Localization and Functional Analysis of the Smoothened Gene in Apis cerana cerana

Smoothened (Smo) is a core component of the Hedgehog signaling pathway and plays an important role in insect tissue growth, but its function in honey bees remains unclear. In this study, we investigated the expression localization of AcerSmo in the antennae of Apis cerana cerana (A. c. cerana) and its impact on olfactory behavior using in situ hybridization, immunohistochemistry, RNA interference, transcriptome sequencing, and behavioral and electrophysiological techniques. The results showed that AcerSmo mRNA is expressed in the sensory neurons beneath trichoid and placoid sensilla, while the protein is localized to the non-neuronal supporting cells of these sensilla, revealing a spatial separation between AcerSmo mRNA and protein localization. Although this pattern suggests cell-type-specific post-transcriptional regulation, the present data do not directly demonstrate intercellular mRNA transport or translation. RNAi-mediated silencing of AcerSmo significantly upregulated the expression of AcerOr1 and AcerOr2. Transcriptome sequencing identified 19 olfaction-related differentially expressed genes involved in signal transduction, substance transport, and various metabolic pathways. Electroantennogram and Y-tube olfactometer assays revealed that after AcerSmo interference, the responses of A. c. cerana to odorants changed significantly: EAG responses to 2-heptanone and hexyl acetate decreased, while avoidance behavior toward 2-heptanone increased; EAG responses to 1-nonanol and eugenol increased, and Y-tube tests showed enhanced attraction to 1-nonanol and increased avoidance of eugenol. Conclusions: These findings indicate that AcerSmo plays an important role in regulating olfactory receptor expression and modulating odorant responses in honey bees, thereby influencing their olfactory behavior.