Structural and Functional Basis of Host-Volatile Binding by Antennal Odorant-Binding Proteins in the Codling Moth <i>Cydia pomonella</i> (Lepidoptera: Tortricidae)

The codling moth (<i>Cydia pomonella</i>) is a major pest of fruit crops and relies on olfaction for host location and oviposition-related behaviors. Here, we prioritized two antennal-enriched odorant-binding proteins, CpomOBP9 and CpomOBP18, and characterized their roles in host-volatile recognition. Fluorescence competitive binding assays with 27 host-associated volatiles revealed ligand-biased binding profiles, with CpomOBP9 showing the highest affinity for pentyl hexanoate and CpomOBP18 preferentially binding α-farnesene. Structure-guided residue prioritization followed by site-directed mutagenesis identified key binding-pocket determinants, with Lys91 being essential for pentyl hexanoate binding to CpomOBP9 and Ile32, Leu35, Phe49, and Leu50 contributing critically to α-farnesene binding to CpomOBP18. RNA interference further supported in vivo function, as knockdown of each OBP significantly reduced Y-tube attraction and antennal electroantennogram (EAG) responses to the corresponding volatiles. Collectively, these results define a structural and functional framework for OBP-mediated host-volatile detection in <i>C. pomonella</i> and provide mechanistic insights relevant to developing and optimizing semiochemical-based monitoring and control tools.