Abstract:
How new mate recognition systems evolve when changes are required in both the male and female components remains a conundrum. Here we investigate the ultrastructure of male and female antennae and the molecular basis of pheromone reception in two pairs of closely-related species of tortricid (leafroller) moth, Ctenopseustis obliquana and C. herana as well as Planotortrix octo and P. excessana. We identified six types of sensilla on the ventral surface of their antennae using scanning electron microscopy: S. trichodea (type I, type II, type III), s. basiconica, s. auricillia, s. chaetica, s. coeloconica and s. styloconica. No major differences in gross antennal morphology or number and type of sensilla were found among the four species. Sexual dimorphism was observed, with sensilla trichodea (type I) only found in males. This finding is consistent with a role for these sensilla in detecting the female sex pheromone reported in other moth. Using a transcriptome sequencing approach from adult male and female antennae, we identified 47 olfactory receptors (ORs) from each C. obliquana, C. herana and P. excessana and 48 ORs from P. octo and assessed their expression levels in male and female antennae using RNA seq-counting and quantitative RT-PCR (qPCR). By using qPCR we identified three male biased and one female biased OR in C. obliquana, four male biased and one female biased OR in C. herana, two male biased and one female biased OR in P. octo and two male biased and one female biased OR in P. excessana. Two ORs were male biased in all four species, OR7 and OR30. Candidate pheromone receptors were tested for their ability to respond to sex pheromone components in a HEK293 cell calcium assay. CoblOR7 and CherOR7 responded to (Z)-8-tetradecenyl acetate (Z8-14:OAc), a pheromone component produced by C. obliquana females. CherOR7 has a reduced sensitivity to Z8-14:OAc and unlike CoblOR7, also responds (Z)-7-tetradecenyl acetate (Z7-14:OAc), which is not a pheromone component in either of the two species, indicating that CherOR7 may be under relaxed constraint compared with CoblOR7. Significantly higher sequence differences were found in the third and the sixth transmembrane domain region of CoblOR7. PAML analysis conducted on orthologous ORs of all four species indicate positive selection acting on the male biased OR7 and another OR, OR64. The fact that OR7 is likely under positive selection, that it is male biased in its expression and that its orthologue in C. obliquana, CoblOR7, responded to sex pheromone components is suggestive of this receptor being important in sex pheromone reception in Planotortrix species also.