Functional characterization of CaspOR20 involved in repellency of 1-penten-3-ol, sulcatone, and methyl salicylate against the rape stem weevil Ceutorhynchus asper.

The rape stem weevil (Ceutorhynchus asper) is a devastating pest of cruciferous crops, and its management heavily relies on insecticides, leading to resistance development and environmental concerns. CaspOR20 is a highly expressed odorant receptor in the antennae of C. asper, yet its functional role remains unclear. In this study, by using the transgenic Drosophila expression system combined with single sensillum recordings, we identified six volatile compounds (1-penten-3-ol, 2-heptanone, sulcatone, methyl salicylate, benzaldehyde, and ethyl acetate) as ligands for CaspOR20. Subsequent electroantennography (EAG) assays revealed that all of these ligands except ethyl acetate elicited significant electrophysiological responses in both male and female C. asper adults. Behavioral assays using an H-tube olfactometer demonstrated that three compounds (1-penten-3-ol, sulcatone, and methyl salicylate) exhibited significant repellent effects on both male and female C. asper. Molecular docking analysis indicated strong binding affinity between CaspOR20 and the three repellent compounds, with key residues (CYS-129, ALA-176, and ASN-179) involved in hydrogen bond interactions. Functional validation via RNA interference targeting CaspOR20 in vivo indicated that CaspOR20-silenced individuals displayed significantly reduced EAG responses to the repellent compounds and lost their behavioral avoidance. This study successfully identified repellent compounds against C. asper, and provided insights into the molecular mechanism underlying C. asper's detection of these compounds. Our findings established a solid foundation for the future development of effective repellents and eco-friendly control strategies against this pest.