Abstract
MicroRNAs (miRNAs) play a crucial role in post-transcriptional gene regulation. However, the regulatory mechanisms of miRNAs in Tribolium castaneum under hypoxic stress remain poorly understood. In this study, we investigated the effect of hypoxia on T. castaneum, and results showed that hypoxia tolerance were adults > pupae > old larvae > young larvae. The eclosion time of both larvae and pupae were delayed in hypoxic environment. After 48 h of hypoxia treatment, the reproductive capacity of the adults was significantly reduced but recovered by the sixth day. Small RNA sequencing identified five miRNAs that were significantly differentially expressed under hypoxia. These included miR-989-3p, miR-279b-5p, miR-2765-5p, miR-750-3p, and miR-309c-3p. Among them, miR-279b-5p plays an important role in growth and development of T. castaneum. We predicted Hexamerin2 (TcHex2) as a target gene of miR-279b-5p. TcHex2 is predominantly expressed in the fat body of old larvae, while miR-279b-5p is expressed across all developmental stages, with higher expression levels in the hemolymph. Both miR-279b-5p and TcHex2 were significantly upregulated in response to hypoxic treatment. Interference with the expression of TcHex2 and miR-279b-5p significantly increased the mortality of T. castaneum under hypoxic conditions. Moreover, overexpression of miR-279b-5p resulted in a significant upregulation of TcHex2 expression. This was further validated by in vitro and in vivo experiments, by using isothermal titration calorimetry (ITC) and dual-luciferase reporter assays. Our study reveals that miR-279b-5p positively regulates TcHex2 expression under hypoxic conditions, highlighting its important role in T. castaneum hypoxic adaptation. These findings provide a theoretical basis for the development of RNAi-based pesticides to control stored-products pests.
