| Long-arm octopus(Octopus minor)is naturally distributed in China,Japan and Korea,especially in Bohai and Yellow sea of China.Recently,it has become a potential commercially and ecologically important species.Because of the shortcoming of artificial breeding technique,the O.minor in the market were captured from inshore fishing mainly.Due to environmental damage,disorderly development and other factors,the population of O.minor was increasing decreased.To protect the continuation of O.minor natural population,it is urgent to take effective strategies and solve the technical bottleneck in breeding industry of O.minor.Unisexual breeding may be an effective way to solve the problem of early ripening and miniaturization in octopods aquaculture industry,while their sexualities are generally hard to distinguish based on morphology appearance at the early development stage.In this regard,this study preliminarily explored the population composition characteristics of O.minor in breeding season;Moreover,the molecular regulation mechanism of sex differentiation in O.minor was studied based on transcriptome sequencing.These results provide a reference for the artificial breeding and resource conservation of O.minor,and provide a certain basis for the sex differentiation and unisexual breeding of cephalopods.Here are the major achievements:1.Study on sex composition and capture strategy of O.minor in breeding seasonTwelve batches of geographical population samples of O.minor collected in Laizhou and Zhifu in 2016 and 2019 were selected,and the stem wrist was used as the sex determination marker,to analyze the relationship between gender proportion and collection time;At the same time,the average body weight of all parents in a single sampling was calculated,and the relationship between gender proportion and average body weight was analyzed.The results showed that there was lower proportion of female octopus during the breeding season,with negative relationship between average body weight and female proportion in the O.minor population,i.e.the higher the proportion of females,the lower the average body weight.The O.minor captured by hands and cage traps had higher survival rate than the O.minor captured by net and purchased from markets,more suitable for catching brood stock octopus for artificial breeding.2.Screening of the sex-determining genes in O.minorSamples of O.minor at different developmental stages were collected in Muping in June and November to prepare mature and immature gonadal tissue samples of O.minor.Subsequently,gonadal transcriptome sequence was performed based on mature and immature O.minor samples.Four groups of 12 gonad tissues were sampled and 11 libraries were retained for biological analysis and a total of 263,749,727 clean reads were obtained.In addition,the clean reads were mapped to the O.minor reference genome,and the percentage of mapped reads ranged from 77.20% to 90.59%.Furthermore,3936 differentially expressed genes(DEGs)were obtained between ovarian and testicular libraries by Venn diagram analysis,including 855 ovarian up-regulated and 3081 testicular up-regulated genes.Subsequently,GO and KEGG enrichment analysis were employed to investigate the biological roles the genes clustered into different groups in the Venn diagram.Then,to the reliability and accuracy of our analysis,10 DEGs were selected randomly for quantitative real-time PCR.Four unigenes(transcription factor Sox-8,fermentation-1,forkhead box protein L2,and ribosomal protein large 24)and one pathway(ECM-receptor interaction)were screened to be candidate molecular markers for gender determining.This results in our study may enhance our understanding of the molecular mechanism underlying sex determination and potentially help to screen the bio-markers for O.minor in different genders.The changes of gender composition in O.minor was attributed to the reproductive migration and egg-protecting characteristics of female O.minor.The difference in body weight between male and female octopus seemed to be attributed to two reasons:K-selected strategy and sperm competition.The O.minor caught by hands had good integrity and was most suitable for breeding.The genes up-regulated in testis such as SOX8 and FEM-1c were selected for the male-biased genes,and the mechanism of male-determining was attributed to the aspects of gene encoding protein regulated by sex chromosome Y and participating in testicular development.Genes up-regulated in the ovaries such as Foxl2 and RPL24 were selected for the female-biased genes,and the mechanism of female determining was attributed to producing specific estrogen and participating in the process of ovarian development.The ECM-receptor interaction pathway was selected as a potential sex determination pathway,and the mechanism was attributed to the perspective of providing basic conditions for the activity of oogenesis cells.According to the results of this study,we look forward to the possibility of establishing a dynamic database of sex composition and average body mass change of O.minor through continuous monitoring,and fitting the model between sex composition and environmental factors,and combining economic fishing and population resource protection of O.minor.Moreover,it was proposed to find out the temporal and spatial differences of gene expression in different developmental stages and different organs of O.minor by means of WGCNA and RNAi,screen the sex-determining hub genes,and reveal the sex determination molecular mechanism of cephalopods represented by O.minor. |
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