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Pheromone-binding proteins (PBPs) play important roles in perception of insect sex pheromones, functioning to recognize and transport pheromone components onto the olfactory receptors of the odorant sensing neurons. Orthaga achatina, a serious pest of camphor trees, uses a mixture of three Type I (Z11-16:OAc, Z11-16:OH and Z11-16:Ald) and one Type II (Z3,Z6,Z9,Z12,Z15-23:H) sex pheromone components in its sex communication, in which Z11-16:OAc is the major component and others are minor components. In this study, we for the first time demonstrated that the three PBPs differentiated in recognition among pheromone components in a moth using mixed-type sex pheromones. First, tissue expression study showed that all three PBPs of O. achatina were expressed only in antennae and highly male-biased, suggesting their involvement in perception of the sex pheromones. Second, the three PBPs were expressed in Escherichia coli and the binding affinities of PBPs to four sex pheromone components and some pheromone analogs were determined by the fluorescence competition binding assays. The results showed that OachPBP1 bound all four sex pheromone components with high binding affinity, while OachPBP2 had high or moderate binding affinity only to three Type I components, and OachPBP3 had high binding affinity only to three minor pheromone components. Furthermore, key amino acid residues that bind to sex pheromone components were identified in three PBPs by 3-D structure modeling and ligand molecular docking, predicting the interactions between PBPs and pheromone components. Our study provides a fundamental insight into the olfactory mechanism in moths that use mixed-type sex pheromones.

Summary Glucosinolates (GSs) are sulfur-containing secondary metabolites characteristic of cruciferous plants [ 1 , 2 ]. Their breakdown products, isothiocyanates (ITCs), are released following tissue disruption by insect feeding or other mechanical damages [ 3 , 4 ]. ITCs repel and are toxic to generalist herbivores, while specialist herbivores utilize the volatile ITCs as key signals for localizing host plants [ 5 , 6 ]. However, the molecular mechanisms underlying detection of ITCs remain open. Here, we report that in the diamondback moth Plutella xylostella, a crucifer specialist, ITCs indeed drive the host preference for Arabidopsis thaliana, and the two olfactory receptors Or35 and Or49 are essential for this behavior. By performing gene expression analyses, we identified 12 (out of 59 in total) female-biased Ors, suggesting their possible involvement in oviposition choice. By ectopically expressing these Ors in Xenopus oocytes and screening their responses with 49 odors (including 13 ITCs, 25 general plant volatiles, and 11 sex pheromone components), we found that Or35 and Or49 responded specifically to three ITCs (iberverin, 4-pentenyl ITC, and phenylethyl ITC). The same ITCs also exhibited highest activity in electroantennogram recordings with female antennae and were the strongest oviposition stimulants. Knocking out either Or35 or Or49 via CRISPR-Cas9 resulted in a reduced oviposition preference for the ITCs, while double Or knockout females lost their ITC preference completely and were unable to choose between wild-type A. thaliana and a conspecific ITC knockout plant. We hence conclude that the ITC-based oviposition preference of the diamondback moth for its host A. thaliana is governed by the cooperation of two highly specific olfactory receptors.