A conserved miR ‐281‐x is associated with self‐grooming behaviour in Apis cerana

Self-grooming behaviour is a crucial adaptation in honey bees for maintaining individual hygiene and combating parasite infestations. The miRNA-mediated influence on self-grooming behaviour has been recently demonstrated in Apis mellifera but remains largely unexplored in Apis cerana. This study combined behavioural analysis, miRNA sequencing and molecular biology techniques to investigate these mechanisms. We first identified two distinct behavioural phenotypes in A. cerana: strong groomers (CS) and weak groomers (CW). miRNA sequencing of bee brains revealed 12 differentially expressed miRNAs between CS and CW groups. Notably, miR-281-x, previously associated with grooming in A. mellifera, was significantly upregulated in CW bees, suggesting a conserved role. Subsequent qPCR and functional experiments confirmed that miR-281-x overexpression suppressed grooming behaviour and reduced brain octopamine (OA) levels in A. cerana. Furthermore, through a dual-luciferase assay, qPCR and fluorescence in situ hybridization (FISH), we identified tyrosine decarboxylase (tdc-1), a key enzyme in OA synthesis, as a direct target of miR-281-x. Finally, RNAi-mediated knockdown of tdc-1 significantly reduced both brain OA levels and grooming behaviour, mirroring the effects of miR-281-x overexpression. Our findings identify a conserved miR-281-x/tdc-1/OA axis that is associated with grooming behaviour in A. cerana, providing new insights into the molecular basis of this essential innate behaviour.