Amfor-Mediated cGMP-PKG Signaling and Transcriptomic Divergence Underlying Division of Labor in Apis mellifera...

Traits such as defense and foraging in social insects depend on the coordinated division of labor (DOL) among workers. However, several aspects of the molecular mechanisms driving behavioral specialization for these tasks remain incompletely characterized. In this study, we examined two forms of DOL in the Western honeybee (Apis mellifera ligustica): foraging (nectar, pollen, and water collection) and defense (guard bees). Using proboscis extension response (PER) assays, gustatory response score (GRS), quantitative PCR, enzyme-linked immunosorbent assay, and transcriptome RNA sequencing of brain tissue, we characterized the behavioral and molecular differences among four task groups. Water foragers showed the highest PER values, gustatory response scores, Amfor expression, and PKG activity, while guard bees showed the lowest PKG activity. Transcriptome analysis identified up to 418 differentially expressed genes (DEGs) between forager subtypes and guard bees. DEGs in water foragers were associated with body surface morphology and water transport, those in pollen and nectar foragers with cGMP synthesis, and those in guard bees with retinol metabolism and olfaction. KEGG enrichment analysis of DEGs from guard-vs-forager pairwise comparisons identified the cGMP-PKG signaling pathway as significantly enriched in both foraging-associated and defense-associated DEG lists, indicating that this pathway serves as a shared outside-hive behavioral enabler rather than a foraging-specific switch. WGCNA further revealed that Nos, Camkii, and Amfor-encoded PKG show correlated expression patterns within the same co-expression module, suggesting that a broader calcium-NO-cGMP signaling network, rather than Amfor alone, may constitute the functional molecular unit underlying task-specific behavior. These findings provide a transcriptomic framework for understanding how the cGMP-PKG pathway and its associated network regulate behavioral DOL in social insects.