Hybridization Between Crassostrea Angulata And C. Sikamea, Saccostrea Cucullata

The Portuguese oyster (Crassostrea angulata) is the subspecies of C. gigas in taxonomy. As an important economic shellfish in our country, C. angulata mainly distributed in the southern coastal of China. The Kumamoto oyster (C. sikamea). which natural distribution is to the south of Jiangsu province, is not yet a mainly farmed oyster specie in our country as an economic shellfish. It is belonging to the same genus Crassostrea with the Portuguese oyster. The hooded oyster, Saccostrea cucullata, is a species of Saccostrea genus distributed mainly in the Indo-Pacific Ocean. In China, it naturally occurs in the Guangdong, Guangxi, and Hainan provinces and the South China Sea. Thus, the distributions of these three species overlap in some natural areas in China.The hybridization between C. angulata and C. sikamea besides the hybridization between C. angulate and Saccostrea cucullata had been studied, include many related experiment. Many detailed and reliable data had been attained, mainly result as follows:(1) To evaluate the gamete compatibility and zygote fertility between Crassostrea angulata and C. sikamea, the fertilization rate and hatching rate of two oysters at different temperature (18,21,24,27,30℃) and salinity (16,20,24,28,32) were studied, and then the F50 critical value were determined under the different sperm concentration. Results clearly demonstrate that hybridization between C. angulata and C. sikamea is achievable in one direction due to asymmetric gamete compatibility. Fertilization and hatching level of SS, AA and SA was raised with temperature increasing, while these max value were severally 99.1±0.3%, 98.4±1.2%,81.3±2.8% for fertilization rate, and 99.1±0.5%,96.8±2.8%、67.1±5.5% for hatching rate at 27℃ , respectively. Fertilization rate and hatching rate of SS, AA and SA at a salinity of 24ppt were severally 98.8±1.3%,94.8±4.5%,81.1±7.4%. and 75.9±3.7%, 86.4±4.2%,66.1 ±4.5%, respectively. Fertilization rate and hatching rate of SS, AA and SA at sperm concentration of 103 ind./μLwere severally 92.5±2.2%,87.2±5.7%,92.4±1.2%, and 79.8±4.3%,78.9±7.8%,82.2±7.0% under temperature of 27℃ and a salinity of 24ppt condition, respectively. The F50 critical value of gamete compatibility and zygote fertility were calculated, and were 58.88 ind./μl and 208.93 ind./μL, respectively. This study provided the theory and practice of reproductive isolation mechanism and interspecific hybridization between C.angulata and C. sikamea.(2) We conducted 2×2 factorial crosses between C. angulata and C. sikamea to determine whether the hybrid progeny can hatch, survive and get heterosis. In this study, fertilization was one direction. Progeny from C. angulata got the fastest growth. Fertilization and hatching success of hybrid was lower than that o of the two intraspecific crosses. This study provided sperms of C. angulata can fertilize eggs from C. sikamea and the hybrid can survive and grow normally. The hybrid larvae have a lower survival and growth rate during the planktonic period compared to progeny of C. sikamea but not significant. The hybrid showed completely hybrid weakness in, growth, survival and weight in juvenile mollusk period.(3) To evaluate the ecological flexibility of hybrids. The effect of salinity on hybrid progenies (SA) and two inbred groups (SS and AA) was investigated using an experimental design incorporating four salinities (15,20,25 and 30 ppt). The results showed that salinities for survival of AA is above 20ppt and for optimal growth is above 25ppt. SS and SA could survive in all four salinities. Both of them have the optimum salinities for survival and optimal growth in 25ppt. Hybrid oyster could endure lower salinities than their parents but have no growth advantage(4)Path analysis of shell morphological traits on weight traits were conducted to the hybrid progenies (SA) and two inbred groups which were sexual maturity in 180 days after hatching. The results showed morphological variation is exist among this three groups.The oysters lived on rock with any shape; they can’t be identified accurately only by external morphology. Meanwhile hybridization in experiment could easily contaminated by other gamete or larvae, so they must be identifide by molecular genetics. ITS2 was used to identify the hybrid progeny of C. angulata and C. sikamea. It got one band in pure oyster, whereas two bands in hybrid progeny through agarose gel electrophoresis, so they were amphimixis hybrid by preliminary inspection.The progenies of hybrid and two inbred groups reached sexual maturity in 180 days after hatching. Microscopic examination, with or without paraffin sectioning, was made of the contents in the gonads. The results showed the gonads of hybrids were as full as purebred oyster. The proportion of male to female of hybrids was 9:1.(5) To determine the feasibility of intergeneric hybridization between Crassostrea angulata and Saccostrea cucullata, which coexist in the South China Sea, we conducted 2×2 artificial crosses between these two species. The fertilization of hybrids was tracked using fluorescence staining of the gametes. Some barriers to sperm recognition of, binding to, and penetrating the egg or forming the pronucleus were observed. Although fertilization success was observed in reciprocal crosses, the overall fertilization level was lower than that of the intraspecific crosses. Hybrid larvae began dying in large numbers 6-8 days after hatching, and the few survivors could not complete metamorphosis. C. angulata♀× S. cucullata♂larvae had a growth rate similar to that of their maternal species, but S. cucullata♀ × C. angulata♂larvae had a lower growth rate. All hybrids were examined using molecular markers, which showed they were true amphimixis hybrids. Our study demonstrates that hybrid embryos can develop normally and that larvae can be produced, but reciprocal hybrids cannot complete metamorphosis to form spat. Thus, post-reproductive isolation between C. angulata and S. cucullata exists.