Abstract
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 the Jhamt gene in Apis mellifera larvae (AmJhamt), thereby mediating JH biosynthesis. Bioinformatics predictions indicate that novel-m0027-3p potentially targets six hormone-related genes (22 mRNAs), including AmJhamt. Dual-luciferase reporter assays and mimics/inhibitor-miRNA feeding confirmed that novel-m0027-3p significantly suppresses the expression of the target gene AmJhamt. 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 genes AmHex70b and AmKr-h1. Conversely, larvae fed with the inhibitors of novel-m0027-3p displayed significantly elevated JH titers and markedly upregulated expression of AmHex70b and AmKr-h1. Our findings provide experimental evidence for the coupling between miRNAs and hormonal pathways in honeybees.