Abstract

Simple SummaryHoneybees play a crucial role in sustaining agricultural production and maintaining ecosystem balance. MicroRNAs (miRNAs) are involved in bee development and caste differentiation by regulating gene transcription and translation. Our findings indicate that hormones act as a key bridge in the miRNA-mediated regulation of honeybee physiology. Specifically, miRNA novel-m0027-3p can directly target juvenile hormone (JH) acid methyl transferase (Jhamt), a key gene involved in JH synthesis, thereby modulating JH titers and signaling pathways during larval development. Our study provides insights into how miRNAs regulate hormone pathways to affect honey bee development.Juvenile hormone (JH) is a key regulator of larval development in honeybees. Its biosynthesis involves multiple enzymatic steps and is modulated by a complex regulatory network that includes microRNAs (miRNAs). Juvenile hormone acid methyltransferase (JHAMT) catalyzes the final step in JH synthesis. This study demonstrates that the miRNA novel-m0027-3p negatively regulates the expression of theJhamtgene inApis melliferalarvae (AmJhamt), thereby mediating JH biosynthesis. Bioinformatics predictions indicate that novel-m0027-3p potentially targets six hormone-related genes (22 mRNAs), includingAmJhamt. Dual-luciferase reporter assays and mimics/inhibitor-miRNA feeding confirmed that novel-m0027-3p significantly suppresses the expression of the target geneAmJhamt. In vivo experiments showed that larvae fed with the mimics of novel-m0027-3p exhibited decreased JH titers and significantly downregulated expression of JH signaling downstream genesAmHex70bandAmKr-h1. Conversely, larvae fed with the inhibitors of novel-m0027-3p displayed significantly elevated JH titers and markedly upregulated expression ofAmHex70bandAmKr-h1. Our findings provide experimental evidence for the coupling between miRNAs and