Chromosomal Studies of Masculinized Hybrids in Bitterlings ( Teleostei : Cypriniformes : Acheilognathinae )

The chromosome analysis of the masculinized hybrid between female Tanakia limbata and male T. signifer in bitterlings (Acheilognathinae) was done. It was presumed that they had intermediate karyotype between the parents, and formed sperms with heteroploidy resulting from the incomplete pairing of homologous chromosomes in meiosis. Due to the abundance of species and the ease of artificial fertilization, the study of the factor of the hybrid sterility in bitterlings would lead to the clarification of the mechanism about species differentiation and karyotype differentiation, and also to developing a new variety.


Introduction
Bitterlings are freshwater fish species ascribed to the subfamily Acheilognathinae (Cyprinidae), and are distributed throughout Eurasia, and more widely in East Asia.Three valid genera, Acheilognathus, Rhodeus, and Tanakia [1], grouping approximately 80 species/subspecies [2], have been recognized.It is known fact that all bitterlings are characterized by peculiar reproductive behavior which involves egg and sperm deposition in the mantle cavity of living freshwater bivalves.
Due to the abundance of species and the ease of artificial fertilization, many hybridization experiments in bitterlings were tried for the purpose of clarification on the phylogenetic relationships of bitterlings, the mechanism of species differentiation, and others.On the one hand [3]- [9] have reported some fertile hybrids, but on the other hand they have found that the sex ratio of bitterling hybrids was biased toward males.[10] observed similar phenomena and made mention of masculinization mechanism of bitterling hybrids.In any case, they had only limited information of chromosomes.
We have been studying hybridization experiments in bitterlings to make clear on the mechanisms of species differentiation and karyotype evolution, and to develop a new variety.
In the present report, the chromosome analysis of the masculinized hybrid between female T. limbata and male T. signifer in bitterlings was done.

F 1 Hybrids between Female Tanakia limbata and Male T. signifer
Thirty eggs from a female of T. limbata and sperms from a male of T. signifer were fertilized artificially.Hatchability was 83% (25 hatched embryos/30 eggs).Survival rate at about one year old after hatching was 32% (8 fishes/25 hatched embryos), and all these 8 hybrids had vivid colors just like a male.
Sperms were gotten from a F 1 hybrid.Chromosomal slides of this fish were made from kidney and testis cells.And also chromosomal slides of other three F 1 hybrids and their parents were obtained from kidney cells.

F 2 Hybrids between Female Rhodeus ocellatus and Male F 1 Hybrid
Thirty-five eggs from a female R. o ocellatus and sperms from a male F 1 hybrid (T.limbata ♀ × T. signifer ♂) were fertilized artificially.Fertilization rate was 57% (20 fertilized eggs/35 eggs used).Chromosomal slides of eight F 2 hybrids were obtained from gastrula cells.Hatching of remaining embryos has not been found.

Meiosis of Rhdeus atremius Fangi
Chromosomal slides of R. a. atremius were made from kidney and testis cells to compare with F 1 hybrid.

Chromosomal Slides
All of these chromosomal slides were made by direct air-drying method and chromosomes were stained with Giemsa.Karyotytpes of F 1 hybrids were analyzed from twenty metaphases in each individual.
The karyotype of F 1 hybrid, shown in Figure 4, had 48 chromosomes and the same chromosomal constitution (8M + 20SM + 20ST/A) of their parents, in all metaphases observed.The distinction on the karyotype among them and the chromosomal aberration were not recognized, and it was estimated that F 1 hybrid had the intermediate karyotype between the parents.And, unusual metaphase chromosomal figures at the first cleavage of meiosis were observed (Figure 5a) compared to a normal figure observed in R. a. fangi (Figure 5b).R. a. fangi (2n = 46, Figure 5c) had 23 bivalent chromosomes in meiosis (Figure 5b).And in the metaphase figure of F 1 hybrids, many univalent chromosomes were found besides some bivalent chromosomes (Figure 5a).And then, the chromosomal number (bivalents + 2 × univalents) in each metaphase was 48.So, the omission of the chromosome was not recognized in that stage.
Metaphase figures from 8 embryos in F 2 hybrid (R. o. ocellatus ♀ × F 1 ♂) were observed (Figure 6).The distribution of the chromosomal numbers in F 2 hybrid is shown in Table 1.All embryos had wide distribution and the mode varied from individual to individual.In some metaphase figures, the structural chromosomal aberration was found (Figure 6b).It is presumed that the variation of the mode was due to the difference of the chromosomal number in each sperm of F 1 , probably resulting from the incomplete pairing of homologous chromosomes in meiosis.In some lethal hybrids of salmon, trout and char, similar phenomena, i.e. the wide distribution of chromosomal numbers and the occurrence of the structural chromosomal aberration, have been observed [11]- [13].It has been speculated that those phenomena were due to the abnormal cell division during early     development, resulting from the chromosomal discord between the parents.The present case can be interpreted in the similar way.When we classify species, the important point is whether or not they can form hybrid.The species differentiation results from an accumulation of more than one reproductive isolation, and the evolution of the hybrid sterility may be the final important factor for the determination of species differentiation.The factor of the species differentiation may be concerned with that of the hybrid sterility.The study of the factor of the hybrid sterility in bitterlings would lead to the clarification of the mechanism about species differentiation and karyotype differentiation, and also to developing a new variety.The investigation at the gene-level is required to clarify the mechanism in addition to the chromosome-level.

Figure 5 .
Figure 5. Two metaphase figures of F 1 hybrid (a) and R. a. fangi (b) at the first cleavage of meiosis, and a metaphase figure from a kidney cell of R. a. fangi (c).

Figure 6 .
Figure 6.Two metaphase figures of F 2 hybrid.Arrows indicate obvious structural chromosomal aberrations.

Table 1 .
Distribution of chromosomal numbers in F 2 hybrid.