Integrated analysis of DNA methylome and RNA transcriptome during high-temperature-induced masculinization in sex-undifferentiated Nile tilapia gonad

Fish sex-determining mechanisms can be classified as genotypic (GSD), temperature (TSD), or genotypic plus temperature effects (GSD + TE). Nile tilapia (Oreochromis niloticus) is a GSD + TE fish species. High-temperature treatment during critical thermosensitive periods (TSPs) can induce the sex reversal of Nile tilapia genetic females into phenotypic males (pseudomales), while the mechanism has been a long-standing mystery. Previous studies have indicated that DNA methylation and gene expression of a large fraction of genes in the gonad in many teleost species were altered after high-temperature treatment. However, genome-wide DNA methylation changes in the sex-undifferentiated fish gonads after high-temperature treatment are unclear. Here, whole-genome bisulfite sequencing (WGBS) was performed to compare the global DNA methylation level of FC (XX Female Control), FT (XX Females after high-temperature treatment during the TSP from 5 dah to 17 dah) and MC (XY Male Control) Nile tilapia sex-undifferentiated gonads. We identified 1196 (FC-vs-FT), 16,559 (FC-vs-MC) and 17,227 (MC-vs-FT) differentially methylated regions (DMRs) in Nile tilapia gonads. A total of 50 DMRs were found to be associated with both high-temperature treatment and sex development, as the DNA methylation levels of these genes differed in both FC-vs-MC and FC-vs-FT. Using previous Nile tilapia gonad transcriptome data from our lab, venn diagram analysis of the differentiation expression genes (DEGs) and DMGs (DMR related genes) obtained 5, 123 and 477 overlapped genes in FC-vs-FT, FC-vs-MC and MC-vs-FT, respectively. Additionally, many important pathways, including neuroactive ligand-receptor interaction, insulin signaling pathway and Calcium signaling pathway were identified. Finally, the Myb (LOC100693304) and Wnt11 (LOC100708759) were selected for further verification using bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qRT-PCR), indicating the accuracy and reliability of the WGBS and RNA-Seq results. This study provided an important foundation to investigate the molecular mechanism of high temperature-induced masculinization in Nile tilapia as well as in other species with GSD + TE effects.