Pheromone receptors for japonilure in Anomala corpulenta and Popillia japonica.

Using gas chromatography-electroantennographic detection (GC-EAD), gas chromatography-mass spectrometry (GC-MS), and chiral capillary GC with synthetic stereoisomers, followed by laboratory and field validation, we identified the sex pheromone of the copper green chafer (Anomala corpulenta) as (R)-japonilure, with its antipode, (S)-japonilure, acting as a behavioral antagonist. Single sensillum recordings (SSR) revealed two types of pheromone-responsive sensilla, one narrowly tuned to the sex pheromone, and another that responded to both (R)- and (S)-japonilure, albeit with lower sensitivity to the latter. Functional characterization of A. corpulenta odorant receptors (ORs) by two-electrode voltage clamp (TEVC) led to the identification of two key receptors, AcorOR18, which was selective for (R)-japonilure, and AcorOR29, which was activated by both enantiomers, and 10 ORs sensitive to plant-derived compounds. SSR combined with fluorescence in situ hybridization (FISH) experiments suggested that AcorOR18 is expressed in pheromone-specific sensilla, whereas AcorOR29 is localized in sensilla responsive to both the pheromone and its antagonist. RNA interference (RNAi) knockdown of either receptor reduced male attraction to (R)-japonilure, while simultaneous knockdown of both genes abolished behavioral responses, confirming that both receptors are required for full pheromone detection. Three orthologous receptors were identified in the Japanese beetle (Popillia japonica) genome. Two of them, PjapOR42 and PjapOR37, exhibited strict enantioselectivity for (R)-japonilure, whereas PjapOR39 showed a relaxed enantiospecificity, responding to both enantiomers and preferentially to the behavioral antagonist. These results suggest that PjapOR39 may mediate detection of (S)-japonilure and contribute to behavioral antagonism, revealing a shared molecular mechanism of enantiomeric discrimination in scarab beetles.