20-Hydroxyecdysone-Responsive miR-2788 Regulates the Larval–Pupal Transition by Targeting Trehalase in Galeruca daurica

Metamorphosis in insects is regulated by hormonal signals and metabolic pathways. In this study, we characterized a 20E-mediated miR-2788/Treh1 regulatory axis involved in the larval-to-pupal transition in the leaf beetle Galeruca daurica. Dual-luciferase assays and expression profiling suggest that miR-2788 directly targets and suppresses trehalase 1 (Treh1), a key enzyme for trehalose hydrolysis. We found that exogenous 20E treatment significantly downregulated miR-2788, leading to the derepression of Treh1 and increased expression of chitin biosynthetic genes (CHI, CHS, and GPI). Conversely, miR-2788 overexpression or Treh1 silencing via RNAi resulted in massive trehalose accumulation, reduced chitin synthesis, and lethal developmental arrest during the metamorphic transition. Furthermore, pharmacological inhibition of Treh1 with Validamycin not only recapitulated these chitin-deficiency phenotypes but was also associated with an upregulation of miR-2788. These results describe a signaling cascade where 20E modulates the conversion of carbohydrate to chitin through the miR-2788/Treh1 axis. This study advances our understanding of hormonal–miRNA–metabolic crosstalk and identifies miR-2788 as a potential target for RNAi-based biopesticide development.