MicroRNA miR-2765-3p regulates reproductive diapause by targeting FoxO in Galeruca daurica

The forkhead box O (FOXO), as a conserved transcription factor, plays an indispensable role in regulating insect diapause. However, how FOXO is regulated to control diapause in insects remains unknown. In this study, we discovered functional binding sites for miR-2765-3p in the 3' untranslated region of FOXO in Galeruca daurica. The luciferase reporter assay showed that miR-2765-3p targeted FOXO and suppressed its expression. The expression profiles of miR-2765-3p and FOXO displayed opposite patterns during the female developmental process. Overexpression of miR-2765-3p by the injection of the miR-2765-3p agomir into adult females reduced FOXO expression, leading to the suppression of lipid accumulation, promotion of ovarian development, and inhibition of reproductive diapause. This is similar to the phenotype that results from the depletion of FOXO by injecting dsFoxO into adult females. In addition, the repression of miR-2765-3p by injecting the miR-2765-3p antagomir increased the FOXO transcript level, leading to the stimulation of lipid accumulation, depression of ovarian development, and induction of reproductive diapause. A hormone injection assay showed that the juvenile hormone (JH) agonist (methoprene) upregulated miR-2765-3p and downregulated FOXO. Notably, injecting methoprene rescued ovarian development defects associated with miR-2765-3p inhibition. These findings indicate that the JH/miR-2765-3p/FOXO axis plays a vital role in the regulation of reproductive diapause in G. daurica.