Involvement of the G-protein-coupled dopamine/ecdysteroid receptor DopEcR in the behavioral response to sex pheromone in an insect

Abstract

Most animals including insects rely on olfaction to find their mating partners. In moths, males are attracted by female-produced sex pheromones inducing stereotyped sexual behavior. The behaviorally relevant olfactory information is processed in the primary olfactory centre, the antennal lobe (AL). Evidence is now accumulating that modulation of sex-linked behavioral output occurs through neuronal plasticity via the action of hormones and/or catecholamines. A G-protein-coupled receptor (GPCR) binding to 20-hydroxyecdysone, the main insect steroid hormone, and dopamine, has been identified in Drosophila (DmDopEcR), and was suggested to modulate neuronal signaling. In the male moth Agrotis ipsilon, the behavioral and central nervous responses to pheromone are age-dependent. To further unveil the mechanisms of this olfactory plasticity, we searched for DopEcR and tested its potential role in the behavioral response to sex pheromone in A. ipsilon males. Our results show that A. ipsilon DopEcR (named AipsDopEcR) is predominantly expressed in the nervous system. The corresponding protein was detected immunohistochemically in the ALs and higher brain centers including the mushroom bodies. Moreover, AipsDopEcR expression increased with age. Using a strategy of RNA interference, we also show that silencing of AipsDopEcR inhibited the behavioral response to sex pheromone in wind tunnel experiments. Altogether our results indicate that this GPCR is involved in the expression of sexual behavior in the male moth, probably by modulating the central nervous processing of sex pheromone through the action of one or both of its ligands.

Figure 1. AipsDopEcR gene expression and protein detection in the brain of A. ipsilon males.

A) Tissue-related AipsDopEcR gene expression. AipsDopEcR was detected mainly in the brain. B–D) 3D projections of confocal optical sections showing immunocytochemical AipsDopEcR protein detection in brains of 5-day-old males. B) Frontal view of the central brain parts. C) Protocerebrum with mushroom body calyx. D) Antennal lobe. Insets show manual reconstructions of labeled cell bodies in the respective series of sections. (MBC) mushroom body calyces; (CC) central complex; (AL) antennal lobes.

Figure 2. Age-related AipsDopEcR transcriptional activity and protein level from brains of 1- to 5-day-old A. ipsilon males.

A) Transcriptional activity of AipsDopEcR. The ribosomal gene RpL8 was used as reference. B) Relative protein expression of AipsDopEcR based on Western blot analysis as shown in (C). C) Protein expression of AipsDopEcR. The control used was the rabbit α-Tubulin protein. AipsDopEcR transcript and protein expression increase with age. Bars represent means ± s.e.m of 6 biological repetitions. Bars with same letters are not significantly different (ANOVA; Tukey test; α = 0.05).

Figure 3. Efficiency of double-stranded RNA-mediated AipsDopEcR silencing in A. ipsilon males.

1-day-old sexually immature males received an injection of saline solution, bacterial LacZ-dsRNA or AipsDopEcR-dsRNA, or no injection (non-injected). For each treatment, the AipsDopEcR mRNA and protein amounts in the brain were evaluated 4 days after the injection by real time qPCR and Western blot respectively. A) Transcriptional activity of AipsDopEcR. The ribosomal gene RpL8 was used as reference. B) Relative protein expression of AipsDopEcR based on Western blot analysis as shown in (C). C) Protein expression of AipsDopEcR. The control used was the rabbit α-Tubulin protein. Bars represent means ± s.e.m of 3 biological repetitions. Bars with same letters are not significantly different (ANOVA; Tukey test; α = 0.05).

Figure 4. Effect of AipsDopEcR silencing by injection of dsRNA on the oriented responses to the sex pheromone (A) and on general flight activity (B) in A. ipsilon males.

1-day-old sexually immature males were injected with saline solution, bacterial LacZ-dsRNA or AipsDopEcR-dsRNA, or received no injection (non-injected). For each treatment, the percentage of oriented or random flight activity was evaluated 4 days after injection in wind tunnel experiments. A) Oriented responses (partial flight+complete flight+landing). AipsDopEcR-dsRNA injection induces a significant inhibition of oriented upwind flight towards the sex pheromone in sexually mature male moths. B) General flight activity (oriented responses+random flights). Locomotor behavior is not affected by AipsDopEcR-dsRNA injection. Numbers in brackets indicate numbers of tested males. Bars with same letters are not significantly different (G-test; P≤0.05).