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 [1] , 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 [2] - [8] have reported some fertile hybrids, but on the other hand they have found that the sex ratio of bitterling hybrids was biased toward males. [9] 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.

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

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 [10] . In this study, chromosome analyses were separated and performed in the

*: 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.

Ueda, T. and Ueda, Y. (2018) Chromosomal Studies of the Hybrid between Female Rhodeus ocellatus ocellatus and Male Rhodeus atremius fangi in Bitterlings (Teleostei: Cypriniformes: Acheilognathinae). Natural Resources, 9, 17-22. https://doi.org/10.4236/nr.2018.81002