MicroRNA-281-X Modulates Self-Grooming Behavior in Honeybees by Targeting Tyrosine Decarboxylase 2 in the...

Self-grooming is a fundamental and evolutionarily conserved behavior and is essential for removing Varroa destructor in honeybees (Apis mellifera). However, its molecular and neural regulation remains poorly understood. This study identifies a microRNA-mediated neuromodulatory pathway that governs the intensity of self-grooming behavior in A. mellifera. Comparative behavioral analyses revealed pronounced differences in grooming intensity between bees exhibiting strong (MS) and weak (MW) grooming phenotypes. Liquid chromatography detection and pharmacological experiments demonstrated that octopamine (OA) levels are significantly different between MS and MW bees, and OA enhances grooming behavior. Small RNA sequencing revealed differentially expressed miRNAs in the brains of MS versus MW bees, leading to the identification of miR-281-x as a candidate associated with OA regulation. Functional validation showed that overexpression of miR-281-x significantly reduces grooming behavior, whereas inhibition of miR-281-x enhances grooming. Mechanistically, bioinformatics prediction and experimental validation confirmed that miR-281-x directly targets the tdc2, which encodes tyrosine decarboxylase 2 in OA biosynthesis. Fluorescence in situ hybridization (FISH) revealed co-localization of miR-281-x and tdc2 in Kenyon cells of the mushroom bodies. RNAi-mediated knockdown of tdc2 significantly reduced grooming intensity and rescue experiments confirmed the miR-281-x-tdc2-OA regulatory axis. Together, our findings uncover a post-transcriptional modulatory mechanism linking a microRNA to neuromodulator-dependent behavioral plasticity, providing insight into the control of grooming intensity in honeybees.