The GPCR membrane receptor, DopEcR, mediates the actions of both dopamine and ecdysone to control sex pheromone perception in an insect

International audienceOlfactory information mediating sexual behavior is crucial for reproduction in many animals, including insects. In male moths, the macroglomerular complex (MGC) of the primary olfactory center, the antennal lobe (AL) is specialized in the treatment of information on the female-emitted sex pheromone. Evidence is accumulating that modulation of behavioral pheromone responses occurs through neuronal plasticity via the action of hormones and/or catecholamines. We recently showed that a G-protein-coupled receptor (GPCR), AipsDopEcR, with its homologue known in Drosophila for its double affinity to the main insect steroid hormone 20-hydroxyecdysone (20E), and dopamine (DA), present in the ALs, is involved in the behavioral response to pheromone in the moth, Agrotis ipsilon. Here we tested the role of AipsDopEcR as compared to nuclear 20E receptors in central pheromone processing combining receptor inhibition with intracellular recordings of AL neurons. We show that the sensitivity of AL neurons for the pheromone in males decreases strongly after AipsDopEcR-dsRNA injection but also after inhibition of nuclear 20E receptors. Moreover we tested the involvement of 20E and DA in the receptor-mediated behavioral modulation in wind tunnel experiments, using ligand applications and receptor inhibition treatments. We show that both ligands are necessary and act on AipsDopEcR-mediated behavior. Altogether these results indicate that the GPCR membrane receptor, AipsDopEcR, controls sex pheromone perception through the action of both 20E and DA in the central nervous system, probably in concert with 20E action through nuclear receptors

Cloning and characterization of a gut-specific cathepsin L from the aphid Aphis gossypii

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An insecticide further enhances experience-dependent increased behavioural responses to sex pheromone in a pest insect

Neonicotinoid insecticides are widely used to protect plants against pest insects, and insecticide residues remaining in the environment affect both target and non-target organisms. Whereas low doses of neonicotinoids have been shown to disturb the behaviour of pollinating insects, recent studies have revealed that a low dose of the neonicotinoid clothianidin can improve behavioural and neuronal sex pheromone responses in a pest insect, the male moth Agrotis ipsilon, and thus potentially improve reproduction. As male moth behaviour depends also on its physiological state and previous experience with sensory signals, we wondered if insecticide effects would be dependent on plasticity of olfactory-guided behaviour. We investigated, using wind tunnel experiments, whether a brief pre-exposure to the sex pheromone could enhance the behavioural response to this important signal in the moth A. ipsilon at different ages (sexually immature and mature males) and after different delays (2 h and 24 h), and if the insecticide clothianidin would interfere with age effects or the potential pre-exposure-effects. Brief pre-exposure to the pheromone induced an age-independent significant increase of sex pheromone responses 24 h later, whereas sex pheromone responses did not increase significantly 2 h after exposure. However, response delays were significantly shorter compared to naive males already two hours after exposure. Oral treatment with clothianidin increased sex pheromone responses in sexually mature males, confirming previous results, but did not influence responses in young immature males. Males treated with clothianidin after pre-exposure at day 4 responded significantly more to the sex pheromone at day 5 than males treated with clothianidin only and than males pre-exposed only, revealing an additive effect of experience and the insecticide. Plasticity of sensory systems has thus to be taken into account when investigating the effects of sublethal doses of insecticides on behaviour

Age-related AipsDopEcR transcriptional activity and protein level from brains of 1- to 5-day-old <i>A. ipsilon</i> males.

<p><b>A)</b> Transcriptional activity of <i>AipsDopEcR</i>. The ribosomal gene <i>RpL8</i> was used as reference. <b>B)</b> Relative protein expression of AipsDopEcR based on Western blot analysis as shown in (C). <b>C)</b> 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).</p

Efficiency of double-stranded RNA-mediated <i>AipsDopEcR</i> silencing in <i>A. ipsilon</i> males.

<p>1-day-old sexually immature males received an injection of saline solution, bacterial <i>LacZ</i>-dsRNA or <i>AipsDopEcR</i>-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. <b>A)</b> Transcriptional activity of <i>AipsDopEcR</i>. The ribosomal gene <i>RpL8</i> was used as reference. <b>B)</b> Relative protein expression of AipsDopEcR based on Western blot analysis as shown in (C). <b>C)</b> 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).</p

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

<p>1-day-old sexually immature males were injected with saline solution, bacterial <i>LacZ</i>-dsRNA or <i>AipsDopEcR</i>-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. <b>A)</b> Oriented responses (partial flight+complete flight+landing). <i>AipsDopEcR</i>-dsRNA injection induces a significant inhibition of oriented upwind flight towards the sex pheromone in sexually mature male moths. <b>B)</b> General flight activity (oriented responses+random flights). Locomotor behavior is not affected by <i>AipsDopEcR</i>-dsRNA injection. Numbers in brackets indicate numbers of tested males. Bars with same letters are not significantly different (G-test; P≤0.05).</p

Effect of the combined treatment of pre-exposure and clothianidin on pheromone responses of 5-day-old <i>A</i>. <i>ipsilon</i> males.

<p>Four-day-old males were pre-exposed with 10 ng of the pheromone blend and subsequently orally treated with 10 ng of clothianidin. Behavioural responses to 1 ng of the pheromone blend were observed in the wind tunnel 24 h later (D5-PE4-CLO). Unexperienced and solvent-treated males, as well as pre-exposure- or clothianidin-treated males were used as controls. <b>(A)</b> Percentage of males showing oriented response. <b>(B)</b> Response delay and standard deviation of males showing oriented responses. The lower whisker presents the minimum, the lower hinge of the box is the first quartile, the line inside the box is the median, the upper hinge is the third quartile, and the extreme of the upper whisker is the maximum. Clothianidin further enhances oriented pheromone responses of <i>A</i>. <i>ipsilon</i> males after brief pre-exposure. Numbers in bars indicate the numbers of males tested (A) and number of males that showed an oriented response (B). Bars with the same letter are not significantly different (G-test for (A), Mann-Whitney test for (B), P < 0.05).</p

Design of experiments.

<p>Treatments were performed on 1-day-, 4-day-, and 5-day-old males, and behavioral tests in wind tunnel were performed either the same day of treatment or 24 h later. Test: behavioral test in wind tunnel.</p