The Identification of Pheromone Receptors from the Lightbrown Apple Moth, Epiphyas postvittana

Abstract

The lightbrown apple moth, Epiphyas postvittana is a horticultural pest native to Australia that has spread to New Zealand, California and Europe. While modern mating disruption tactics have proven to be efficacious for controlling the pest they have not become well adopted, primarily due to health concerns related to releasing large amounts of pheromones into the environment. A better understanding of odorant and pheromone reception in the moth could facilitate the development of novel pest control tools targeting the moth’s olfactory system that could be used in place of pheromones for mating disruption. Here we undertake a bioinformatic approach to identify odorant receptors (ORs) and other genes associated with olfactory reception in E. postvittana. Of the 70 OR genes that we identified, eight are phylogenetically related to known pheromone receptors from other moths. In addition, we found two male-biased ORs that did not group with previously described pheromone receptors. The development of rapid and reliable assays to characterize insect ORs and pheromone receptors (PRs) remains a challenge for the field. Typically insect ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. We have developed a Human Embryonic Kidney (HEK) cell-based assay system for the expression and functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Here, we express candidate EposPRs in HEK293 cells along with EposOrco and EposSNMP1 and test for responsiveness to a panel of 62 pheromone-related compounds. We found that EposOR1, EposOR6 and EposOR45 all respond to certain E. postvittana pheromone components and to pheromones used by other moths, some of which have known function as behavioral antagonists in this species. Finally, we show that activation of EposOR6 with the major pheromone component, E11-14:OAc, or the behavioral antagonist, Z11-14:OAc, prevents subsequent re-activation by either compound in this cell-based system, implying a pheromonedegrading enzyme may be required to reset the system. These results lead us to propose that in E. postvittana behavioral agonism and antagonism may be mediated through some sort of peripheral molecular mechanism.