바다 송사리 Oryzias dancena와 자바 송사리 O. javanicus 간 잡종의 세포유전학적 및 생식생물학적 특징

Abstract

Marine medaka Oryzias dancena and javanese medaka O. javanicus are distributed in estuarine waters of East to Southeast Asia. In the present study, induction of hybrid and reciprocal hybrid between O. dancena and O. javanicus (ODJ and OJD) were performed for biological and cytogenetic analysis and to induce sex reversal. Back cross hybrid between female O. dancena and male ODJ were produced for examine its effect on early embryogenesis. The egg development and morphological changes of larvae, juveniles and adults of O. dancena, O. javanicus and their hybrids showed no difference in the parents group and hybrids. However, there were time differences from the development stage. Fertilized eggs were incubated at 25 ± 1°C and embryonic development process was observed by light microscopy based on diagnostic features of the developing embryos. The time to blastodisc stage was almost identical in all experiment groups. The development time to the formation of tubular heart stage was 2 days 1 hour 30 minutes which were similar between O. dancena and ODJ. The time difference began to be observed from the development stage which is the heart beating stage, in O. dancena and ODJ. The time to reach this stage was 2 days (d) 4 hours (hr) and 2 d 5 hr 30 min for O. dancena and ODJ respectively. In O. javanicus and OJD, it took 22 hr 30 min from late gastrula stage which was similar in both species. However, the development of OJD becomes slower from the next stage to the beginning of early neurula stage. It took 1 d 6 hr 30 min for O. javanicus whereas, 1 d 7 hr for OJD. The time of the development stage was the fastest in O. dancena and ODJ, O. javanicus and OJD followed next. The O. dancena was hatched 11 d after fertilization, ODJ at 13 d, O. javanicus at 14 d and OJD at 15 d. Osteological development of O. dancena was investigated using cartilage and bone staining. Newly hatched larvae attained a mean of 4.4 ± 0.2 mm in total length (TL) and, at this stage, the lateral ethmoid, frontal, asupraoccipital and exoccipital already showed ossification. At this moment, ossification of the maxilla, premailla, pterotic was on process. In pariental and pterotic, the ossification took place 4 days after hatching at 4.6 ± 0.2 mm TL. Calcification in the lateral ethmoid, frontal, supraoccipital, exoccipital and vertebrae began 9 weeks after hatching (mean = 22.6 ± 2.7 mm in TL). Calcification in maxilla, premaxiila and pterotic began 9 weeks after hatching at 32.1 ± 2.4 mm TL. Simultaneously, calcification of caudal fin, dorsal fin and anal fin were observed. The calcified pectoral fin was first observed at 9 week after hatching and reached complete calcification at 12 week. Aggregate bone ossification and calcification of O. dancena was completed at week 12 after hatching. Sex differentiation and gonad development were investigated in a marine medaka species, O. dancena. Primordial germ cell (PGC) was first observed at 5 dpf and migrated to presumptive gonadal area between the gut and pronephric duct at 9 dpf. The morphological differentiation between male and female gonads was identifiable at 12 days post-hatching (dph). Early oocytes at perinucleolus stage as well as the formation of spermatid and efferent duct were observed at 28 dph. At 6 weeks of age, the ovary exhibited the yolk granulation in many oocytes while testis possessed a considerable number of spermatogonium and spermatid. The first ovulation was observed in 9-week-old females, and at the same age, males contained fully-matured spermatoza. Data obtained from this study indicates that the gonad differentiation of O. dancena is the typical type of differentiated gonochorism. Cytogenetic analysis was performed to obtain basic information for genetic identification of O. dancena, O. javanicus and their hybrids. The karyotype of all experimental groups showed 2n=48 chromosomes consisting of 24 pairs of acrocentrics and fundamental number (NF) was 48 and the first pair carried secondary constrictions near the centromeric regions. Erythrocyte area and volume of O. dancena were 9.8 ± 0.5 μm2 and 18.2 ± 1.0 μm3 respectively, 8.3 ± 0.5 μm2 and 15.8 ± 1.5 μm3 in O. javanicus, and 18.3 ± 0.5 μm2 and 15.7 ± 1.3 μm3 in ODJ. The erythrocyte area and volume of ODJ were similar to those of O. javanicus. In the flow cytometric analysis, cellular DNA content was 1.8 ± 0.1 pg/cell in O. dancena. Frequency distribution of erythrocyte nucleolar organizer regions (NORs) of O. dancena and O. javanicus showed 1 per cell (O. dancena: 20.5%, O. javanicus: 12.2%) or 2 per cell (O. dancena: 78.0%, O. javanicus: 88.8%) respectively. The rate of abnormality in ODJ and OJD was 10.6% and 90.5%. OJD showed high degree of abnormality such as growth retardation and edema. The proportion of males was 90.0% and 31.3% for ODJ and OJD, respectively. Although the morphological characteristics of hybrids were similar to those of O. javanicus, there were no differences in the number of ray nodes and vertebrae between hybrids and parental species. As a result of observing early egg development of the back cross between female O. dancena and ODJ hybrids, all the embryos were dead at late gastrula stage. Bisphenol A (BPA) is mainly used in the production of epoxy resins and polycarbonate plastics, which is a known endocrine disruptor and acutely toxic to aquatic organisms. In this study, estrogenic effect of BPA was investigated on ODJ. ODJ were exposed to various concentrations (eg. 2.5 mg/L, 5.0 mg/L and 10.0 mg/L) for 56 days. The growth rate, abnormality and the ratio of female and male were observed in test group and control group. As a result, the growth was 14.7 ± 2.0 mm in TL in 2.5 mg/L, 13.7 ± 2.5 mm in 5.0 mg/L, 12.8 ± 2.5 mm in 10.0 mg/L in test group while it was 18.0 ± 1.2 mm in TL in control group which was not treated with bisphenol A. The result showed that the growth decreased as the concentration of BPA increased. The abnormality rate was 13.6% in control group, 65.4% in 2.5 mg/L, 81.3% in 5.0 mg/L and 98.1% in 10.0 mg/L which showed increase in abnormality as an increase of BPA concentration. As a result of analyzing ratio of sex in the test group and control group, 6.0% was examined to be interspecific in controls, 76.9% in 2.5 mg/L and 100.0% in 5.0 mg/L and 10.0 mg/L. These results suggest that BPA has estrogenic effect on hybrids of O. dancena and O. javanicus.