Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species

Abstract

Sex pheromone communication, acting as a prezygotic barrier to mating, is believed to have contributed to the speciation of moths and butterflies in the order Lepidoptera. Five decades after the discovery of the first moth sex pheromone, little is known about the molecular mechanisms that underlie the evolution of pheromone communication between closely related species. Although Asian and European corn borers (ACB and ECB) can be interbred in the laboratory, they are behaviorally isolated from mating naturally by their responses to subtly different sex pheromone isomers, (E)-12- and (Z)-12-tetradecenyl acetate and (E)-11- and (Z)-11-tetradecenyl acetate (ACB: E12, Z12; ECB; E11, Z11). Male moth olfactory systems respond specifically to the pheromone blend produced by their conspecific females. In vitro, ECB(Z) odorant receptor 3 (OR3), a sex pheromone receptor expressed in male antennae, responds strongly to E11 but also generally to the Z11, E12, and Z12 pheromones. In contrast, we show that ACB OR3, a gene that has been subjected to positive selection (ω = 2.9), responds preferentially to the ACB E12 and Z12 pheromones. In Ostrinia species the amino acid residue corresponding to position 148 in transmembrane domain 3 of OR3 is alanine (A), except for ACB OR3 that has a threonine (T) in this position. Mutation of this residue from A to T alters the pheromone recognition pattern by selectively reducing the E11 response ∼14-fold. These results suggest that discrete mutations that narrow the specificity of more broadly responsive sex pheromone receptors may provide a mechanism that contributes to speciation.

Fig. 1.

Evidence of positive selection acting on the coding sequence of ACB OR3. A total of 54 nucleotide sequences representing eight species and five OR gene lineages were analyzed for evidence of selection. Bootstrap values for major branches are shown as a percentage of n = 1,000 replications. The normalized nonsynonymous to synonymous substitution rate (ω) is shown for significant groupings. ORs 1, 4, and 5 have one uniform ω for all branches in the gene lineage, whereas ORs 3 and 6 have two or more rates (ω) for branches within the gene lineage. ω > 1 was observed only for ACB OR3, suggesting that positive selection has acted on this branch. OR nomenclature follows their original publication (13, 14). O. furnacalis (ACB), O. latipennis, O. ovalipennis, O. nubilalis [ECB(E)] and [ECB(Z)], O. palustralis, O. scapulalis, O. zaguliaevi, and O. zealis.

Fig. 2.

ACB OR3 responds preferentially to the (E)-12- and (Z)-12-tetradecenyl acetate pheromones produced by ACB females. (A and B) Representative traces show membrane currents in Xenopus oocytes coexpressing OR3 receptors from ECB(Z) (A) and ACB (B) with the obligate partner Orco. A saturating dose of E11 or E12 (1 μM) was applied at the end of each experiment for ECB(Z) OR3 or for ACB OR3, respectively, to normalize the response of each pheromone. (C and D) Concentration-dependence of ECB(Z) OR3 (C) and ACB OR3 (D) responses to E11 (diamonds), Z11 (triangles), Z12 (circles), and E12 (squares) pheromones. The responses were normalized to saturating (1 μM) concentrations of E11 [ECB(Z)] or E12 (ACB) pheromones applied at the end of the experiment.

Fig. 3.

Identity of amino acid residue 148 of ACB and ECB(Z) OR3 determines differential affinity for E11. (A) Representative traces show membrane currents in oocytes coexpressing ACB OR3, ECB(Z) OR3, and ECB(Z) OR3^A148T with Orco, in respose to 10-nM doses of E11, Z11, E12, and Z12. (B) Concentration-dependence of ECB(Z) OR3 (filled squares), ACB OR3 (filled circles), ECB(Z) OR3^A148T (open squares), and ACB OR3^T148A (open circles) receptors to E11. The responses were normalized to a saturating 1-μM concentration of E11 applied at the end of the experiment.

Fig. 4.

Ostrinia OR3 sequences showing the unique threonine (T) for alanine (A) substitution at position 148 within TMD3 of ACB OR3. (A) A TOPCONS (topcons.net) model of ACB OR3 with position 148 illustrated by a black circle. The residues included in the alignment in Fig. 4B are highlighted in gray. (B) Clustal alignment of the predicted TMD3 of OR3 from eight Ostrinia species. O. furnacalis (ACB), O. latipennis, O. ovalipennis, O. nubilalis [ECB(E) and ECB(Z)], O. palustralis, O. scapulalis, O. zaguliaevi, and O. zealis.