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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.

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Background: MicroRNAs (miRNAs) are a class of small noncoding RNAs of approximately 22 nt in length, which regulate gene expression at the post-transcriptional level. Although the regulatory roles of miRNAs in various physiological processes throughout insect development have been investigated, it is almost unknown about the roles of miRNAs involved in the regulation of diapause in insects.Results: We constructed 12 small RNA libraries from Galeruca daurica adults at different diapause stages: pre-diapause (PD), diapause (D), post-diapause 1 (TD1), and post-diapause 2 (TD2). Using Illumina sequencing, a total of 95.06 million valid reads was obtained, and 230 miRNAs, including 143 conserved and 87 novel miRNAs, were identified from G. daurica. The expression profiles of these miRNAs were assessed across different diapause stages and miRNAs that were highly expressed at different diapause stages were identified. Comparative analysis of read counts indicated that both conserved and novel miRNAs were differently expressed among the four different diapause stages, and the differential expression was validated via qRT-PCR. The 25, 11, 15, 14, 26, and one miRNAs were differentially expressed in D/PD, D/TD1, D/TD2, TD1/PD, TD2/PD, and TD2/TD1, respectively. The KEGG and GO analysis of the predicted target genes suggested the essential roles of miRNAs in the regulation of summer diapause in G. daurica, especially via the juvenile hormone, ribosome, MAPK signaling, mTOR signaling, Ca2+ signaling, and G-protein coupled receptor signaling pathways.Conclusion: Our research results indicate that miRNAs may be involved in the regulation of summer diapause in G. daurica, and these results also provide an important new small RNA genomics resource for further studies on insect diapause.