| Rock bream(Oplegnathus fasciatus)is an important marine economic fish in China,with a unique multiple sex chromosomes system(♀X1X1X2X2/♂X1X2Y),presenting distinct karyotype characteristics:Male contains an exceptionally large heterotypic chromosome and possesses chromosomes one less than females.To date,over 50 cases with multiple sex chromosomes have been reported in teleost phylogeny,which distributed in 25 families of 13 orders,indicating its crucial role in the formation and evolution of sex chromosome in fish.However,little is known about the molecular sex determination mechanism of multiple sex chromosome system in fish.Meanwhile,the study of sex control can not only be used in mono-sex breeding to increase economic value of the aquaculture industry,but also be a feasible strategy for investigating sex determination and differentiation.In this study,homologous sperm inactivation combined with cold shock was used to induce gynogenesis in rock bream,and the parameters of gynogenesis procedure were optimized,including the UV inactivation conditions of homologous sperm,and the onset and duration of cold shock induction.Histological,cytological,and molecular biological methods were used to identify the gynogenetic rock breams.Sex reversal were further conducted,where gynogenetic rock breams with undifferentiated gonads(40 days post hatching,dph)were fed with diets containing 10 mg/kg of letrozole(LTZ)or 17αmethyltestosterone(MT)respectively for 60 days,while 40 dph normal rock breams were fed with diets containing 10 mg/kg of 17β-estradiol(E2)for 60 days.Samples were taken at 100,130 and 400 dph to investigate the effects of LTZ,MT and E2 on the growth,gonadal development,and differentiation of the rock bream respectively,by methods of statistical analysis,molecular identification,and histological observation.The main results are as follows:1.The results showed that the optimal UV irradiation of the O.fasciatus sperm for gynogenesis was at 80 mJ/cm2.Cold shock optima for gynogenesis were 10 min of 3～4℃treatment at 2 min after fertilization.Results of morphological observation and larvae DNA content detection by flow cytometry proved that the gynogenetic larvae were diploid fish.2.The genetic analysis by microsatellite revealed exclusively maternal inheritance in all gynogenetic fry and no paternal inheritance were detected.The average heterozygote ratio of the nine pairs of microsatellite loci used was 0.796,and two lines(60 offspring)did not have all pure genotypes at the nine microsatellite loci tested.The results of sex ratio detection and growth performance of gynogenetic and the control fish suggested that gynogenetic fish were all females,and grew faster than the control after 15 months post hatching.3.The results of sex reversal experiments at 400 dph showed that LTZ and MT were able to induced 100%masculinization of genotype-females(neo-males).MT had negative effects on fish growth.Meanwhile,LTZ and MT could accelerate gonadal development of the neo-males.4.The results of E2 induced sex reversal experiments at 400 dph showed that E2 was able to induce only 62.5%feminization of genotype-males.E2 had negative effects on fish growth,while inhibited gonadal development in genotype-females.In this study,we investigated optimal procedures of artificial gynogenesis of rock bream,and explored the sex plasticity of the multiple sex chromosomes fish represented by the rock bream.The results of the study can not only provide new ideas and guidance for the monosex breeding of rock bream,but also lay the research foundation for revealing the mechanism of sex determination and differentiation of fish with multiple sex chromosomes. |
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