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.

Food wanting in honeybees is closely associated with the neurotransmitter dopamine; however, the regulatory role of non-coding RNAs in this process remains unclear. In this study, using the honeybee (Apis mellifera) as a model organism, we systematically investigated the molecular network and functional mechanisms by which long non-coding RNAs (lncRNAs) regulate the dopaminergic signaling pathway to mediate food wanting. By establishing two appetite-state models, fed honeybees (FB) and starved honeybees (SB), and combining brain dopamine quantification with behavioral assays, we found that dopamine levels in the honeybee brain were significantly elevated during starvation. Using transcriptome sequencing, we identified 1146 lncRNAs in the honeybee brain, among which 174 were differentially expressed long noncoding RNAs (DElncRNAs) between the two states, predominantly upregulated. Cis- and trans-acting analyses revealed that these DElncRNAs could target multiple genes involved in neural signal transmission, synaptic function, and dopaminergic pathways. KEGG enrichment analysis showed that their target genes were significantly enriched in pathways such as taste transduction, dopaminergic synapse, and neuroactive ligand–receptor interaction. Furthermore, a ceRNA network revealed that several DElncRNAs may regulate dopamine synthesis genes, including DOPA decarboxylase (Ddc), by competing for dopamine-associated miRNAs such as miR-375-3p, influencing food wanting in honeybees. Overall, our findings provide a foundation for uncovering the potential regulatory mechanisms of DElncRNAs in honeybee food wanting and offer new insights into the connection between neural regulation and behavioral manifestation in insects.

Varroa destructor is a major ectoparasitic threat to honeybee (Apis mellifera) colonies, compromising individual and colony health by transmitting viral pathogens and suppressing the host immune system. Grooming behavior is a critical defense mechanism against Varroa destructor infestation in honeybees. However, the molecular regulation underlying this behavior remains poorly understood. This study identifies differentially expressed small RNAs associated with self-grooming intensity in weak and strong groomers. Specifically, we demonstrate that miR-281-x negatively regulates octopamine production by directly targeting the tyrosine decarboxylase 2 (Tdc2) gene, modulating the intensity of self-grooming behavior. Knockdown of miR-281-x in weak groomers (MW) and overexpression in strong groomers (MS) alter grooming intensity. RNA interference of Tdc2 in MS bees reduces octopamine levels, which decreases grooming. Pharmacological modulation of octopamine signaling in MW bees increases the time to first grooming response and grooming frequency. We identify a miR-281-x-Tdc2-octopamine pathway regulating self-grooming in A. mellifera, showing that miRNAs play a key role in octopamine signaling and offering new insights into the molecular mechanisms of grooming against Varroa destructor.

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.