In insects, juvenile hormone (JH) is essential for orchestrating reproductive events. For example, in the male moth Agrotis ipsilon, the behavioral response to female sex pheromone is linked to neuronal sensitivity in the primary olfactory centers (antennal lobes, ALs), and the maturation of accessory sex glands (ASGs) are known to be age- and JH-dependent. The molecular basis of this regulatory action of JH is not fully deciphered, and we show here that the heterodimerizing partner of Methoprene-tolerant called Taiman (Tai) is essential for the sexual maturation of male A. ipsilon. Tai expression in ALs and ASGs is elevated from the third day of adult life and is responsible for the acquisition of behavioral responsiveness to the sex pheromone and ASG maturation. Tai-deficient old males exhibited altered sexual behavior and delayed ASG maturation. Moreover, the expression levels of Tai and Krüppel homolog 1 (Kr-h1), an early JH-induced transcription factor, were reduced in ALs and ASGs of JH-deprived and Tai-deficient old males, respectively. Exogenous JH injection into young males resulted in precocious sexual maturation and this JH induction was suppressed by Tai silencing. Our results demonstrate that Tai is an actor of the JH signaling pathway that operates in ALs and ASGs to promote pheromone information processing and consequently the display of sexual behavior in synchrony with ASG maturation, ultimately optimizing male reproductive success. Thus, this study provides additional insights into the molecular mechanisms underlying hormonal regulation of sexual maturation in insects.

In animals, sexual maturation is marked by the development of reproductive behaviors in synchronism with the acquisition of fertility, and this timing is influenced by chemosensory experiences. In naïve and immature individuals, exposure to sex pheromones may accelerate sexual development, and mechanisms underlying this pheromone induction are not fully identified. Using the moth Agrotis ipsilon, we showed that pre-exposure of immature males to female sex pheromones led to early increases in the performance of sex pheromone-triggered oriented flight as well as in the maturation of accessory sex glands (ASGs) producing seminal proteins. Conjointly, biosynthesis and circulating amounts of juvenile hormone (JH) raised with an upregulation of the expression of JH receptor, Methoprene-tolerant 1 (Met1) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in ASGs and the primary olfactory centers, the antennal lobes (ALs). In the sex pheromone pre-exposed immature males, the loss of function of Met1 or Kr-h1 caused a reduction in the induction of the sex pheromone behavioral responsiveness and the ASG secretory activity. Taken together, our results showed that the accelerated effects of sex pheromone pre-exposure on male sexual maturation are mediated by increased JH biosynthesis. This ultimately leads to early induction of JH signaling in ASGs for seminal protein production and in ALs for the central processing of pheromone information, which causes the display of sexual behavior in male A. ipsilon. Finally, this study expands our understanding of endocrine mechanisms by which animals can modulate their fitness according to past olfactory experiences.

In animals, sexual maturation coincides with the development of sexual behaviors and reproductive system. These developmental events are influenced by diet and governed by endocrine signals. Here, for the first time in insects, we explored functional links between nutrition and juvenile hormone (JH) in the male reproductive physiology through the insulin signaling pathway (ISP) acting as a transducer of nutritional signals. We turned to the male moth Agrotis ipsilon for which sexual maturation, including accessory sex glands (ASGs) development concomitantly with antennal lobes (ALs) maturation for female sex pheromone processing and display of sexual behavior, is known to be JH- and diet-dependent. Indeed, a diet rich in sugars with sodium was previously shown to accelerate sexual maturation, which was achieved from the third day of adult life. In this study, we demonstrated that such a diet raised i) the expression of JH signaling actors (Methoprene-tolerant, Taiman, and Krüppel homolog 1) in ALs and ASGs, ii) the biosynthesis and circulating levels of JH, and iii) the expression of both insulin receptor (InR) and insulin-like peptides (ILPs) in corpora allata (CAs) and brain respectively. Insulin injection raised JH biosynthesis following increased HMG-CoA reductase expression in CAs; opposite effects were induced in InR-deficient males. Thus, we highlighted that promoting effects of a diet composed of sugars with sodium on male sexual maturation results from an early induction of ISP causing an increase in JH biosynthesis followed by a potentiation of JH actions on the development of ASGs and ALs in A. ipsilon.

To extract any adaptive benefit, the circadian clock needs to be synchronized to the 24-h day-night cycles. We have investigated if it is a general property of the brain's circadian clock to recognize social interactions as external time givers. Sociosexual interactions with the opposite sex are universal, prevalent even in the lives of solitary animals. The solitary adult life of the Spodoptera littoralis moth is singularly dedicated to sex, offering an ideal context for exploring the impact of sociosexual cues on circadian timekeeping. We have identified specific olfactory cues responsible for social entrainment, revealing a surprisingly strong influence of pheromone-mediated remote sociosexual interactions on circadian rhythms. Males' free-running rhythms are induced and synchronized by the sex pheromone that the female releases in a rhythmic fashion, highlighting a hierarchical relation between the female and male circadian oscillators. Even a single pulse of the sex pheromone altered clock gene expression in the male brain, surpassing the effect of light on the clock. Our finding of a daytime-dependent, lasting impact of pheromone on male's courtship efficacy indicates that circadian timing in moths is a trait under sexual selection. We have identified specific components of the sex-pheromone blend that lack mate-attractive property but have powerful circadian effects, providing rationale for their continued retention by the female. We show that such volatiles, when shared across sympatric moth species, can trigger communal synchronization. Our results suggest that the sex pheromone released by female moths entrains males' behavioral activity rhythm to ensure synchronized timing of mating.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

Many priority pollutants are concentrated in the environment due to human activity. Most are highly toxic to various organisms, including endocrine disruptors EDCs, aromatic polycyclic hydrocarbons PAHs, pesticides. While the effects of single and binary exposure have been widely explored, several pollutants can be simultaneously present at the same time in the environment, in in more or less polluted matrices. Effective pollution control requires the presence and sources of contamination to be identified. Previously we used Drosophila melanogaster to investigate metal pollution. Here, we re-used Drosophila to identify the biomarkers of pollution, and to determine if they can be used for specific types of pollution. Single and combined exposure of Bis(2-ethylhexyl) phthalate (DEHP), bisphenol A, nonylphenol, benzo(a)pyrene, and glyphosate was investigated. The impact of these pollutants on post-embryonic development and the expression pattern of 38 molecular targets were examined using qPCR. During single exposure, different profiles were observed at the molecular level. In complex mixtures, the expression profile resembled that of bisphenol A. In contrast, relatively specific gene expression profiles were obtained for the effects of each pollutant separately. While direct pollutant-gene profiling remains difficult in mixtures, molecular biology analyses enhance pollution monitoring, and should be incorporated in toxicological studies.

In the moth Agrotis ipsilon, the behavioral response of males to the female-emitted sex pheromone increases throughout adult life and following a prior exposure to sex pheromone, whereas it is temporally inhibited after the onset of mating. This behavioral flexibility is paralleled with changes in neuronal sensitivity to pheromone signal within the primary olfactory centers, the antennal lobes. In the present study, we tested the hypothesis that neuroligins, post-synaptic transmembrane proteins known to act as mediators of neuronal remodeling, are involved in the olfactory modulation in A. ipsilon males. We cloned a full-length cDNA encoding neuroligin 1, which is expressed predominantly in brain and especially in antennal lobes. The level of neuroligin 1 expression in antennal lobes gradually raised from day-2 until day-4 of adult life, as well as at 24 h, 48 h and 72 h following pre-exposure to sex pheromone, and the temporal dynamic of these changes correlated with increased sex pheromone responsiveness. By contrast, there was no significant variation in antennal lobe neuroligin 1 expression during the post-mating refractory period. Taken together, these results highlight that age- and odor experience-related increase in sex pheromone responsiveness is linked to the overexpression of neuroligin 1 in antennal lobes, thus suggesting a potential role played by this post-synaptic cell-adhesion molecule in mediating the plasticity of the central olfactory system in A. ipsilon.

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.

Heavy metals, like many other chemical elements, are naturally present in the environment; however, the concentrations of these metals in various environmental matrices have increased through their intensive use in many human activities (such as industry, mining and agriculture). Among the heavy metals, cadmium (Cd) and mercury (Hg) induce a wide variety of defects in animals. While the effects of these heavy metals have been widely documented, a single exposure paradigm is typically used. Few studies have focused on evaluating combined exposure to these metals. However, in the environment, animals are confronted with a plethora of substances simultaneously; thus, the presence and origin of such substances must be determined to reduce the sources of contamination. Using the model of the fruit fly Drosophila melanogaster, for which many tools are readily available, we investigated how different concentrations of Cd and Hg in single and combined exposures impact post-embryonic development. In parallel, we evaluated the extended expression pattern of 38 molecular targets used as potential biomarkers of exposure through qPCR. Our results showed that both metals caused developmental delays and mortality in dose-dependent responses. Both metals were able to deregulate genes involved in hormonal control, general stress, and oxidative stress. Importantly, we confirmed synergistic interactions between Cd and Hg. Our results indicate the importance of assessing several biomarkers and their kinetics in mixtures. Drosophila represents a useful model for monitoring the toxicity of substances in polluted environments.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

Most animal species, including insects, are able to modulate their responses to sexual chemosignals and this flexibility originates from the remodeling of olfactory areas under the influence of the dopaminergic system. In the moth Agrotis ipsilon, the behavioral response of males to the female-emitted sex pheromone increases throughout adult life and after a prior exposure to pheromone signal, and this change is accompanied by an increase in neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs). To identify the underlying neuromodulatory mechanisms, we examined whether this age- and experience-dependent olfactory plasticity is mediated by dopamine (DA) through the Dop1 receptor, an ortholog of the vertebrate D1-type dopamine receptors. which is positively coupled to adenylyl cyclase. We cloned A. ipsilon Dop1 (AiDop1), which is expressed predominantly in brain and especially in ALs; its knockdown induced a decrease in AL cAMP and altered sex pheromone-orientated flight. The levels of DA, AiDop1 expression and cAMP in ALs increased from the third day of adult life and at 24 and 48 h following pre-exposure to sex pheromone, and the dynamic of these changes correlated with the increased responsiveness to sex pheromone. These results demonstrate that Dop1 is required for the display of male sexual behavior and that age- and experience-related neuronal and behavioral changes are sustained by DA-Dop1 signaling that operates within ALs, probably through cAMP-dependent mechanisms in A. ipsilon. Thus, this study expands our understanding of the neuromodulatory mechanisms underlying olfactory plasticity, mechanisms that appear to be highly conserved between insects and mammals.

In the male moth, Agrotis ipsilon, the behavioural response and neuron sensitivity within the olfactory centres, the antennal lobes (ALs), to female sex pheromone increase with age, in correlation with the maturation of sex accessory glands (SAGs). By contrast, newly mated males cease to be attracted to sex pheromone and remate when their SAGs are refilled during the next night. The insect hormone receptor 38 (HR38), an ortholog of the vertebrate NR4A receptors, is a component of ecdysteroid signalling pathway which controls adult male physiology and behaviour. Here, we cloned the A. ipsilon HR38 (AiHR38) and explored its function in the coordination of reproductive events in the male. AiHR38 was detected in SAGs and ALs, and where its amount raised with age, in parallel with SAG protein content and sex pheromone responsiveness. By contrast, the AL and SAG AiHR38 expressions declined at 0-2 h after mating, in linking with depletion of SAG protein reserves and loss of sensitivity to sex pheromone. The increased AL and SAG AiHR38 expressions at 20-24 h postmating coincided with replenishing of SAGs and recovery of sensitivity to sex pheromone for a new mating. Moreover, AiHR38 knockdown resulted in reduction in SAG protein amount and disruption of sex pheromone-orientated flight. These results show that the insect HR38 is essential both for SAG activity, probably by controlling the protein synthesis, and display of male sexual behaviour, and that the concomitant regulation of its expression within SAGs and olfactory centres contributes to synchronisation between fertility and sexual activity. DATABASE: The nucleotide sequence of Agrotis ipsilon HR38 is available in the DDBJ/EMBL/GenBank databases under the accession number MF402845.

Di (2-ethylhexyl) phthalate (DEHP) is recognized in vertebrates as an Endocrine Disrupting Chemical (EDC). DEHP can alter steroid hormones production, development, reproduction and behavior in vertebrates. Only few studies investigated DEHP effects on insects. However, some recent studies on aquatic insects showed that DEHP could also act as an EDC by interfering with the signaling pathways of ecdysteroids, the main hormones involved in the control of insect post-embryonic development and physiology. The aim of the study was to investigate (1) the fate of DEHP within a terrestrial insect species by exposing larvae to food containing a wide range of DEHP concentrations and (2) the effects of this chemical on their post-embryonic development and metamorphosis, by using a multi-level approach. DEHP was shown to be present both in larvae and resulting stages, with higher concentrations in chrysalises and adults than in larvae. DEHP concentrations also decreased at the end of the last larval instar, suggesting the metabolic transformation or excretion of this chemical during this time. Only the two highest DEHP doses induced higher insect mortality, whereas low and intermediate concentrations increased larval food consumption without affecting body weight. Metabolic profiles showed that in control insects, the last three days before metamorphosis correspond to a metabolic transition, but with time-dependent changes in treated insects. Interestingly, DEHP treatments also alter both hemolymphatic ecdysteroid titers and expression levels of ecdysteroid response genes. These results confirm that DEHP can alter insect post-embryonic development and metamorphosis, by interfering with ecdysteroid pathways.

Glutathione-S-transferases (GSTs) are conjugating enzymes involved in the detoxification of a wide range of xenobiotic compounds. The expression of GSTs as well as their activities have been also highlighted in the olfactory organs of several species, including insects, where they could play a role in the signal termination and in odorant clearance. Using a transcriptomic approach, we identified 33 putative GSTs expressed in the antennae of the cotton leafworm Spodoptera littoralis. We established their expression patterns and revealed four olfactory-enriched genes in adults. In order to investigate the evolution of antennal GST repertoires in moths, we re-annotated antennal transcripts corresponding to GSTs in two moth and one coleopteran species. We performed a large phylogenetic analysis that revealed an unsuspected structural—and potentially functional—diversity of GSTs within the olfactory organ of insects. This led us to identify a conserved clade containing most of the already identified antennal-specific and antennal-enriched GSTs from moths. In addition, for all the sequences from this clade, we were able to identify a signal peptide, which is an unusual structural feature for GSTs. Taken together, these data highlight the diversity and evolution of GSTs in the olfactory organ of a pest species and more generally in the olfactory system of moths, and also the conservation of putative extracellular members across multiple insect orders.

Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called "juvenile hormone esterase duplication (JHEdup)" has been previously reported to hydrolyse different fruit esters in vitro. Here we functionally characterize JHEdup in vivo. We show that the jhedup gene is highly expressed in large basiconic sensilla that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. Our results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids.

Carboxylesterases are enzymes widely distributed within living organisms. In insects, they have been mainly involved in dietary metabolism and detoxification function. Interestingly, several members of this family called carboxylesterase-like adhesion molecules (CLAMs) have lost their catalytic properties and are mainly involved in neuro/developmental functions. CLAMs include gliotactins, neurotactins, glutactins, and neuroligins. The latter have for binding partner the neurexin. In insects, the function of these proteins has been mainly studied in Drosophila central nervous system or neuromuscular junction. Some studies suggested a role of neuroligins and neurexin in sensory processing but CLAM expression within sensory systems has not been investigated. Here, we reported the identification of 5 putative CLAMs expressed in the olfactory system of the model pest insect Spodoptera littoralis. One neuroligin, Slnlg4-yll and its putative binding partner neurexin SlnrxI were the most expressed in the antennae and were surprisingly associated with olfactory sensilla. In addition, both transcripts were upregulated in male antennae after mating, known to modulate the sensitivity of the peripheral olfactory system in S. littoralis, suggesting that these molecules could be involved in sensory plasticity.

Pesticides have long been used as the main solution to limit agricultural pests, but their widespread use resulted in chronic or diffuse environmental pollutions, development of insect resistances, and biodiversity reduction. The effects of low residual doses of these chemical products on organisms that affect both targeted species (crop pests) but also beneficial insects became a major concern, particularly because low doses of pesticides can induce unexpected positive—also called hormetic—effects on insects, leading to surges in pest population growth at greater rate than what would have been observed without pesticide application. The present study aimed to examine the effects of sublethal doses of deltamethrin, one of the most used synthetic pyrethroids, known to present a residual activity and persistence in the environment, on the peripheral olfactory system and sexual behavior of a major pest insect, the cotton leafworm Spodoptera littoralis. We highlighted here a hormetic effect of sublethal dose of deltamethrin on the male responses to sex pheromone, without any modification of their response to host-plant odorants. We also identified several antennal actors potentially involved in this hormetic effect and in the antennal detoxification or antennal stress response of/to deltamethrin exposure.