Streptococcus agalactiae is an important pathogenic bacterium causing great economic loss in Nile tilapia (Oreochromis niloticus) culture. Resistant and susceptible groups sharing the same genome showed significantly different resistance to S. agalactiae in the genetically improved farmed tilapia strain of Nile tilapia. The resistance mechanism is unclear. We determined genome-wide DNA methylation profiles in spleen of resistant and susceptible O. niloticus at 5 h postinfection with S. agalactiae using whole-genome bisulfite sequencing. The methylation status was higher in the spleen samples from resistant fish than in the susceptible group. A total of 10,177 differentially methylated regions were identified in the two groups, including 3725 differentially methylated genes (DMGs) (3129 hyper-DMGs and 596 hypo-DMGs). The RNA sequencing showed 2374 differentially expressed genes (DEGs), including 1483 upregulated and 891 downregulated. Integrated analysis showed 337 overlapping DEGs and DMGs and 82 overlapping DEGs and differentially methylated region promoters. By integrating promoter DNA methylation with gene expression, we revealed four immune-related genes (Arnt2, Nhr38, Pcdh10, and Ccdc158) as key factors in epigenetic mechanisms contributing to pathogen resistance. Our study provided systematic methylome maps to explore the epigenetic mechanism and reveal the methylation loci of pathogen resistance and identified methylation-regulated genes that are potentially involved in defense against pathogens. Key Points Whole-genome bisulfite sequencing distinguishes between resistant and susceptible O. niloticus. Promoter methylation regulates gene expression in immune-related genes.

Chinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism. We used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17β-estradiol exposed group. This is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation.

Background: Monopterus albusis a hermaphroditic and economically farmed fish that undergoes sex reversal from ovary to testis via ovotestis during gonadal development. The epigenetic changes that are associated with gonadal development in this species remain unclear.Methods: We produced DNA methylome, transcriptome, and chromatin accessibility maps of the key stages of gonad development: ovary, ovotestis, and testis. The expression of the key candidate genes was detected using qRT-PCR and in situ hybridization and the methylation levels were analysed using bisulphite sequencing PCR. Promoter activity and regulation were assessed using dual-luciferase reporter assays.Results: Gonadal development exhibits highly dynamic transcriptomic, DNA methylation, and chromatin accessibility changes. We found that DNA methylation status, especially of the transcription start site, was significantly negatively correlated with gene expression while chromatin accessibility exhibited no correlation with gene expression during gonadal development. The epigenetic signatures revealed many novel regulatory elements and genes involved in sex reversal, which were validated. DNA methylation detection and site mutation of plastin-2 promoter, as a candidate gene, revealed that DNA methylation could impact the binding of transcription factor dmrt1 and foxl2 through methylation and demethylation to regulate plastin-2 expression during gonadal development.Conclusions: These data provide novel insights into epigenetic modification and help elucidate the potential molecular mechanism by which dynamic modification of DNA methylation plays a crucial role in gonadal development.

Background: Monopterusalbus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism of the sex reversal was unknown. Methods: We produced transcriptomes containing mRNAs and lncRNAs in the crucial stages of the gonad, including the ovary, ovotestis and testis. The expression of the crucial lncRNAs and their target genes was detected using qRT‒PCR and in situ hybridization. The methylation level and activity of the lncRNA promoter were analysed by applying bisulfite sequencing PCR and dual-luciferase reporter assays, respectively. Results: This effort revealed that gonadal development was a dynamic expression change. Regulatory networks of lncRNAs and their target genes were constructed through integrated analysis of lncRNA and mRNA data. The expression and DNA methylation of the lncRNAs MSTRG.38036 and MSTRG.12998 and their target genes Psmβ8 and Ptk2β were detected in developing gonads and sex reversal gonads. The results showed that lncRNAs and their target genes exhibited consistent expression profiles and that the DNA methylation levels were negatively regulated lncRNA expression. Furthermore, we found that Ptk2β probably regulates cyp19a1 expression via the Ptk2β/EGFR/STAT3 pathway to reprogram sex differentiation. Conclusions: This study provides novel insight from lncRNA to explore the potential molecular mechanism by which DNA methylation regulates lncRNA expression to facilitate target gene transcription to reprogram sex differentiation in M.albus, which will also enrich the sex differentiation mechanism of teleosts.