A time course transcriptome analysis of brains from sex-undifferentiated Nile tilapia discloses genes associated with high-temperature-induced masculinization

Abstract

Fish sex-determining mechanisms can be classified as genotypic (GSD), temperature (TSD), or genotypic plus temperature (GSD + TE) effects. Water temperature during thermosensitive periods (TSPs) could affect the expression levels of many genes in the gonad or the brain in various teleost species. However, studies on gene expression changes in the brain of Nile tilipia from the start to the end of high-temperature during TSP have not been investigated. In this study, we performed RNA-Seq on brain samples from 5 dah (days after hatching) Nile tilapia that underwent high temperature treatment for 6 h (5 dah +6 h) and 24 h (5 dah +24 h). The results showed that 307 and 933 differential expressed genes (DEGs) were present in the female controls (FC) vs high-temperature-treated females (FT) comparison group and that 606 and 762 DEGs were in the FC vs male controls (MC) comparison group at 5 dah +6 h and 5 dah +24 h, respectively. To validate the DEGs obtained by RNA-Seq, 10 genes were randomly selected to perform qRT-PCR. The results showed that the expression levels of each gene among the 10 genes were similar between the RNA-Seq and qRT-PCR results at the two time points and that there was a strong correlation between the RNA-Seq and qRT-PCR data (r = 0.532, P < .01). Using previous Nile tilapia brain transcriptome data at 5 dah +12 d from our lab, profiling analysis of the Nile tilapia brain transcriptome data at three time points (5 dah +6 h, 5 dah +24 h and 5 dah +12 d) indicated that six genes associated with sex differentiation and high-temperature treatment, including Kdm6bb, were significantly upregulated, while 13 genes, including cytochrome P450 2 J6, were significantly downregulated in the FC vs FT comparison group at two or three examined time points. Meanwhile, the expression levels of four genes (Dnaja, Dnajb1a, heat shock protein 70, and heat shock 70 kDa protein 1) in heat shock protein family were significantly upregulated after high-temperature treatment at two examined time points, and Hspa8b were significantly upregulated after high-temperature treatment at three examined time points. Finally, the Kdm6bb was selected for further verification using in situ hybridization. This study found many genes associated with high-temperature induced masculinization and provided new insights into the molecular mechanisms in Nile tilapia as well as in other species with GSD + TE effects.