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

Simple SummaryThe Hedgehog signaling pathway is highly conserved across species, yet its role in insect olfaction remains poorly understood. Here, we characterized the Smoothened (Smo) gene—a core component of this pathway—in the eastern honey beeApis cerana cerana (A. c. cerana). We found thatAcerSmoexhibits a unique spatial segregation pattern: its mRNA is transcribed in sensory neurons beneath antennal trichoid and placoid sensilla, while the protein localizes to adjacent non-neuronal supporting cells, revealing a spatial separation betweenAcerSmomRNA and protein localization, suggesting cell-type-specific regulation ofAcerSmoexpression during olfactory function. RNA interference-mediated silencing ofAcerSmoupregulated the expression of key olfactory receptor genesAcerOr1andAcerOr2, and altered bees’ electrophysiological and behavioral responses to ecologically relevant odorants including 2-heptanone, 1-nonanol, and eugenol. Our findings demonstrate thatAcerSmomodulates honey bee olfactory recognition by regulating olfactory receptor expression, expanding the known physiological functions of the Hedgehog pathway beyond development into adult sensory regulation.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 ofAcerSmoin the antennae ofApis 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 thatAcerSmomRNA 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 betweenAcerSmomRNA 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 ofAcerSmosignificantly upregulated the expression ofAcerOr1andAcerOr2. 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 afterAcerSmointerference, the responses ofA. c. ceranato 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 thatAcerSmoplays an important role in regulating olfactory receptor expression and modulating odorant responses in honey bees, thereby influencing their olfactory behavior.