A single point mutation causes one-way alteration of pheromone receptor function in two Heliothis species

The sex pheromone processing system of moths has been a major focus of research on olfaction and speciation, as it is highly specific and closely related to reproductive isolation. The two noctuid moths Heliothis virescens and Heliothis subflexa have been used as a model for deciphering the mechanisms underlying differentiation in pheromone communication, but no information exist regarding the functions of the pheromone receptors (PRs) of H. subflexa. Here, we functionally characterized all candidate PRs of H. subflexa, and found that only the response profile of OR6 differed between the two species. Through domain swapping and site-directed mutation followed by functional characterization, we identified a critical amino acid in OR6 caused a one-way alteration in specificity. This result suggests HsubOR6 evolved from an ancestral OR6 gene with a HvirOR6-like function and implies that the evolutionary direction of the receptor specificity was from the H. virescens-like pattern to H. subflexa-like pattern.