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Background: Carboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/ pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified. Methodology: In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components. Conclusion: We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant- Degrading Enzyme active towards a host plant volatile.

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.

Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis, a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

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

Responses of insect olfactory receptor neurons (ORNs) involve an entry of Ca²⁺ through olfactory heterodimeric receptor complexes. In moths, the termination of ORN responses was found to strongly depend on the external Ca²⁺ concentration through the activation of unknown Ca²⁺-dependent Cl⁻ channels. We thus investigated the molecular identity of these Cl⁻ channels. There is compelling evidence that bestrophins form Cl⁻ channels when expressed in heterologous systems. Here we provide evidence that antennae of the moth Spodoptera littoralis express three transcripts encoding proteins with hallmarks of bestrophins. One of these transcripts, SlitBest1b, is expressed in ORNs. The heterologous expression of SlitBest1b protein in CHO-K1 cells yielded a Ca²⁺-activated Cl⁻ current that shares electrophysiological properties with the native Ca²⁺-activated Cl⁻ current of ORNs. Both currents are anionic, present similar dependence on the intracellular Ca²⁺ concentration, partly inactivate over time, have the same anion permeability sequence, the same sequence of inhibitory efficiency of blockers, the same almost linear I-V relationships and finally both currents do not depend on the cell volume. Therefore, our data suggest that SlitBest1b is a good candidate for being a molecular component of the olfactory Ca²⁺-activated Cl⁻ channel and is likely to constitute part of the insect olfactory transduction pathway. A different function (e.g. regulation of other proteins, maintenance of the anionic homeostasis in the sensillar lymph) and a different role (e.g. involvement in the olfactory system development) cannot be excluded however.

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 response of insect olfactory receptor neurons (ORNs) involves an increase in intracellular Ca2+ concentration, as in vertebrate ORNs. In order to decipher the Ca2+ clearance mechanisms in insect ORNs, we have investigated the presence of a plasma membrane Ca2+ ATPase (PMCA) in the peripheral olfactory system of the moth Spodoptera littoralis. From an analysis of a male antennal expressed-sequence-tag database combined with a strategy of 5'/3' rapid amplification of cDNA ends plus the polymerase chain reaction, we have cloned a full-length cDNA encoding a PMCA. In adult males, the PMCA transcript has been found in various tissues, including the antennae in which its presence has been detected in the sensilla trichodea, and in cultured ORNs. The PMCA gene is slightly expressed at the end of the pupal stage, reaches a maximum at emergence and is maintained at a high level during the adult period. Taken together, these results provide, for the first time, molecular evidence for the putative participation of a PMCA in signalling pathways responsible for the establishment and functioning of the insect peripheral olfactory system.

To dissect the steroid hormone signaling pathway involved in insect cell morphological differentiation, we extended the application of the double-stranded RNA-mediated interference (dsRNAi) method to the epidermal IAL-PID2 cell line from Plodia interpunctella Lepidoptera. We first demonstrated that dsRNA was capable of efficiently blocking the steroid hormone 20-hydroxyecdysone (20E) inducibility of proteins that belong. to the nuclear receptor superfamily, including the ecdysone receptor (EcR), its partner Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor and the HR3 transcription factor. We then showed that inhibiting the 20E induction of EcR, USP or HR3 proteins prevented the increased synthesis of P tubulin and consequently the morphological transformation of cells. Thanks to this functional approach, we have shown, for the first time, the participation of EcR, USP and HR3 in a 20E signaling pathway that directs morphological differentiation in insect cells by regulating beta tubulin expression.

The response of insect olfactory receptor neurons (ORNs) involves an increase in intracellular [Ca.sup.2+] concentration, as in vertebrate ORNs. In order to decipher the [Ca.sup.2+] clearance mechanisms in insect ORNs, we have investigated the presence of a plasma membrane [Ca.sup.2+] ATPase (PMCA) in the peripheral olfactory system of the moth Spodoptera littoralis. From an analysis of a male antennal expressed-sequence-tag database combined with a strategy of 5'/3' rapid amplification of cDNA ends plus the polymerase chain reaction, we have cloned a full-length cDNA encoding a PMCA. In adult males, the PMCA transcript has been found in various tissues, including the antennae in which its presence has been detected in the sensilla trichodea, and in cultured ORNs. The PMCA gene is slightly expressed at the end of the pupal stage, reaches a maximum at emergence and is maintained at a high level during the adult period. Taken together, these results provide, for the first time, molecular evidence for the putative participation of a PMCA in signalling pathways responsible for the establishment and functioning of the insect peripheral olfactory system. Keywords Plasma membrane calcium ATPase Olfactory transduction * Calcium clearance * Olfactoryreceptor neuron * Spodoptera littoralis (Insecta)

The response of insect olfactory receptor neurons (ORNs) involves an increase in intracellular Ca super(2+) concentration, as in vertebrate ORNs. In order to decipher the Ca super(2+) clearance mechanisms in insect ORNs, we have investigated the presence of a plasma membrane Ca super(2+) ATPase (PMCA) in the peripheral olfactory system of the moth Spodoptera littoralis. From an analysis of a male antennal expressed-sequence-tag database combined with a strategy of 5'/3' rapid amplification of cDNA ends plus the polymerase chain reaction, we have cloned a full-length cDNA encoding a PMCA. In adult males, the PMCA transcript has been found in various tissues, including the antennae in which its presence has been detected in the sensilla trichodea, and in cultured ORNs. The PMCA gene is slightly expressed at the end of the pupal stage, reaches a maximum at emergence and is maintained at a high level during the adult period. Taken together, these results provide, for the first time, molecular evidence for the putative participation of a PMCA in signalling pathways responsible for the establishment and functioning of the insect peripheral olfactory system.