Induction and Survival of Diploid Gynogenesis With Ultraviolet Light-Irradiated Sperm in the Manila Clam Ruditapes philippinarum

In order to produce gynogenetic diploid of Manila clam, Ruditapes philippinarum, we studied the optimal conditions of gynogenetic diploid induced by ultraviolet (UV). The irradiance of UV-induced sperm was 230 μW (cm²·s), and the irradiation time was 0, 3, 6, 9, 12, 15, 18, 21, 24, and 27 s. After the egg fertilized with genetically inactivated sperm, the fertilized eggs were treated with cytochalasin B (CB) at different concentrations of 0, 0.25, 0.50, 0.75, and 1.0 mg/L for 20 min. The ploidy of larvae under different induction concentrations were analyzed by flow cytometry, and the chromosome division process during sperm entry and cleavage was observed by fluorescence microscope. In addition, the ultrastructure of sperm, embryonic development, and chromosome were observed. The results showed that the fertilization rate and cleavage rate decreased gradually with the prolongation of UV irradiation time. We found UV irradiation for 9 s was the optimal time for haploid formation, with an induction rate of 100%. The CB treatment at 0.75 mg/L was the optimal concentration for the formation of D-larvae, and the induction rate was 9.60% ± 5.10%. The results showed that the optimal conditions for inducing gynogenetic diploid of R. philippinarum were irradiation with UV intensity of 230 μW (cm²·s) for 9 s and inhibited second polar body release by CB at concentration of 0.75 mg/L. In this study, the conditions for inducing Manila clam gynogenetic diploid were obtained, which lays the basis for further genetic research on the gynogenesis of R. philippinarum.