A tale of two copies: Evolutionary trajectories of moth pheromone receptors

Abstract

Pheromone communication is an essential component of reproductive isolation in animals. As such, evolution of pheromone signaling can be linked to speciation. For example, the evolution of sex pheromones is thought to have played a major role in the diversification of moths. In the crop pests Spodoptera littoralis and S. litura, the major component of the sex pheromone blend is (Z,E)-9,11-tetradecadienyl acetate, which is lacking in other Spodoptera species. It indicates that a major shift occurred in their common ancestor. It has been shown recently in S. littoralis that this compound is detected with high specificity by an atypical pheromone receptor, named SlitOR5. Here, we studied its evolutionary history through functional characterization of receptors from different Spodoptera species. SlitOR5 orthologs in S. exigua and S. frugiperda exhibited a broad tuning to several pheromone compounds. We evidenced a duplication of OR5 in a common ancestor of S. littoralis and S. litura and found that in these two species, one duplicate is also broadly tuned while the other is specific to (Z,E)-9,11-tetradecadienyl acetate. By using ancestral gene resurrection, we confirmed that this narrow tuning evolved only in one of the two copies issued from the OR5 duplication. Finally, we identified eight amino acid positions in the binding pocket of these receptors whose evolution has been responsible for narrowing the response spectrum to a single ligand. The evolution of OR5 is a clear case of subfunctionalization that could have had a determinant impact in the speciation process in Spodoptera species.

Keywords: ancestral gene resurrection; evolution; pheromone receptor.

Fig. 1.

Or5 orthologs and paralogs in four Spodoptera species. (A) Evolution of the number of Or genes (blue boxes) in the genus Spodoptera as determined by the phylogenetic analysis (SI Appendix, Fig. S1). Gene gains are shown in green and losses in red. Phylogenetic relationships and divergence times are from ref. . (B) Synteny of Or5 orthologs and paralogs in the four species. Exons are shown with dark colors, introns with light colors. Arrows indicate the 5′ end of the ORF and the direction of transcription. (C) Phylogeny of ORs encoded by genes shown in B. The scale bar shows the expected number of amino acid substitutions per site. (D) Identity matrix between amino acid sequences encoded by Or5 and Or75 genes. (E) Expression level of Or5 and Or75 genes in olfactory organs of the four species as determined by RT-qPCR. Reference genes were ATPs for S. exigua, Actin for S. frugiperda, and Rpl13 for S. litura and S. littoralis.

Fig. 2.

Tuning breadth of OR5 orthologs and paralogs in Spodoptera species. (A) Action potential frequency recorded in Drosophila at1 OSNs expressing Or5 and Or75 genes of the four species after stimulation with 26 sex pheromone compounds (10 μg loaded in the stimulus cartridge). Boxes show the first and third quartiles, whispers show the distribution, and dots show outliers. Dark purple shows responses significantly different from the response to solvent (P < 0.05, one-way ANOVA followed by a Dunnett’s post-hoc test). (B) Dose–response curves of the four OR5 orthologs. For SexiOR5 and SfruOR5, four compounds were tested. For SlituOR5 and SlitOR5, which were specific to (Z,E)-9,11-14:OAc, only this compound was tested.

Fig. 3.

Tuning breadth of resurrected ancestral ORs before and after the Or5 duplication in the ancestor of S. littoralis and S. litura. (A) Phylogeny of OR5 orthologs and paralogs (purple). The ancestral sequences that were resurrected are indicated on the nodes (blue). (B) Amino acid sequence identity matrix between the three ancestral and the six extant ORs. (C) Action potential frequency recorded in Drosophila at1 OSNs expressing Or5/Or75 ancestors after stimulation with 26 sex pheromone compounds (10 μg loaded in the stimulus cartridge). Boxes show the first and third quartiles, whispers show the distribution, and dots show outliers. Dark blue shows responses significantly different from the response to solvent (P < 0.05, one-way ANOVA followed by a Dunnett’s post-hoc test). (D) Dose–response curves of the three ancestral ORs. (E) Heatmap summarizing the tuning breadth of OSNs expressing ancestral (blue) and extant Or5/Or75 genes (purple). Receptors were clustered using Manhattan distances (dendrogram on the left).

Fig. 4.

Structural basis for the evolution of OR5 tuning breadth. (A) 3D structure of AncOR5_75 as predicted by AlphaFold2. The ligand (Z,E)-9,11-14:OAc is shown in yellow. The 28 amino acid positions predicted to interact with the ligand (distance <5 Å) are shown in blue. (B) Binding pockets of AncOR5_75 and AncOR5 showing the eight amino acids differing between the two ancestors. S2, 3, 4, and 5: transmembrane helices; ECL2: extracellular loop 2. (C) Inward current measured in Xenopus oocytes coexpressing AncOR5, AncOR5_75, or AncOR5_75_mut8x and SlitOrco after stimulation with a panel of six components from the S. littoralis sex pheromone (10⁻⁵ M solution). Responses to the negative control (1× Ringer’s buffer containing 0.01% DMSO) were 0. (D) Principal component analysis based on the responses of AncOR5, AncOR5_75, and its related mutants. (E) Dose–response curves of AncOR5, AncOR5_75, and AncOR5_75_mut8x expressed in Xenopus oocytes.