Search

Background: Galeruca daurica has become a new pest on the Inner Mongolia grasslands since an abrupt outbreak in 2009 caused serious damage. As a pupa indicator during insect metamorphosis, the early response gene of the ecdysone signaling pathway, Broad-Complex (Br-C), plays a vital role in the growth and development of insects. MicroRNAs (miRNAs) are small non-coding RNAs which mediate various biological activities, but it is unknown whether and how Br-C is regulated by miRNAs., Results: Temporal expression profiles revealed that miR-285 and Br-C basically displayed an opposite trend during larval-adult development, and Br-C was sharply up-regulated on the last day of final-instar larvae while miR-285 was significantly down-regulated. Both dual-luciferase reporter assay and miRNA-mRNA interaction assay indicated that miR-285 interacts with the coding sequence of Br-C and represses its expression. Not only overexpression but also downexpression of miR-285 led to the failure of larval to pupal to adult metamorphosis. In addition, both overexpression of miR-285 and silence of Br-C inhibited the expression of Br-C and other ecdysone signaling pathway genes, including E74, E75, ECR, FTZ-F1, and HR3. On the contrary, suppressing miR-285 obtained opposite results. Further experiments showed that 20-hydroxyecdysone down-regulated miR-285 and up-regulated Br-C and above-mentioned genes, whereas juvenile hormone alalogue (JHA) resulted in opposite effects., Conclusion: Our results reveal that miR-285 is involved in mediating the metamorphosis in G. daurica by targeting Br-C in the ecdysone signaling pathway. miR-285 and its target Br-C could be as a potential target for G. daurica management. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3' UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 Ca 2+ 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)