence of the influence of recombination or crossing over.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae).

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis.

Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin.

Aneuploid progenies of triploid hybrids between diploid and tetraploid loach Misgurnus anguillicaudatus in China.

Triploid Chinese loach, Misgurnus anguillicaudatus, hybrids between tetraploids from Hubei Province and diploids from Liaoning Province were mated with either diploid wild-type or triploid hybrids to analyze viability and ploidy of the resultant progenies. Both triploid males and females generated fertile gametes, but progenies from the crosses using gametes of triploid hybrids did not survive beyond the larval stages. In crosses between wild-type diploid females and triploid hybrid males, embryos ranging from 2.2n to 2.6n were predominant with a mode of either 2.4n (chromosome numbers 59, 60, 61) or 2.5n (chromosome numbers 62, 63). Those from the crosses between triploid hybrid females and diploid males gave a modal ploidy level at approximately 2.5n in one case, but a shift to a higher ploidy level was observed in other embryos. In the progenies between triploid hybrid females and males, the ploidy level at approximately 3.0n (chromosome numbers 74, 75, 76) was most frequent. The cytogenetic results of the progenies suggest the production of aneuploid gametes with a modal ploidy level at approximately 1.5n in triploid hybrids. However, a shift to higher chromosome numbers in gametes was observed in certain cases, suggesting the involvement of mortality selection of gametes and/or zygotes with lower chromosome numbers.

Meiotic chromosome configurations in triploid progeny from reciprocal crosses between wild-type diploid and natural tetraploid loach Misgurnus anguillicaudatus in China.

Here, we showed meiotic chromosome configurations prepared from oocyte germinal vesicles and spermatocytes of triploid loaches produced from reciprocal crosses between wild-type diploids (2n = 50) obtained from Dalian, Liaoning Province, China and natural tetraploids (4n = 100) from Chibi, Hubei Province, China. Major meiotic cells in triploids comprised 25 bivalents and 25 univalents, but cells with one to five trivalents were also observed. When three homologous chromosomes bearing nucleolar organizing regions (NOR) were identified with the detection of signals or positive sites by silver staining, chromomycin A3 staining and fluorescence in situ hybridization with a 5.8S + 28S rDNA probe, two third of selected triploid cells gave a configuration including one bivalent with two NORs (association of two homologous chromosomes) and one univalent with one NOR. However, other triploid cells showed three univalent each of which had one NOR, suggesting a failure of synapsis between homologous chromosomes. These results suggested that triploid female and male should produce aneuploid gametes with the theoretical mode at 1.5n (37 or 38 chromosomes).

Germ cells are not the primary factor for sexual fate determination in goldfish.

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.

Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish.

Germ cell-deficient fish usually develop as phenotypic males. Thus, the presence of germ cells is generally considered to be essential for female gonadal differentiation or the maintenance of ovarian structure. However, little is known of the role of germ cells in the determination of the sexual fate of gonadal somatic cells. We have established an inducible germ cell deficiency system in the loach (Misgurnus anguillicaudatus, Cypriniformes: Cobitidae), a small freshwater fish, using knockdown of the dead end gene with a morpholino antisense oligonucleotide. Interestingly, loach lacking germ cells could develop as either phenotypic males or females, as characterized morphologically by the presence or absence of bony plates in the pectoral fins, respectively. The phenotypic males and females had testicular and ovarian structures, respectively, but lacked germ cells. Gene expression patterns in these male and female germ cell-deficient gonads were essentially the same as those in gonads of normal fish. Our observations indicate that sexually dimorphic gonads can develop in germ cell-deficient loach. In contrast to the situation in other model fish species, the gonadal somatic cells in phenotypic females autonomously differentiated into ovarian tissues and also played a role in the maintenance of gonadal structure. On the basis of our observations, we propose two possible models to explain the role of germ cells in sex determination in fish.

Diploid clone produces unreduced diploid gametes but tetraploid clone generates reduced diploid gametes in the Misgurnus loach.

Most individuals of the loach Misgurnus anguillicaudatus reproduce bisexually, but cryptic clonal lineages reproduce by natural gynogenesis of unreduced diploid eggs that are genetically identical to maternal somatic cells. Triploid progeny often occur by the accidental incorporation of a sperm nucleus into diploid eggs. Sex reversal from a genetic female to a physiological male is easily induced in this species by androgen treatment and through environmental influences. Here, we produced clonal tetraploid individuals by two methods: 1) fertilization of diploid eggs from a clonal diploid female with diploid sperm of a hormonally sex-reversed clonal diploid male and 2) artificial inhibition of the release of the second polar body in eggs of clonal diploid females just after initiation of gynogenetic development. There is no genetic difference between the clonal diploid and tetraploid individuals except for the number of chromosome sets or genomes. Clonal tetraploid males never produced unreduced tetraploid sperm, only diploid sperm that were genetically identical to those of a clonal diploid. Likewise, clonal tetraploid females did not form unreduced tetraploid eggs, just diploid eggs. However, the eggs' genotypes were identical to those of the original clone, and almost all the eggs initiated natural gynogenesis. Thus, gametogenesis of the clonal tetraploid loach is controlled by the presence of two chromosome sets to pair, thereby preserving the normal meiotic process, i.e., the formation of bivalents and subsequently two successive divisions.

Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus), a teleost fish.

BACKGROUND: Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, Misgurnus anguillicaudatus (a teleost fish). RESULTS: When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing. CONCLUSION: In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.

The origin of natural tetraploid loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inferred from meiotic chromosome configurations.

In the loach, or Oriental weatherfish Misgurnus anguillicaudatus (Teleostei: Cobitidae), diploid (2n = 50) and tetraploid individuals (4n = 100) are often sympatric in central China. The evolutionary mechanism of this tetraploidization was analyzed with the observation of meiotic behavior of chromosomes in both the germinal vesicles of mature oocytes and the primary spermatocytes in diploid and tetraploid loaches. Whereas diploid specimens usually showed 25 bivalents in meiotic cells, tetraploid loaches exhibited 0-6 quadrivalents and 38-50 bivalents in both sexes, with the modal number of quadrivalents as three in females and four in males. In the diploid specimens, the two largest metacentric chromosomes bearing nucleolar organizing regions (NORs) identified by chromomycin A(3) staining and fluorescence in situ hybridization with a 5.8S + 28S rDNA probe formed one bivalent with terminal association. In the tetraploids, four NOR-bearing chromosomes never formed a quadrivalent, but were organized into two terminally-associated bivalents. These findings suggest an autotetraploid origin of the natural tetraploid loach and subsequent rediploidization of whole genome. The latter process, however, seems still in progress as inferred from the concurrence of up-to several quadrivalents and the majority of bivalents.

Diploid and aneuploid sperm in tetraploid ginbuna, Carassius auratus langsdorfii.

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratusauratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types.

Control of Developmental Speed in Zebrafish Embryos Using Different Incubation Temperatures.

The zebrafish is a valuable model organism that is widely used in studies of vertebrate development. In the laboratory, zebrafish embryonic development is normally carried out at 28.5°C. In this study, we sought to determine whether it was possible to modify the speed of embryonic development through the use of short- and long-term variations in incubation temperature. After incubation at 20°C-32°C, most early-stage embryos survived to the epiboly stage, whereas more than half of the embryos died at <20°C or >32°C. The rate of development differed between embryos incubated at the lowest (18°C) and highest (34°C) temperatures: a difference of 60 min was observed at the 2-cell stage and 290 min at the 1k-cell stage. When blastulae that had developed at 28°C were transferred to a temperature lower than 18°C for one or more hours, they developed normally after being returned to the original 28°C. Analyses using green fluorescent protein-buckyball mRNA and in situ hybridization against vasa mRNA showed that primordial germ cells increase under low-temperature culture; this response may be of use for studies involving heterochronic germ cell transplantation. Our study shows that embryonic developmental speed can be slowed, which will be of value for performing time-consuming, complicated, and delicate microsurgical operations.

Unisexual hybrids break through an evolutionary dead end by two-way backcrossing.

Unisexual vertebrates (i.e., those produced through clonal or hemiclonal reproduction) are typically incapable of purging deleterious mutations, and, as a result, are considered short-lived in evolutionary terms. In hemiclonal reproduction (hybridogenesis), one parental genome is eliminated during oogenesis, producing haploid eggs containing the genome of a single parent. Hemiclonal hybrids are usually produced by backcrossing hemiclonal hybrids with males of the paternal species. When hemiclonal hybrids from a genus of greenlings (Hexagrammos) are crossed with males of the maternal species, the progeny are phenotypically similar to the maternal species and produce recombinant gametes by regular meiosis. The present study was conducted to determine if the hemiclonal genome is returned to the gene pool of the maternal species in the wild. Using a specific cytogenetic marker to discriminate between such progeny and the maternal species, we observed that Hexagrammos hybrids mated with maternal and paternal ancestors at the same frequency. This two-way backcrossing in which clonal genomes are returned to the gene pool where they can undergo recombination plays an important role in increasing the genetic variability of the hemiclonal genome and reducing the extinction risk. In this way, hybrid lineages may have survived longer than predicted through occasional recombinant generation.

Hybrid between Danio rerio female and Danio nigrofasciatus male produces aneuploid sperm with limited fertilization capacity.

In the Danio species, interspecific hybridization has been conducted in several combinations. Among them, only the hybrid between a zebrafish (D. rerio) female and a spotted danio (D. nigrofasciatus) male was reported to be fertile. However, beyond these investigations, by means of reproductive biology, gametes of the hybrid have also not been investigated genetically. For this study, we induced a hybrid of the D. rerio female and D. nigrofasciatus male in order to study its developmental capacity, reproductive performance and gametic characteristics. Its hybrid nature was genetically verified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rhodopsin gene. Almost all the hybrids (36/37) were males, and only one was female. Developing oocytes were observed in the hybrid female, but ovulated eggs have not been obtained thus far. Microscopic observation revealed various head sizes of sperm in the hybrid males. Flow cytometry showed that the hybrid males generated aneuploid sperm with various ploidy levels up to diploidy. In backcrosses between D. rerio females and hybrid males, fertilization rates were significantly lower than the control D. rerio, and most resultant progeny with abnormal appearance exhibited various kinds of aneuploidies ranging from haploidy to triploidy, but only one viable progeny, which survived more than four months, was triploid. This suggested the contribution of fertile diploid sperm of the hybrid male to successful fertilization and development.