Sensory responsiveness and transcriptomic signatures of water foragers reveal multilayered regulation of division of labor in Apis cerana cerana

Division of labor defines colony organization in eusocial insects. However, behavioral differences among forager types, especially water foragers, and their molecular regulatory mechanisms remain poorly understood. Using Apis cerana cerana as the study organism, we integrated proboscis extension reflex (PER) and gustatory response score (GRS) behavioral assays, reverse transcription quantitative polymerase chain reaction (RT-qPCR), transcriptome sequencing, weighted gene co-expression network analysis (WGCNA), and gene set enrichment analysis to compare behavioral characteristics and gene expression profiles among guard bees, pollen foragers, nectar foragers, and water foragers. Behavioral analyses showed that water foragers exhibited significantly higher GRS than nectar foragers (P < 0.05), supporting the response threshold model in water-foraging behavior. RT-qPCR analysis showed that expression of Acfor (PKG) and AcOct (octopamine receptor β2R) was significantly higher in guard bees than in all forager types, whereas AcInR (insulin receptor) expression was lowest in water foragers. AcOct expression showed a significant negative correlation with GRS in nectar foragers (r = −0.554, P = 0.049) and water foragers (r = −0.606, P = 0.028), indicating that octopamine receptor signaling regulates division of labor through modulation of sensory thresholds. Transcriptomic analysis showed that genes upregulated in all forager types relative to guard bees were enriched in oxidative phosphorylation, including mitochondrial NADH dehydrogenase subunits ND1, ND4, and ND5, indicating enhanced flight energy metabolism as a shared feature. Among water-forager-specific upregulated genes, functional candidates included an RYamide receptor, cilia-associated proteins, and M7BP. These findings suggest that neuropeptide signaling drives water-foraging motivation, chemosensory structures are enhanced, and suppression of the AcInR-M7BP axis supports specialization. WGCNA identified a positive correlation trend between the MEturquoise module and the water-forager phenotype (r = 0.53, P = 0.0763). Together, these findings reveal multilayered regulatory mechanisms underlying division of labor in A. cerana cerana water foragers and provide experimental evidence for the molecular basis of division of labor in eusocial insects.