Nicolas Montagné

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world’s worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains (“C” and “R”) that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis : a resource for olfactory and pheromone detection research

BackgroundNocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.ResultsBy targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.ConclusionsOur project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.

Putative Chemosensory Receptors of the Codling Moth, Cydia pomonella, Identified by Antennal Transcriptome Analysis

The codling moth, Cydia pomonella, is an important fruit pest worldwide. As nocturnal animals, adults depend to a large extent on olfactory cues for detection of food and mates, and, for females, oviposition sites. In insects, odor detection is mediated by odorant receptors (ORs) and ionotropic receptors (IRs), which ensure the specificity of the olfactory sensory neuron responses. In this study, our aim was to identify chemosensory receptors in the codling moth as a means to uncover new targets for behavioral interference. Using next-generation sequencing techniques, we identified a total of 43 candidate ORs, one gustatory receptor and 15 IRs in the antennal transcriptome. Through Blast and sequence similarity analyses we annotated the insect obligatory co-receptor ORco, five genes clustering in a conserved clade containing sex pheromone receptors, one homolog of the Bombyx mori female-enriched receptor BmorOR30 (but no homologs of the other B. mori female-enriched receptors) and one gene clustering in the sugar receptor family. Among the candidate IRs, we identified homologs of the two highly conserved co-receptors IR8a and IR25a, and one homolog of an IR involved in phenylethyl amine detection in Drosophila. Our results open for functional characterization of the chemosensory receptors of C. pomonella, with potential for new or refined applications of semiochemicals for control of this pest insect.

A predicted sex pheromone receptor of codling moth Cydia pomonella detects the plant volatile pear ester

Plant volatiles mediate host discrimination and host finding in phytophagous insects. Understanding how insects recognize these signals is a current challenge in chemical ecology research. Pear ester, ethyl (E,Z)-2,4-decadienoate, is a powerful, bisexual attractant of codling moth Cydia pomonella (Lepidoptera, Tortricidae) and strongly synergizes the male response to female-produced sex pheromone. We show here that the codling moth odorant receptor (OR) CpomOR3 is dedicated to detecting this plant volatile. Heterologous expression of CpomOR3 in Drosophila T1 trichoid and ab3A basiconic sensilla, followed by a screening with codling moth pheromone compounds and known plant volatile attractants, confirms that CpomOR3 binds to pear ester. Although CpomOR3 does not respond to any of the pheromone components tested, a phylogenetic analysis of lepidopteran chemosensory receptor genes reveals a close relationship of CpomOR3 with pheromone receptors (PRs) in moths. This corroborates the interaction of ecological and social chemosensory cues during premating communication. The finding that a plant volatile compound, pear ester, is a specific ligand for a PR-like lepidopteran receptor adds to our understanding of insect-plant interactions and emphasizes the interaction of natural and sexual selection during the phylogenetic divergence of insect herbivores.

A comparison of the olfactory gene repertoires of adults and larvae in the noctuid moth Spodoptera littoralis.

To better understand the olfactory mechanisms in a lepidopteran pest model species, the cotton leafworm Spodoptera littoralis, we have recently established a partial transcriptome from adult antennae. Here, we completed this transcriptome using next generation sequencing technologies, namely 454 and Illumina, on both adult antennae and larval tissues, including caterpillar antennae and maxillary palps. All sequences were assembled in 77,643 contigs. Their analysis greatly enriched the repertoire of chemosensory genes in this species, with a total of 57 candidate odorant-binding and chemosensory proteins, 47 olfactory receptors, 6 gustatory receptors and 17 ionotropic receptors. Using RT-PCR, we conducted the first exhaustive comparison of olfactory gene expression between larvae and adults in a lepidopteran species. All the 127 candidate olfactory genes were profiled for expression in male and female adult antennae and in caterpillar antennae and maxillary palps. We found that caterpillars expressed a smaller set of olfactory genes than adults, with a large overlap between these two developmental stages. Two binding proteins appeared to be larvae-specific and two others were adult-specific. Interestingly, comparison between caterpillar antennae and maxillary palps revealed numerous organ-specific transcripts, suggesting the complementary involvement of these two organs in larval chemosensory detection. Adult males and females shared the same set of olfactory transcripts, except two male-specific candidate pheromone receptors, two male-specific and two female-specific odorant-binding proteins. This study identified transcripts that may be important for sex-specific or developmental stage-specific chemosensory behaviors.

A diversity of putative carboxylesterases are expressed in the antennae of the noctuid moth Spodoptera littoralis.

Recent studies have suggested that pheromone‐degrading enzymes belonging to the carboxylesterase family could play a role in the dynamics of the olfactory response to acetate sex pheromones in insects. Bioinformatic analyses of a male antennal expressed sequence tag library allowed the identification of 19 putative esterase genes expressed in the antennae of the moth Spodoptera littoralis. Phylogenetic analysis revealed that these genes belong to different insect esterase clades, defined by their putative cellular localization and substrate preferences. Interestingly, two of the 19 genes appeared to be antennal specific, suggesting a specific role in olfactory processing. This high esterase diversity suggested that the antennae are the location for intense esterase‐based metabolism, against potentially a large range of exogenous and endogenous molecules.

Candidate chemosensory Genes in Female Antennae of the Noctuid Moth Spodoptera littoralis

Chemical senses are crucial for all organisms to detect various environmental information. Different protein families, expressed in chemosensory organs, are involved in the detection of this information, such as odorant-binding proteins, olfactory and gustatory receptors, and ionotropic receptors. We recently reported an Expressed Sequence Tag (EST) approach on male antennae of the noctuid moth, Spodoptera littoralis, with which we could identify a large array of chemosensory genes in a species for which no genomic data are available. Here we describe a complementary EST project on female antennae in the same species. 18,342 ESTs were sequenced and their assembly with our previous male ESTs led to a total of 13,685 unigenes, greatly improving our description of the S. littoralis antennal transcriptome. Gene ontology comparison between male and female data suggested a similar complexity of antennae of both sexes. Focusing on chemosensation, we identified 26 odorant-binding proteins, 36 olfactory and 5 gustatory receptors, expressed in the antennae of S. littoralis. One of the newly identified gustatory receptors appeared as female-enriched. Together with its atypical tissue-distribution, this suggests a role in oviposition. The compilation of male and female antennal ESTs represents a valuable resource for exploring the mechanisms of olfaction in S. littoralis.

Functional characterization of a sex pheromone receptor in the pest moth Spodoptera littoralis by heterologous expression in Drosophila

Moth sex pheromone communication is recognised as a long‐standing model for insect olfaction studies, and a widespread knowledge has been accumulated on this subject thanks to numerous chemical, electrophysiological and behavioural studies. A key step has been the identification of candidate sex pheromone receptors, opening new routes to understanding the specificity and sensitivity of this communication system, but only few of these receptors have as yet been functionally characterised. In this context, we aim at unravelling the molecular bases of pheromone reception in the noctuid moth Spodoptera littoralis. Taking advantage of a collection of antennal‐expressed sequence tags, we previously identified three fragments of candidate pheromone receptors in this species. Here, we report full‐length cloning of one of these receptors, named SlitOR6. Both sequence and expression pattern analyses were consistent with its annotation as a pheromone receptor, which we further confirmed by functional characterization. Using Drosophila antennae as a heterologous expression system, we identified a single component of the pheromone blend of S. littoralis, (Z,E)‐9,12‐tetradecadienyl acetate, as the ligand of SlitOR6. Two strategies were employed: (i) expressing SlitOR6 in the majority of Drosophila olfactory neurons, in addition to endogenous receptors, and monitoring the responses to pheromone stimuli by electroantennography; (ii) replacing the Drosophila pheromone receptor OR67d with SlitOR6 and monitoring the response by single sensillum recordings. Results were fully congruent and responses to (Z,E)‐9,12‐tetradecadienyl acetate were highly specific in both heterologous systems. This approach appears to be efficient and reliable for studying moth pheromone receptors in an in vivo context.

Molecular evolution of the crustacean hyperglycemic hormone family in ecdysozoans

BackgroundCrustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated.ResultsCHH family peptides were characterized by molecular cloning in a branchiopod crustacean, Daphnia magna, and in a collembolan, Folsomia candida. Genes encoding such peptides were also rebuilt in silico from genomic sequences of another branchiopod, a chelicerate and two nematodes. These sequences were included in updated datasets to build phylogenies of the CHH family in pancrustaceans. These phylogenies suggest that peptides found in Branchiopoda and Collembola are more closely related to insect ITPs than to crustacean CHHs. Datasets were also used to support a phylogenetic hypothesis about pancrustacean relationships, which, in addition to gene structures, allowed us to propose two evolutionary scenarios of this multigenic family in ecdysozoans.ConclusionsEvolutionary scenarios suggest that CHH family genes of ecdysozoans originate from an ancestral two-exon gene, and genes of arthropods from a three-exon one. In malacostracans, the evolution of the CHH family has involved several duplication, insertion or deletion events, leading to neuropeptides with a wide variety of functions, as observed in decapods. This family could thus constitute a promising model to investigate the links between gene duplications and functional divergence.

Candidate Chemosensory Genes in the Stemborer Sesamia nonagrioides

The stemborer Sesamia nonagrioides is an important pest of maize in the Mediterranean Basin. Like other moths, this noctuid uses its chemosensory system to efficiently interact with its environment. However, very little is known on the molecular mechanisms that underlie chemosensation in this species. Here, we used next-generation sequencing (454 and Illumina) on different tissues from adult and larvae, including chemosensory organs and female ovipositors, to describe the chemosensory transcriptome of S. nonagrioides and identify key molecular components of the pheromone production and detection systems. We identified a total of 68 candidate chemosensory genes in this species, including 31 candidate binding-proteins and 23 chemosensory receptors. In particular, we retrieved the three co-receptors Orco, IR25a and IR8a necessary for chemosensory receptor functioning. Focusing on the pheromonal communication system, we identified a new pheromone-binding protein in this species, four candidate pheromone receptors and 12 carboxylesterases as candidate acetate degrading enzymes. In addition, we identified enzymes putatively involved in S. nonagrioides pheromone biosynthesis, including a ∆11-desaturase and different acetyltransferases and reductases. RNAseq analyses and RT-PCR were combined to profile gene expression in different tissues. This study constitutes the first large scale description of chemosensory genes in S. nonagrioides.