Bitterlings are freshwater fish species ascribed to the subfamily Acheilognathinae (Cyprinidae), and are distributed throughout East Asia, and more widely in Eurasia. About 80 species/subspecies  , have been recognized. It is a known fact that all bitterlings have a unique ecology in that they lay their eggs in freshwater bivalves.
Bitterlings illustrate the symbiosis between humans and nature, because the habitats of them are closely related to human activities. They have decreased with a change in human activity. And, many kinds of them are endangered species. Preservation of such an environment as “Satoyama” is an important issue; a study of bitterlings would provide a good example to grasp the biodiversity and ecosystems of this environmentally sensitive area. One of the decrease causes of rare native species will be introduction of alien species of close relation. Genetic disturbance by reduction in habitat environment and hybrid formation is considered as bad influence.
Due to the abundance of species and easy doing of artificial fertilization, many cytogenetic experiments in bitterlings were tried for the purpose of clarification on the phylogenetic relationships of bitterlings, the mechanism of species differentiation, and others. And, many hybridization experiments have been tried equally. On the one hand  -  have reported some fertile hybrids, but on the other hand they have found that the sex ratio of bitterling hybrids was biased toward males.  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. On the one hand there is fear of a decline of the procreative power of the native species and hereditary disturbance by hybrid formation. And, a hybrid study will give useful suggestion in environment preservation.
In the present report, the chromosome analysis of the masculinized hybrid between female Rhodeus ocellatus ocellatus and male Rhdeus atremius fangi in bitterlings was done.
2. Materials and Methods
2.1. F1 Hybrids between Female R. o. ocellatus and Male R. a. fangi
R. ocellatus ocellatus and of R. atremius fangi were collected in Tochigi-prefecture, Japan and Fujian-province, China, respectively. Thirty eggs from a female of R. o. ocellatus and sperms from a male of R. a. fangi were fertilized in a plastic petri dish at 20C artificially. Then, fertilized eggs were kept at 20C.
Hatchability was 87% (26 hatched embryos/30 eggs). Obvious form abnormality wasn’t found in the individuals. Survival rate at about one year old after hatching was 35% (9 fishes/26 hatched embryos), and all these 9 hybrids had vivid colors just like a male.
Sperms were gotten from a F1 hybrid. Chromosomal slides of this fish were made from kidney and testis cells. And also chromosomal slides of other two F1 hybrids and their parents were obtained from kidney cells.
2.2. F2 Hybrids between Female R. o. ocellatus and Male F1 Hybrid
Forty-eight eggs from a female R. o. ocellatus and sperms from a male F1 hybrid (R. o. ocellatus ♀ × R. a. fangi ♂) were fertilized artificially. Fertilization rate was 52% (25 fertilized eggs/48 eggs used). Chromosomal slides of fifteen F2 hybrids were obtained from early- and late-gastrula cells. Hatching of remaining embryos has not been found.
In addition, chromosomal slides of four F2 hybrids between female R. a. fangi and F1 hybrid were obtained from late-gastrula cells.
2.3. Meiosis of R. a. fangi
Chromosomal slides of R. a. fangi were made from kidney and testis cells to compare with F1 hybrid.
2.4. Chromosomal Slides
All of these chromosomal slides were made by direct air-drying method and chromosomes were stained with Giemsa. Each slide was observed by 600 times of optical microscope. Karyotytpes of F1 hybrids were analyzed from twenty metaphases in each individual.
3. Results and Discussion
Karyotypes of R. o. ocellatus, as shown in Figure 1, had 2n = 48 including 8 metacentrics (M), 20 submetacentrics (SM) and 20 subtelocentrics (ST) or acrocentrics (A). Karyotype of R. a. fangi had 2n = 46 including 4 SM, 42 ST or A (Figure 2).
The karyotype of F1 hybrid, shown in Figure 3, had 47 chromosomes including 4 M, 12 SM and 31 ST or A, in all metaphases observed. The distinction on the karyotype among them and the chromosomal aberration were not recognized, and it was estimated that F1 hybrid had the intermediate karyotype
Figure 1. Karyotypes of R. o. ocellatus.
Figure 2. Karyotype of R. a. fangi.
Figure 3. Karyotype of F1 hybrid (R. o. ocellatus ♀ × R. a. fangi ♂).
Figure 4. Two metaphase figures of F1 hybrid (a) and R. a. fangi (b) at the first cleavage of meiosis. They were stained with Giemsa. The scales indicate 5 micrometer.
between the parents. And, unusual metaphase chromosomal figures at the first cleavage of meiosis were observed (Figure 4(a)) compared to a normal figure observed in R. a. fangi (Figure 4(b)). R. a. fangi (2n = 46, Figure 2) had 23 bivalent chromosomes in meiosis (Figure 4(b)). And in the metaphase figure of F1 hybrids, many univalent chromosomes were found besides some bivalent chromosomes (Figure 4(a)). And then, the chromosomal number (bivalents + 2 × univalents) in each metaphase was 47. So, the omission of the chromosome was not recognized in that stage.
Metaphase figures from 9 embryos (at early-gastrula stage) and 6 embryos (at late-gastrula stage) in F2 hybrid (R. o. ocellatus ♀ × F1 ♂) were observed (Figure 5(a)). Besides, metaphase figures from 4 embryos (at late-gastrula stage) in F2 hybrid (R. a. fangi ♀ × F1 ♂) were observed.
The distribution of the chromosomal numbers in F2 hybrid is shown in Table 1. Clear form abnormality wasn’t admitted in most metaphase figures. The embryo showed wide distribution, and modes were observed in each embryo. And, the mode varied from individual to individual. In some metaphase figures, the structural chromosomal aberration was found (Figure 5(b)). It is presumed that the variation of the mode was due to the difference of the chromosomal number in each sperm of F1, probably resulting from the incomplete pairing of homologous chromosomes in meiosis.
Besides, a similar result to this study was reported in another bitterling hybrid  . In this study, chromosome analyses were separated and performed in the
Figure 5. Two metaphase figures of F2 hybrid. Some obvious structural chromosomal aberrations were observed (b). They were stained with Giemsa. The scales indicate 10 micrometer.
Table 1. Distribution of chromosomal numbers in F2 hybrid.
*: Some cells had structural chromosomal aberrations (SCA).
early-gastrula and the late-gastrula. And, indefiniteness of the mode and the tendency for which a structural chromosomal aberration appearance is conspicuous were seen in the late-gastrula. Cell division seems developed mechanically until early-gastrula period. Various genes begin to function after a blastula period. So, after gastrula period genetic manifested harmonization is disordered, and cell division and a morphogenesis will be warped. As a result, hatching of remaining embryos of F2 wouldn't be found.
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 develop a new variety. On the other hand, introduction of foreign related species is regarded as a problem in environment preservation. Genetic disturbance by reduction in habitat environment and hybrid formation is considered as bad influence. A hybrid study will be to give useful suggestion in environment preservation.