Sex pheromone receptor specificity in the European corn borer moth, Ostrinia nubilalis

Abstract

Background: The European corn borer (ECB), Ostrinia nubilalis (Hubner), exists as two separate sex pheromone races. ECB(Z) females produce a 97ratio3 blend of Z11- and E11-tetradecenyl acetate whereas ECB(E) females produce an opposite 1ratio99 ratio of the Z and E isomers. Males of each race respond specifically to their conspecific female's blend. A closely related species, the Asian corn borer (ACB), O. furnacalis, uses a 3ratio2 blend of Z12- and E12-tetradecenyl acetate, and is believed to have evolved from an ECB-like ancestor. To further knowledge of the molecular mechanisms of pheromone detection and its evolution among closely related species we identified and characterized sex pheromone receptors from ECB(Z).

Methodology: Homology-dependent (degenerate PCR primers designed to conserved amino acid motifs) and homology-independent (pyrophosphate sequencing of antennal cDNA) approaches were used to identify candidate sex pheromone transcripts. Expression in male and female antennae was assayed by quantitative real-time PCR. Two-electrode voltage clamp electrophysiology was used to functionally characterize candidate receptors expressed in Xenopus oocytes.

Conclusion: We characterized five sex pheromone receptors, OnOrs1 and 3-6. Their transcripts were 14-100 times more abundant in male compared to female antennae. OnOr6 was highly selective for Z11-tetradecenyl acetate (EC(50) = 0.86+/-0.27 microM) and was at least three orders of magnitude less responsive to E11-tetradecenyl acetate. Surprisingly, OnOr1, 3 and 5 responded to all four pheromones tested (Z11- and E11-tetradecenyl acetate, and Z12- and E12-tetradecenyl acetate) and to Z9-tetradecenyl acetate, a behavioral antagonist. OnOr1 was selective for E12-tetradecenyl acetate based on an efficacy that was at least 5-fold greater compared to the other four components. This combination of specifically- and broadly-responsive pheromone receptors corresponds to published results of sensory neuron activity in vivo. Receptors broadly-responsive to a class of pheromone components may provide a mechanism for variation in the male moth response that enables population level shifts in pheromone blend use.

Figure 1. Male-biased expression of five ECB(Z) sex pheromone receptor genes.

Ratios of male to female expression (M:F) are presented below each bar. Gene expression, determined by real-time quantitative PCR with SYBR green, is reported relative to the reference gene OnRPS3 on a logarithmic scale. Expression values are presented as averages (with standard error bars) of four biological replicates and three nested technical replicates. Sex-biased expression is supported by nested ANOVA analyses of the normalized CT values, P = 0.03, 0.04, 0.001, 0.06, 0.001 and 0.003, OnOrs1-6 respectively.

Figure 2. Functional screen of candidate ECB(Z) pheromone receptors.

Oocytes expressing OnOr2 and either OnOr1 (A), OnOr3 (B), OnOr4 (C), OnOr5, (D) or OnOr6 (E) were challenged with 20 sec applications (arrowheads) of various ECB and ACB pheromones (at 10 µM): Z9–14:OAc (Z9), Z12–14:OAc (Z12), E12–14:OAc (E12), Z11–14:OAc (Z11), and E11–14:OAc (E11). Each application was separated by 10 min washing in ND96 (4.6 ml/min). Pheromone-induced currents were measure by two-electrode voltage clamp electrophysiology.

Figure 3. Dose-response relationships for Z11-14:OAc and E11-14:OAc activation of OnOr6/2.

Pheromone-induced currents were measure by two-electrode voltage clamp electrophysiology. Refer to Table 1 for EC₅₀, Hill slope, and relative efficacy values. Data is presented as means ± SEM (Z11–14:OAc, n = 4; E11–14:OAc, n = 5).

Figure 4. Dose-response relationships for E12–14:OAc, Z12–14:OAc, Z11–14:OAc, E11–14:OAc and Z9–14:OAc activation of OnOr1/2.

Left and right graphs have different y-axis scales of the same data points. Pheromone-induced currents were measure by two-electrode voltage clamp electrophysiology. Refer to Table 1 for EC₅₀, Hill slope, and relative efficacy values. Data is presented as means ± SEM (E12–14:OAc, n = 5; Z12–14:OAc, n = 6; Z11–14:OAc, n = 7; E11–14:OAc, n = 5; and Z9–14:OAc, n = 5).

Figure 5. Phylogenetic relatedness of OnOrs1-6 to the Lepidoptera sex pheromone receptor subfamily, neighbor-joining (corrected distance) tree.

Bootstrap values are presented as a percentage of n = 1000 replicates at significant branch points. The tree is rooted with lepidopteran orthologs of DmOr83b. The responses of receptors that have been functionally characterized are indicated by numbers corresponding to the 15 pheromone compounds listed, bolded numbers indicate the strongest response. Bm, Bombyx mori; Di, Diaphania indica; Ep, Epiphyas postvittana; Hv; Heliothis virescens; Msex, Manduca sexta; On, Ostrinia nubilalis; Px, Plutella xylostella; Msep, Mythimna separata. Superfamily taxonomies are delineated by vertical bars. ECB receptors reported in this study are bolded; OnOr1* was reported in and is identical to OnOr5 in this study. Pheromone ligands: 1) E11–14:OH; E11-tetradecen-1-ol, 2) Z9–14:Al; Z9-tetradecenal, 3) Z9–14:OAc; Z9-tetradecenyl acetate, 4) Z11–14:OAc; Z11-tetradecenyl acetate, 5) E11–14:OAc; E11-tetradecenyl acetate, 6) Z12–14:OAc; Z12-tetradecenyl acetate, 7) E12–14:OAc; E12-tetradecenyl acetate, 8) Z11–16:OH; Z11-hexadecen-1-ol, 9) Z9–16:Al; Z9-hexadecenal, 10) Z11–16:Al; Z11-hexadecenal,11) E11–16:Al; E11-hexadecenal, 12) Z11–16:OAc; Z11-hexadecenyl acetate, 13) E11–16:OAc; E11-hexadecenyl acetate, 14) E10, Z12–16:OH; E10,Z12-hexadecadien-1-ol, and 15) E10, Z12–16:Al; E10,Z12-hexadecadienal.