Ame‐miR‐2161 affects the survival and development of honeybee larvae through the juvenile hormone acid methyltransferase gene

Abstract MicroRNAs (miRNAs) are endogenous small non‐coding RNAs that play crucial regulatory roles in insect growth and development. However, the coordinated regulation of honeybee development by miRNAs and hormones remains poorly understood. In this study, the regulatory network of target genes for Apis mellifera miRNA‐2161 (ame‐miR‐2161) was constructed, and its association with the survival and development of worker larvae was investigated. The results showed that ame‐miR‐2161 potentially targets 22 mRNAs, with particular emphasis on the target gene juvenile hormone acid methyltransferase ( Jhamt ), a key rate‐limiting enzyme in the final step of the juvenile hormone (JH) biosynthesis. RT‐qPCR analysis showed concordant expression patterns between ame‐miR‐2161 and Jhamt across larval developmental stages. Dual‐luciferase assays confirmed that Jhamt is a direct target of ame‐miR‐2161. Functional studies revealed that overexpression of ame‐miR‐2161 upregulated the Jhamt expression, leading to a significant increase in JH titre in 4‐ to 6‐day‐old larvae, accompanied by a gradual upregulation of the JH downstream response gene Kr ‐ h 1. Conversely, inhibition of ame‐miR‐2161 downregulated the Jhamt expression, reducing JH titre and markedly suppressing Kr ‐ h 1 expression, indicating that ame‐miR‐2161 positively regulates the expression of Jhamt . Furthermore, ame‐miR‐2161 overexpression enhanced larval survival, whereas its inhibition decreased survival rates. Although pupation rates remained unaffected, ame‐miR‐2161 modulation influenced larval body weight changes. These results suggest that ame‐miR‐2161 regulates JH levels by targeting Jhamt , thereby modulating larval survival and development in honeybees. Our findings provide novel insights into the miRNA‐mediated regulation of hormone signalling and metamorphic development in honeybees.