Pheromone receptor evolution in the cryptic leafroller species, Ctenopseustis obliquana and C. herana.

How new mate recognition systems evolve when changes are required in both the male and female components remains a conundrum. Here, we investigated the molecular basis of pheromone reception in two species of tortricid (leafroller) moth, Ctenopseustis obliquana and C. herana. Male C. obliquana are attracted to a 90:10 blend of (Z)-8-tetradecenyl acetate (Z8-14:OAc) and (Z)-5-tetradecenyl acetate (Z5-14:OAc), whereas C. herana males are attracted to Z5-14:OAc alone. We used a transcriptome sequencing approach from adult male and female antennae to identify 47 olfactory receptors (ORs) from each species and assessed their expression levels in male and female antennae using RNA-Seq counting and quantitative RT-PCR. Three male-biased and one female-biased OR were identified in C. obliquana by quantitative RT-PCR, and four male-biased and one female-biased receptor in C. herana. The male-biased receptors, CoblOR7, CoblOR30, CherOR7, CherOR30, CherOR1a and CherOR1b were tested for their ability to respond to sex pheromone components in a HEK293 cell calcium assay. CoblOR7 and CherOR7 responded to Z8-14:OAc, however, no receptor for Z5-14:OAc was identified. In addition to Z8-14:OAc, CherOR7 also responded to Z7-14:OAc, indicating that this receptor may be under relaxed constraint. Of the 29 amino acid differences between CoblOR7 and CherOR7, significantly more are located in the third and the sixth transmembrane domain regions. Overall, these findings are consistent with studies revealing the presence of neurons tuned to both Z8-14:OAc and Z5-14:OAc in both species, but that for C. herana males, the ability to detect Z8-14:OAc is currently not required.