Molecular mechanisms based on peripheral level of vanillin recognition in Orthaga achatina (Lepidoptera: Pyralidae).

Orthaga achatina (Lepidoptera Pyralidae) is a specialist pest of the camphor tree Cinnamomum camphora. Vanillin is a volatile compound found in many plants, and its effects on insects can be either attractive or repellent, depending on the species. However, the behavioral response of Orthaga achatina to vanillin, a volatile compound emitted by camphor trees, remains unexplored. In this study, we found that vanillin attracts both male and female O. achatina adults. Fluorescence competitive binding assays further revealed that among the five odorant-binding proteins (OBPs) highly expressed in both male and female antennae, OachOBP7 exhibited the most prominent binding affinity with vanillin. Furthermore, by employing the Xenopus oocyte expression and two-electrode voltage clamp recording system (XOE-TEVC) to conduct a functional characterization of 40 ORs, vanillin was the optimal ligand for OachOR7 among all tested ligands. In addition, with the 3D structure modeling and molecular docking techniques, it was revealed that OachOR7 displayed a relatively high binding affinity (-5.5 kcal/mol), and Gln84 and Asn189 were predicted to be key amino acid residues for binding vanillin. Finally, the two amino acids were verified by site-specific mutagenesis followed by XOE-TEVC, showing that the binding ability of OR7 to vanillin was significantly reduced to 9.23 × 10 μM after the mutation of two amino acids. This study demonstrate vanillin's behavioral attraction to O. achatina and reveal its molecular basis, offering new possibilities for targeted pest management using this compound.