Identification of sex-biased MiRNA markers informative of heat-past events

Elevated temperatures during early developmental stages play a pivotal role in the fate of the adult sexual phenotype of fish populations, usually leading to male-skewed sex ratios. This is the case with European sea bass (Dicentrarchus labrax), one of the most important species in the European aquaculture industry. To unveil informative markers of the past thermal events, we investigated changes in the miRNome within the gonads of this species. Consequently, we exposed European sea bass to elevated temperatures (21ºC) during early development (from 7 to 68 days post fertilization). After one-year post-heat treatment growing at natural temperature, a miRNA-sequencing analysis was conducted in the ovaries and testes of juvenile fish. The examination of miRNA expression levels identified three and twelve miRNAs in ovaries and testes, respectively, reflecting past thermal events. To assess the evolutionary conservation of these identified miRNAs in gonads, we cross-referenced our data with miRNome public information from ovaries and testes in nine additional fish species from the FishmiRNA database. This analysis uncovered 33 potential sex-biased markers present in at least five studied species along the evolutionary timeline. For instance, miR-155, miR-429, and miR-140 were consistently female-skewed, while miR-143, miR-499, and miR-135b-3p were consistently male-skewed. In addition, among these markers, three conserved sex-skewed miRNAs proved to be informative regarding past thermal events in the ovaries (e.g., miR-192-5p, miR-146a-5p and miR-143-3p) and four in the testes (miR-129-5p, miR-724-5p, miR-143-3p, and miR-223-3p). Notably, miR-223-3p was conserved female-skewed, but showed upregulation in males exposed to high temperature, and miR-143-3p was inhibited in both heated females and males. These miRNAs could serve as markers of heat-induced masculinization. This research broadens the inventory of sex-specific miRNAs across evolution in fish, and elucidates thermosensitive miRNAs in the gonads.