Identification of odorant‐binding proteins in Ceutorhynchus asper and functional analyses of CaspOBP9

BACKGROUND: The rape stem weevil (Ceutorhynchus asper) represents a significant threat to economically-important cruciferous crops. The main aim was to characterize CaspOBPs (odorant-binding proteins) and the functions of CaspOBP9. RESULTS: We performed transcriptomic sequencing on C. asper tissues and identified 21 CaspOBPs. A majority of CaspOBPs were relatively highly expressed in the antennae; for these OBPs, CaspOBP9, 12, 16, 19 and 20 were female-biased, whereas CaspOBP5, 8 and 10 were male-biased. Recombinant CaspOBP9 showed good affinity for four plant volatiles and two pesticides, suggesting that CaspOBP9 was involved in perception of both pesticides and host volatiles. Behavioral experiments showed that C. asper was significantly attracted to decanal, benzyl isothiocyanate (ITC) and phenylethyl isothiocyanate. Molecular simulations suggest that Van der Waals forces serve as the main driving force for the formation of CaspOBP9-ligand complexes. Residues LEU:71, THR:79 and TYR:105 were identified to be critical in the formation of the complexes. Site-directed mutagenesis experiments showed that mutated proteins at the above-mentioned three sites had reduced or even no binding affinity for the corresponding compounds. RNAi experiments targeting CaspOBP9 confirmed its essential roles in olfactory perception of C. asper. CONCLUSIONS: CaspOBP9 could play important roles in perception of particular plant volatiles (e.g. benzyl ITC) and pesticides (e.g. chlorpyrifos) by C. asper, and its key residues LEU:71, THR:79 and TYR:105 were identified. This research provides valuable insights into the molecular mechanisms underlying olfactory perception of C. asper, and a foundation for the development of novel control techniques for this weevil. © 2025 Society of Chemical Industry.