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.

Transcriptome-wide identification of microRNAs in response to 20-hydroxyecdysone in Galeruca daurica.

Both 20-hydroxyecdysone (20E) and miRNAs have multiple functions in the regulation of various physiological processes in insects. However, little is known about the interaction between 20E and miRNAs. In this study, six small RNA libraries were constructed from the adult Galeruca daurica treated with 20E and dimethyl sulfoxide (DMSO), respectively. Using small RNA sequencing, a total of 183 miRNAs, including 140 known and 43 novel miRNAs, were identified. Compared with the control (DMSO), 52 miRNAs (21 up-regulated and 31 down-regulated) were significantly differentially expressed after 20E treatment. The KEGG and GO analysis of the predicted genes targeted by 20E-responsive miRNAs indicate that 20E may influence the metabolic change during reproductive diapause in G. daurica via regulating miRNAs.

MicroRNA let-7-5p targets the juvenile hormone primary response gene Krüppel homolog 1 and regulates reproductive diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate various biological processes in insects. However, their roles in the regulation of insect diapause remain unknown. In this study, we address the biological function of a conserved miRNA, let-7-5p in the regulation of a juvenile hormone primary response gene, Krüppel homolog 1 (Kr-h1), which modulates reproductive diapause in Galeruca daurica. The dual luciferase reporter assay showed that let-7-5p depressed the expression of Kr-h1. The expression profiles of let-7-5p and Kr-h1 displayed opposite patterns in the adult developmental stage. Injection of let-7-5p agomir in pre-diapause adult females inhibited the expression of Kr-h1, which consequently led to delay ovarian development, increase lipid accumulation, expand fat body, and induce reproductive diapause just as depleting Kr-h1 did. Conversely, injection of let-7-5p antagomir resulted in opposite effects by reducing fat storage and stimulating reproduction. Moreover, JH receptor agonist methoprene reduced the expression of let-7-5p, and rescued the ovarian development defects associated with let-7-5p overexpression. These results indicate that let-7-5p plays an important role in the regulation of reproductive diapause and development of G. daurica adults through its target gene Kr-h1.

Identification and functional analysis of microRNAs in the regulation of summer diapause in Galeruca daurica.

MicroRNAs (miRNAs) 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 regulation of diapause in insects. We constructed nine small RNA libraries from Galeruca daurica adults at different diapause stages: pre-diapause (PD), diapause (D), and post-diapause (TD). Using Illumina sequencing, a total of 95.06 million valid reads was obtained, and 222 miRNAs, including 135 conserved and 87 novel miRNAs, were identified from G. daurica. The expression profiles of these miRNAs were analyzed across different diapause stages. The 30 and 13 miRNAs were differentially expressed in the D/PD and TD/D comparisons, 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, and Ca2+ signaling pathways. 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.