Transcriptomic analysis combined with RNAi reveal defense strategies and molecular mechanisms in Western and Eastern honeybees against attacks by the yellow-legged hornet

Attack and defense are critical to animal survival, yet the defensive strategies of Western honeybees (Apis mellifera) and Eastern honeybees (Apis cerana) against the yellow-legged hornet (Vespa velutina), as well as the molecular basis for their differences, remain poorly understood. This study combined behavioral, pharmacological, transcriptomic, and RNAi approaches to compare the defensive responses in both bee species. We found that V. velutina preferentially attacks A. mellifera and less frequently target A. cerana colony, reflecting distinct opportunistic predation strategies. Behaviorally, A. cerana initiated defense earlier and displayed a higher level of alertness, whereas A. mellifera reacted at closer ranges. Pharmacological interventions demonstrated that octopamine (OA) significantly modulated defensive responses in both species. Critically, transcriptomic analysis identified a key molecular divergence: under hornet threat, A. mellifera exhibited upregulated expression of the Octβ2R octopamine receptor, while A. cerana did not. RNA interference (RNAi) mediated suppression of tyramine beta-hydroxylase (Tbh), a key OA synthesis gene, significantly reduced defensive behavior in both species, confirming the important role of this pathway. Furthermore, we revealed that A. mellifera defense involved activation of energy metabolism pathways such as the TCA cycle and oxidative phosphorylation. In contrast, A. cerana employed broader metabolic regulation, including unsaturated fatty acid biosynthesis. The findings not only provide new insights into the evolution of honeybee defensive behaviors but also offer important references for exploring the relationship between gene regulation and behavioral evolution in social insects