Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids such as clothianidin. The residual accumulation of low concentrations of these insecticides can have positive effects on target pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction and olfactory synaptic transmission is cholinergic, neonicotinoid residues could indeed modify chemical communication. We recently showed that treatments with low doses of clothianidin could induce hormetic effects on behavioral and neuronal sex pheromone responses in the male moth, Agrotis ipsilon. In this study, we used high-throughput RNAseq and proteomic analyses from brains of A. ipsilon males that were intoxicated with a low dose of clothianidin to investigate the molecular mechanisms leading to the observed hormetic effect. Our results showed that clothianidin induced significant changes in transcript levels and protein quantity in the brain of treated moths: 1229 genes and 49 proteins were differentially expressed upon clothianidin exposure. In particular, our analyses highlighted a regulation in numerous enzymes as a possible detoxification response to the insecticide and also numerous changes in neuronal processes, which could act as a form of acclimatization to the insecticide-contaminated environment, both leading to enhanced neuronal and behavioral responses to sex pheromone.

The confused flour beetle, Tribolium confusum, is a common and severe pest of stored products. Here, using fumigation tests during four different exposure times, we evaluated the toxicity of different doses of essential oil of spike lavender, Lavandula spica and one of its major constituents, linalool, on different life stages of T. confusum under laboratory conditions. The toxicity of the L. spica oil and linalool varied as a function of the developmental stage and treatment duration. Young larvae (L1) were the most susceptible to toxic effects, with LC50 = 19.535 mu l/L of air for L. spica oil and LC50 = 14.198 mu l/L of air for linalool after 24 h of exposure, whereas older larvae (L8) were affected only very little by fumigation. Linalool caused higher egg mortality than L. spica oil at equivalent doses, but lower mortality in pupae and adults. Emergence of intact adult insects from surviving eggs, larvae and pupae was further reduced as a function of dose and exposure time to both L. spica oil and linalool compared to control-treated insects. Our results show that L. spica oil and linalool might be suitable for biological control of T. confusum, but tests at a larger scale are necessary to confirm our results.

SPECIALTY GRAND CHALLENGE article Front. Physiol., 18 July 2011Sec. Invertebrate Physiology volume 2 - 2011 | https://doi.org/10.3389/fphys.2011.00038

Many aphid species reproduce parthenogenetically throughout most of the year, with individuals having identical genomes. Nevertheless, aphid clones display a marked polyphenism with associated behavioural differences. Pea aphids (Acyrthosiphon pisum), when crowded, produce winged individuals, which have a larger dispersal range than wingless individuals. We examined here if brain structures linked to primary sensory processing and high-order motor control change in size as a function of wing polyphenism. Using micro-computing tomography (micro-CT) scans and immunocytochemical staining with anti-synapsin antibody, we reconstructed primary visual (optic lobes) and olfactory (antennal lobes) neuropils, together with the central body of winged and wingless parthenogenetic females of A. pisum for volume measurements. Absolute neuropil volumes were generally bigger in anti-synapsin labelled brains compared to micro-CT scans. This is potentially due to differences in rearing conditions of the used aphids. Independent of the method used, however, winged females consistently had larger antennal lobes and optic lobes than wingless females in spite of a larger overall body size of wingless compared to winged females. The volume of the central body, on the other hand was not significantly different between the two morphs. The larger primary sensory centres in winged aphids might thus provide the neuronal substrate for processing different environmental information due to the increased mobility during flight.

Essential oils of aromatic plants and their individual volatile components have been tested in pest management strategies for their toxic and often repellent effects on target insects. When evaluating their possible effects on crucial behaviours of the pest insects, the olfactory environment including intraspecific communication cues has to be considered. We used the flour beetle Tribolium confusum du Val (Coleoptera: Tenebrionidae), a common stored-product pest, to investigate the influence of oil of spike lavender, Lavandula spica Medik. (Labiaceae), and its main component, linalool, at various doses on olfactory-guided behaviour. Using four-way olfactometers, a dose-dependent repellent effect of L. spica oil and linalool alone was revealed. On the other hand, we confirmed that T. confusum is attracted by conspecifics, by means of an aggregation pheromone and by 10 ng of one of its components, 1-tetradecene. Twenty-four hour pre-exposure to 10 mu l of L. spica oil abolished subsequent attraction to 1-tetradecene and reduced attraction to five conspecifics. Simultaneous exposure to L. spica oil or linalool and five conspecifics reduced the repellent effect of the volatiles in a dose-dependent manner, whereas simultaneous exposure to 1-tetradecene at 10 ng abolished the repellent effect of L. spica oil only at a dose of 0.01 mg. These results indicate a dose-dependent trade-off between attractive and plant-derived repellent volatiles, which may influence the effectiveness of such volatiles in their potential use in alternative pest management strategies.

Acibenzolar-S-methyl (ASM) is a chemical compound, which is able to induce resistance in several model and non-model plants, but the end-players of this induced defense remain ill-defined. Here, we test the hypothesis that treatment with ASM can protect apple (Malus × domestica) against the rosy apple aphid (Dysaphis plantaginea) and investigate the defense molecules potentially involved in resistance. We measured aphid life traits and performed behavioral assays to study the effect of ASM on plant resistance against the aphid, and then combined transcriptomic, bioinformatics, metabolic and biochemical analyses to identify the plant compounds involved in resistance. Plants treated with ASM negatively affected several life traits of the aphid and modified its feeding and host seeking behaviors. ASM treatment elicited up-regulation of terpene synthase genes in apple and led to the emission of (E,E)-α-farnesene, a sesquiterpene that was repellent to the aphid. Several genes encoding amaranthin-like lectins were also strongly up-regulated upon treatment and the corresponding proteins accumulated in leaves, petioles and stems. Our results link the production of specific apple proteins and metabolites to the antibiosis and antixenosis effects observed against Dysaphis plantaginea, providing insight into the mechanisms underlying ASM-induced herbivore resistance.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

In insects, olfaction plays a crucial role in many behavioral contexts, such as locating food, sexual partners, and oviposition sites. To successfully perform such behaviors, insects must respond to chemical stimuli at the right moment. Insects modulate their olfactory system according to their physiological state upon interaction with their environment. Here, we review the plasticity of behavioral responses to different odor types according to age, feeding state, circadian rhythm, and mating status. We also summarize what is known about the underlying neural and endocrinological mechanisms, from peripheral detection to central nervous integration, and cover neuromodulation from the molecular to the behavioral level. We describe forms of olfactory plasticity that have contributed to the evolutionary success of insects and have provided them with remarkable tools to adapt to their ever-changing environment.

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids, leaving residues in the environment. There is now evidence that low doses of insecticides can have positive effects on pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction, and olfactory synaptic transmission is cholinergic, neonicotinoid residues could modify chemical communication. We recently showed that treatments with different sublethal doses of clothianidin could either enhance or decrease behavioural sex pheromone responses in the male moth, Agrotis ipsilon. We investigated now effects of the behaviourally active clothianidin doses on the sensitivity of the peripheral and central olfactory system. We show with extracellular recordings that both tested clothianidin doses do not influence pheromone responses in olfactory receptor neurons. Similarly, in vivo optical imaging does not reveal any changes in glomerular response intensities to the sex pheromone after clothianidin treatments. The sensitivity of intracellularly recorded antennal lobe output neurons, however, is upregulated by a lethal dose 20 times and downregulated by a dose 10 times lower than the lethal dose 0. This correlates with the changes of behavioural responses after clothianidin treatment and suggests the antennal lobe as neural substrate involved in clothianidin-induced behavioural changes.

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.

In moths, which include many agricultural pest species, males are attracted by female-emitted sex pheromones. Although integrated pest management strategies are increasingly developed, most insect pest treatments rely on widespread use of neurotoxic chemicals, including neonicotinoid insecticides. Residual accumulation of low concentrations of these insecticides in the environment is known to be harmful to beneficial insects such as honey bees. This environmental stress probably acts as an "info-disruptor" by modifying the chemical communication system, and therefore decreases chances of reproduction in target insects that largely rely on olfactory communication. However, low doses of pollutants could on the contrary induce adaptive processes in the olfactory pathway, thus enhancing reproduction. Here we tested the effects of acute oral treatments with different low doses of the neonicotinoid clothianidin on the behavioral responses to sex pheromone in the moth Agrotis ipsilon using wind tunnel experiments. We show that low doses of clothianidin induce a biphasic effect on pheromone-guided behavior. Surprisingly, we found a hormetic-like effect, improving orientation behavior at the LD20 dose corresponding to 10 ng clothianidin. On the contrary, a negative effect, disturbing orientation behavior, was elicited by a treatment with a dose below the LD0 dose corresponding to 0.25 ng clothianidin. No clothianidin effect was observed on behavioral responses to plant odor. Our results indicate that risk assessment has to include unexpected effects of residues on the life history traits of pest insects, which could then lead to their adaptation to environmental stress.

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.

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.

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.

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.

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.

SUMMARY Male moths are confronted with complex odour mixtures in a natural environment when flying towards a female-emitted sex pheromone source. Whereas synergistic effects of sex pheromones and plant odours have been observed at the behavioural level, most investigations at the peripheral level have shown an inhibition of pheromone responses by plant volatiles, suggesting a potential role of the central nervous system in reshaping the peripheral information. We thus investigated the interactions between sex pheromone and a behaviourally active plant volatile, heptanal, and their effects on responses of neurons in the pheromone-processing centre of the antennal lobe, the macroglomerular complex, in the moth Agrotis ipsilon. Our results show that most of these pheromone-sensitive neurons responded to the plant odour. Most neurons responded to the pheromone with a multiphasic pattern and were anatomically identified as projection neurons. They responded either with excitation or pure inhibition to heptanal, and the response to the mixture pheromone + heptanal was generally weaker than to the pheromone alone, showing a suppressive effect of heptanal. However, these neurons responded with a better resolution to pulsed stimuli. The other neurons with either purely excitatory or inhibitory responses to all three stimuli did not exhibit significant differences in responses between stimuli. Although the suppression of the pheromone responses in AL neurons by the plant odour is counter-intuitive at first glance, the observed better resolution of pulsed stimuli is probably more important than high sensitivity to the localization of a calling female.

Most animals, including pest insects, live in an "odor world" and depend strongly on chemical stimuli to get information on their biotic and abiotic environment. Although integrated pest management strategies including the use of insect growth regulators (IGRs) are increasingly developed, most insect pest treatments rely on neurotoxic chemicals. These molecules are known to disrupt synaptic transmission, affecting therefore sensory systems. The wide-spread use of neurotoxic insecticides and the growing use of IGRs result in residual accumulation of low concentrations in the environment. These insecticide residues could act as an "info-disruptor" by modifying the chemical communication system, and therefore decrease chances of reproduction in target insects. However, residues can also induce a non-expected hormesis effect by enhancing reproduction abilities. Low insecticide doses might thus induce adaptive processes in the olfactory pathway of target insects, favoring the development of resistance. The effect of sublethal doses of insecticides has mainly been studied in beneficial insects such as honeybees. We review here what is known on the effects of sublethal doses of insecticides on the olfactory system of insect pests.