Abstract

MicroRNAs (miRNAs) are short and conserved noncoding RNAs which could regulate the target gene expression by inhibiting the translation of mRNA. In this study, high-throughput sequencing was employed to investigate the miRNA expression profiles in Litopenaeus vannamei under freshwater conditions. A total of 100 known and 374 novel miRNAs were identified, among which 23 miRNAs were significantly differentially expressed. Additionally, bioinformatic analysis predicted 287 target genes of the differentially expressed miRNAs (DEMs), which were functionally associated with a diverse array of biological processes and signaling pathways. Furthermore, the functional role of miR-252a, a prominent DEM identified via the sequencing, was investigated through an overexpression assay to assess its regulatory impact on expression of the osmoregulation-related genes, including cytoplasmic carbonic anhydrase ( CAc ), glycosyl-phosphatidylinositol-linked carbonic anhydrase ( CAg ), Na + /K + -ATPase , bestrophin-2 ( BEST2 ), and calcium-activated chloride channel regulator 1 ( CLCA1 ), in shrimp under freshwater stress. As salinity decreased, the expression levels of CAc , BEST2 , Na + /K + -ATPase , and CLCA1 were obviously up-regulated ( P  < 0.05) in shrimp from each treatment group compared to their levels under 30‰ salinity. Notably, an obvious up-regulation of CAg expression was detected in each treatment only at 96 h post injection (hpi) under 0.5‰ salinity, compared to its level at 24 hpi under 30‰ salinity. Additionally, the expression of all tested genes in agomiR-252a-treated shrimp remained markedly lower ( P  < 0.05) than that in the agomiR-252a-NC groups throughout the entire salinity reduction process (30‰ to 0.5‰). These findings suggest that miR-252a may act as a post-transcriptional repressor of osmoregulatory genes in L. vannamei and potentially play a cr