Marie-Christine François

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.

A new member of the PBAN family in Spodoptera littoralis: molecular cloning and immunovisualisation in scotophase hemolymph

In this article, we report evidence suggesting that the immunoreactive factor previously detected in Spodoptera littoralis scotophase hemolymph is PBAN, which supports a humoral route of the hormone to the pheromone gland. Western blot after native-PAGE of prepurified scotophase hemolymph extracts yielded an immunoreactive band with the same mobility as S. littoralis Br-SOG factor and the expected mobility for a noctuid PBAN. This band was not detected in photophase hemolymph extract. The identity of S. littoralis Br-SOG factor as PBAN was obtained from cDNA cloning using RT-PCR strategy. This allowed us to deduce the amino acid sequence of Spl-PBAN, which is highly homologous to other known PBANs. Moreover, we found that the PBAN encoding cDNA also encoded four other putative amidated peptides (Spl-DH homologue, Spl-alpha-NP, Spl-beta-NP and Spl-gamma-NP) that are identical or highly conserved among noctuids, and two non amidated peptides of unknown function. This cDNA organization is common to all known cDNAs encoding PBANs, leading to the release of different peptides after putative enzymatic cleavage of the preprohormone.

Candidate chemosensory ionotropic receptors in a Lepidoptera

A new family of candidate chemosensory ionotropic receptors (IRs) related to ionotropic glutamate receptors (iGluRs) was recently discovered in Drosophila melanogaster. Through Blast analyses of an expressed sequenced tag library prepared from male antennae of the noctuid moth Spodoptera littoralis, we identified 12 unigenes encoding proteins related to D. melanogaster and Bombyx mori IRs. Their full length sequences were obtained and the analyses of their expression patterns suggest that they were exclusively expressed or clearly enriched in chemosensory organs. The deduced protein sequences were more similar to B. mori and D. melanogaster IRs than to iGluRs and showed considerable variations in the predicted ligand‐binding domains; none have the three glutamate‐interacting residues found in iGluRs, suggesting different binding specificities. Our data suggest that we identified members of the insect IR chemosensory receptor family in S. littoralis and we report here the first demonstration of IR expression in Lepidoptera.

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.

An antennal circadian clock and circadian rhythms in peripheral pheromone reception in the moth Spodoptera littoralis.

Circadian rhythms are observed in mating behaviors in moths: females emit sex pheromones and males are attracted by these pheromones in rhythmic fashions. In the moth Spodoptera littoralis, we demonstrated the occurrence of a circadian oscillator in the antenna, the peripheral olfactory organ. We identified different clock genes, period (per), cryptochrome1 (cry1) and cryptochrome2 (cry2), in this organ. Using quantitative real-time PCR (qPCR), we found that their corresponding transcripts cycled circadianly in the antenna as well as in the brain. Electroantennogram (EAG) recordings over 24 h demonstrated for the first time a circadian rhythm in antennal responses of a moth to sex pheromone. qPCR showed that out of one pheromone-binding protein (PBP), one olfactory receptor (OR), and one odorant-degrading enzyme (ODE), all putatively involved in the pheromone reception, only the ODE transcript presented a circadian rhythm that may be related to rhythms in olfactory signal resolution. Peripheral or central circadian clock control of olfaction is then discussed in light of recent data.

cDNA cloning of biotransformation enzymes belonging to the cytochrome P450 family in the antennae of the noctuid moth Mamestra brassicae.

The involvement of cytochrome P450 enzymes in olfaction was demonstrated in vertebrates some time ago. In insects these enzymes are well known for their role in insecticide resistance, but the involvement of P450 in pheromone degradation was only recently demonstrated. Using a PCR strategy, we have isolated two cDNAs from the antennae of the cabbage armyworm Mamestra brassicae–CYP4L4 and CYP4S4– which encode microsomal P450s. CYP4S4 expression is restricted to the antennae, whereas CYP4L4 is also found in the proboscis and legs. Moreover, the two genes are strongly expressed in one type of sensory unit of the antennae – the sensilla trichodea – which are tuned to the detection of odourants. The putative function of the corresponding enzymes is discussed with regard to their respective expression patterns.

Molecular cloning of two pheromone binding proteins in the cabbage armyworm Mamestra brassicae

Two cDNA clones encoding pheromone binding proteins (PBPs) were isolated from antennal cDNA of Mamestra brassicae by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE-PCR) performed with specific primers deduced from the N-terminal sequences of two PBPs previously reported. The deduced protein sequences of the two PBPs showed a strong relationship between primary structures and functional properties of the corresponding mature proteins.

Molecular identification and characterization of two new Lepidoptera chemoreceptors belonging to the Drosophila melanogaster OR83b family

In insect antennae, olfaction depends on olfactory receptors (ORs) that function through heterodimerization with an unusually highly conserved partner orthologue to the Drosophila melanogaster DOR83b. Here, we report the identification of two cDNAs encoding new DOR83b orthologues that represent the first members, although nonconventional, of the OR families of two noctuid crop pests, the cotton leafworm Spodoptera littoralis and the cabbage armyworm Mamestra brassicae. They both displayed high protein sequence conservation with previously identified DOR83b orthologues. Transcripts were abundantly detected in adult chemosensory organs as well as in fifth instar larvae heads. In adult antennae, the expression patterns of both genes revealed common features with other members of the OR83b subfamily: they appeared to be expressed at the bases of numerous olfactory sensilla belonging to different functional categories, suggesting that both receptors may be co‐expressed with yet unidentified conventional ORs. Bioinformatic analyses predicted the occurrence of seven transmembrane domains and an unusual topology with intracellular N‐termini and extracellular C‐termini, extending to Lepidoptera the hypothesis of an inverted topology for DOR83b orthologues, demonstrated to date only in D. melanogaster.

Molecular Cloning and in Situ Expression Patterns of Two New Pheromone-Binding Proteins from the Corn Stemborer Sesamia nonagrioides

We describe the identification and characterization of two new cDNAs encoding pheromone-binding proteins (PBPs) from the male antennae of Sesamia nonagrioides, a species where no PBPs have been identified to date. Because PBPs are thought to participate in the first step of odor detection in a specific manner, we focused our investigation on this olfactory protein family using reverse transcription–polymerase chain reaction strategies. The deduced amino acid sequences of SnonPBP1 and SnonPBP2 revealed mature proteins of 142 and 143 amino acids, respectively, with six cysteine residues in conserved positions relative to other known PBPs. The alignment of the two mature S. nonagrioides PBPs with other noctuid PBPs showed high sequence identity (70–80%) with other full-length sequences from GenBank. Sequence identity between SnonPBP1 and SnonPBP2 was only 46%, suggesting that the two proteins belong to different classes of PBPs already described from the Noctuidae. Furthermore, analyses of expression patterns of SnonPBP1 and SnonPBP2 were performed by in situ hybridization on antennae of both sexes, and these studies revealed the expression of the two PBPs at the bases of olfactory sensilla (basiconica or trichodea) from both sexes. The possible binding properties of these two new PBPs are discussed according to their homologies with other known PBPs and S. nonagrioides pheromone components.