The grooming behavior of honeybees serves as a crucial auto-protective mechanism against Varroa mite infestations. Compared to Apis mellifera, Apis cerana demonstrates more effective grooming behavior in removing Varroa mites from the bodies of infested bees. However, the underlying mechanisms regulating grooming behavior remain elusive. In this study, we evaluated the efficacy of the auto-grooming behavior between A. cerana and A. mellifera and employed RNA-sequencing technology to identify differentially expressed genes (DEGs) in bee brains with varying degrees of grooming behavior intensity. We observed that A. cerana exhibited a higher frequency of mite removal between day 5 and day 15 compared to A. mellifera, with day-9 bees showing the highest frequency of mite removal in A. cerana. RNA-sequencing results revealed the differential expression of the HTR2A and SLC17A8 genes in A. cerana and the CCKAR and TpnC47D genes in A. mellifera. Subsequent homology analysis identified the HTR2A gene and SLC17A8 gene of A. cerana as homologous to the HTR2A gene and SLC17A7 gene of A. mellifera. These DEGs are annotated in the neuroactive ligand–receptor interaction pathway, the glutamatergic synaptic pathway, and the calcium signaling pathway. Moreover, CCKAR, TpnC47D, HTR2A, and SLC17A7 may be closely related to the auto-grooming behavior of A. mellifera, conferring resistance against Varroa infestation. Our results further explain the relationship between honeybee grooming behavior and brain function at the molecular level and provide a reference basis for further studies of the mechanism of honeybee grooming behavior.

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.

CSBV is a major pathogen threatening the health of A. cerana, but the mechanisms underlying natural resistance in its larval host remain poorly understood. To elucidate this, we established resistant (R) and susceptible (S) larval phenotypes through controlled CSBV inoculation and combined untargeted metabolomics with biochemical assays. Metabolomic profiling revealed a markedly disrupted metabolic state in S larvae, with 8272 differential metabolites compared to controls, versus a more moderate response in R larvae 4208 metabolites. Key pathways, including galactose and glycerophospholipid metabolism, were significantly perturbed. Crucially, R larvae exhibited a profoundly enhanced antioxidant defense in the gut, with activities of catalase, glutathione S-transferase, and superoxide dismutase being 2.5-fold, 1.5-fold, and 2.0-fold higher, respectively, than in S larvae. Our findings demonstrate that CSBV resistance is linked to a modulated metabolic response and a robust, activated antioxidant system, providing new insights into host–pathogen interactions and potential strategies for safeguarding pollinator health.