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 naïve 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.

Synaptotagmin I, a molecular target for steroid hormone signaling controlling the maturation of sexual behavior in an insect.

UNLABELLED: As in vertebrates, the insect steroid hormones, especially 20-hydroxyecdysone (20E), initiate and regulate sexual behavior by acting on the central nervous system. This 20E action is, in part, triggered by transcriptional events mediated through the binding of 20E to a heterodimer comprising the ecdysone receptor (EcR) and ultraspiracle (USP). However, to date, our knowledge about this genomic steroid pathway remains incomplete. In moths, males detect female sex pheromones, eliciting stereotyped sexual behavior. In Agrotis ipsilon males, the behavioral response and the neuronal sensitivity to sex pheromone in the olfactory center, the antennal lobe (AL), increase with age. We recently showed that 20E controlled this age-dependent olfactory plasticity via the activation of an EcR/USP-dependent pathway in the AL. Here, we cloned the gene encoding A. ipsilon synaptotagmin I (AisytI), a presynaptic vesicle protein known to act as a calcium sensor in neurotransmitter release. AisytI was expressed in the AL, where its amount increased with age, whereas its knockdown inhibited the sex pheromone-oriented flight of males. 20E administration to males induced AL AisytI expression in a dose-dependent and time-dependent manner. Moreover, A. ipsilon EcR silencing caused decreases in AL AisytI expression and the behavioral response to sex pheromone. Our results show that the synaptotagmin I gene is a target gene for the genomic steroid signaling that controls the expression of insect sexual behavior by acting on central sex pheromone processing. This study thus represents a significant advance in our understanding of the steroid actions that influence neural functions, and thereby behavioral plasticity, in various organisms. DATABASE: The nucleotide sequence of Agrotis ipsilon synaptotagmin I is available in the DDBJ/EMBL/GenBank databases under the accession number KJ863735.

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

Olfactory 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.

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

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.

Is the rapid post-mating inhibition of pheromone response triggered by ecdysteroids or other factors from the sex accessory glands in the male moth Agrotis ipsilon?

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.

Steroid hormone signaling is involved in the age-dependent behavioral response to sex pheromone in the adult male moth Agrotis ipsilon.

In most animals, including insects, male reproduction depends on the detection and processing of female-produced sex pheromones. In the male moth, Agrotis ipsilon, both behavioral response and neuronal sensitivity in the primary olfactory center, the antennal lobe (AL), to female sex pheromone are age- and hormone-dependent. In many animal species, steroids are known to act at the brain level to modulate the responsiveness to sexually relevant chemical cues. We aimed to address the hypothesis that the steroidal system and in particular 20-hydroxyecdysone (20E), the main insect steroid hormone, might also be involved in this olfactory plasticity. Therefore, we first cloned the nuclear ecdysteroid receptor EcR (AipsEcR) and its partner Ultraspiracle (AipsUSP) of A. ipsilon, the expression of which increased concomitantly with age in ALs. Injection of 20E into young sexually immature males led to an increase in both responsiveness to sex pheromone and amount of AipsEcR and AipsUSP in their ALs. Conversely, the behavioral response decreased in older, sexually mature males after injection of cucurbitacin B (CurB), an antagonist of the 20E/EcR/USP complex. Also, the amount of AipsEcR and AipsUSP significantly declined after treatment with CurB. These results suggest that 20E is involved in the expression of sexual behavior via the EcR/USP signaling pathway, probably acting on central pheromone processing in A. ipsilon.

The transcription factor Krüppel homolog 1 is linked to the juvenile hormone-dependent maturation of sexual behavior in the male moth, Agrotis ipsilon.

In the male moth, Agrotis ipsilon, the behavioral response and neuronal sensitivity in the primary olfactory center, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. Although JH has been shown to control this age-dependent plasticity, the underlying signaling pathway remains obscure. In this context, we cloned a full cDNA encoding the Krüppel homolog 1 transcription factor (AipsKr-h1) of A. ipsilon, which was found to be predominantly expressed in ALs, where its amount increased concomitantly with age and sex pheromone responses. Conversely, the expression of AipsKr-h1 protein in the antenna was age-independent. Moreover, the administration of JH in immature males or fluvastatin, an inhibitor of JH biosynthesis, in mature males induced an increase or a decline of the AipsKr-h1 protein level in ALs, respectively. This effect was suppressed with a combined injection of fluvastatin and JH. Our results showed that Aipskr-h1 is a JH-upregulated gene that might mediate JH action on central pheromone processing, modulating sexual behavior in A. ipsilon.

A female-biased expressed elongase involved in long-chain hydrocarbon biosynthesis and courtship behavior in Drosophila melanogaster.

Drosophila melanogaster produces sexually dimorphic cuticular pheromones that are a key component of the courtship behavior leading to copulation. These molecules are hydrocarbons, with lengths of 23 and 25 carbons in males (mainly with one double bond) and 27 and 29 carbons in females (mainly with two double bonds). Here, we describe an elongase gene, eloF, with female-biased expression. The 771-bp ORF encodes a 257-aa protein that shows the highest sequence identity with mouse SSC1 elongase (33%). The activity of the cDNA expressed in yeast was elongation of saturated and unsaturated fatty acids up to C30. RNAi knockdown in Drosophila led to a dramatic modification of female hydrocarbons, with decreased C29 dienes and increased C25 dienes accompanied by a modification of several courtship parameters: an increase in copulation latency and a decrease in both copulation attempts and copulation. Feminization of the hydrocarbon profile in males by using targeted expression of the transformer gene resulted in high expression levels of eloF, suggesting that the gene is under the control of the sex-determination hierarchy. There is no expression of eloF in Drosophila simulans, which synthesize only C23 and C25 hydrocarbons. These results strongly support the hypothesis that eloF is a crucial enzyme for female pheromone biosynthesis and courtship behavior in D. melanogaster.

A new elongase selectively expressed in Drosophila male reproductive system.

We have identified an elongase gene, elo68alpha, which is specifically transcribed in males. We have characterized the elo68alpha open reading frame, expressed it in fasDelta elo1Delta yeast and showed that it could elongate myristoleic and palmitoleic acids, therefore sharing an Elo1 specificity. This elongase was found to be exclusively expressed in male genital system (testis and ejaculatory bulb). Northern blot analysis showed that the elo68alpha gene was inducible at low temperatures. One P-strain mutant for elo68alpha and three excision lines for this P-element were subsequently studied. The excision line with only 1% elo68alpha expression showed decreased levels of vaccenyl acetate, a male pheromone produced in the ejaculatory bulb. The induction of elo68alpha expression at 21 degrees C was also paralleled with higher vaccenyl acetate production. These results strongly suggest that elo68alpha is involved in the elongation of short unsaturated fatty acids in males and might play a role in vaccenyl acetate biosynthesis.

Female-specific regulation of cuticular hydrocarbon biosynthesis by dopamine in Drosophila melanogaster.

The role of dopamine (DA) is investigated in cuticular hydrocarbon biosynthesis in Drosophila melanogaster with three different approaches: use of DA-deficient mutants (dopa decarboxylase temperature sensitive mutants reared at restrictive temperature, and rescued by dopamine ingestion or by pale mutants partially rescued by a tyrosine hydroxylase construction), pharmacological treatments (tyrosine hydroxylase inhibitors) and topical application on decapitated flies. We report that DA specifically regulates diene hydrocarbon biosynthesis, which is female specific. Our results suggest that DA acts in adult flies within the first hours of imaginal life and that DA production from the brain is crucial for this process. Thus, DA contributes to reproduction in D. melanogaster by acting during a critical period during development of young adults.