Christelle Monsempes

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.

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.

Mating-induced differential coding of plant odour and sex pheromone in a male moth

Innate behaviours in animals can be influenced by several factors, such as the environment, experience, or physiological status. This behavioural plasticity originates from changes in the underlying neuronal substrate. A well‐described form of plasticity is induced by mating. In both vertebrates and invertebrates, males experience a post‐ejaculatory refractory period, during which they avoid new females. In the male moth Agrotis ipsilon, mating induces a transient inhibition of responses to the female‐produced sex pheromone. To understand the neural bases of this inhibition and its possible odour specificity, we carried out a detailed analysis of the response characteristics of the different neuron types from the periphery to the central level. We examined the response patterns of pheromone‐sensitive and plant volatile‐sensitive neurons in virgin and mated male moths. By using intracellular recordings, we showed that mating changes the response characteristics of pheromone‐sensitive antennal lobe (AL) neurons, and thus decreases their sensitivity to sex pheromone. Individual olfactory receptor neuron (ORN) recordings and calcium imaging experiments indicated that pheromone sensory input remains constant. On the other hand, calcium responses to non‐pheromonal odours (plant volatiles) increased after mating, as reflected by increased firing frequencies of plant‐sensitive AL neurons, although ORN responses to heptanal remained unchanged. We suggest that differential processing of pheromone and plant odours allows mated males to transiently block their central pheromone detection system, and increase non‐pheromonal odour detection in order to efficiently locate food sources.

Heritable genome editing with CRISPR/Cas9 induces anosmia in a crop pest moth

Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species. Here we demonstrate that the CRISPR/Cas9 system is highly efficient for genome editing in a non-model crop pest Lepidoptera, the noctuid moth Spodoptera littoralis. We knocked-out the olfactory receptor co-receptor Orco gene to investigate its function in Lepidoptera olfaction. We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation. CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals. Our work genetically defines Orco as an essential OR partner for both host and mate detection in Lepidoptera, and demonstrates that CRISPR/Cas9 is a simple and highly efficient genome editing technique in noctuid pests opening new routes for gene function analysis and the development of novel pest control strategies.

Heterogeneity and convergence of olfactory first-order neurons account for the high speed and sensitivity of second-order neurons.

In the olfactory system of male moths, a specialized subset of neurons detects and processes the main component of the sex pheromone emitted by females. It is composed of several thousand first-order olfactory receptor neurons (ORNs), all expressing the same pheromone receptor, that contact synaptically a few tens of second-order projection neurons (PNs) within a single restricted brain area. The functional simplicity of this system makes it a favorable model for studying the factors that contribute to its exquisite sensitivity and speed. Sensory information—primarily the identity and intensity of the stimulus—is encoded as the firing rate of the action potentials, and possibly as the latency of the neuron response. We found that over all their dynamic range, PNs respond with a shorter latency and a higher firing rate than most ORNs. Modelling showed that the increased sensitivity of PNs can be explained by the ORN-to-PN convergent architecture alone, whereas their faster response also requires cell-to-cell heterogeneity of the ORN population. So, far from being detrimental to signal detection, the ORN heterogeneity is exploited by PNs, and results in two different schemes of population coding based either on the response of a few extreme neurons (latency) or on the average response of many (firing rate). Moreover, ORN-to-PN transformations are linear for latency and nonlinear for firing rate, suggesting that latency could be involved in concentration-invariant coding of the pheromone blend and that sensitivity at low concentrations is achieved at the expense of precise encoding at high concentrations.

Shifts in sensory neuron identity parallel differences in pheromone preference in the European corn borer

Pheromone communication relies on highly specific signals sent and received between members of the same species. However, how pheromone specificity is determined in moth olfactory circuits remains unknown. Here we provide the first glimpse into the mechanism that generates this specificity in Ostrinia nubilalis. In Ostrinia nubilalis it was found that a single locus causes strain-specific, diametrically opposed preferences for a 2-component pheromone blend. Previously we found pheromone preference to be correlated with the strain and hybrid-specific relative antennal response to both pheromone components. This led to the current study, in which we detail the underlying mechanism of this differential response, through chemotopically mapping of the pheromone detection circuit in the antenna. We determined that both strains and their hybrids have swapped the neuronal identity of the pheromone-sensitive neurons co-housed within a single sensillum. Furthermore, neurons that mediate behavioral antagonism surprisingly co-express up to five pheromone receptors, mirroring the concordantly broad tuning to heterospecific pheromones. This appears as possible evolutionary adaptation that could prevent cross attraction to a range of heterospecific signals, while keeping the pheromone detection system to its simplest tripartite setup.

The use of the sex pheromone as an evolutionary solution to food source selection in caterpillars

Sex pheromones are released by adults of a species to elicit a sexual interaction with the other sex of the same species. Here we report an unexpected effect of a moth sex pheromone on the caterpillars of the same species. We demonstrate that larvae of the cotton leafworm Spodoptera littoralis are attracted by the moth sex pheromone and that this phenomenon is independent of sex determination. In addition, we show that the olfactory sensilla carried by the caterpillar antennae are sensitive to the pheromone and that the caterpillar sensilla express pheromone-binding proteins that are used by adult antennae to bind pheromone components. Finally, we demonstrate that the larvae are preferentially attracted to a food source when it contains the sex pheromone main component. A possible interpretation of these results is that the sex pheromone is used to promote food search in caterpillars, opening potential new routes for insect pest management.

Experience-dependent modulation of antennal sensitivity and input to antennal lobes in male moths (Spodoptera littoralis) pre-exposed to sex pheromone.

SUMMARY Sex pheromones are intraspecific olfactory signals emitted by one sex to attract a potential mating partner. Behavioural responses to sex pheromones are generally highly stereotyped. However, they can be modulated by experience, as male moths previously exposed to female sex pheromone respond with a lower threshold upon further detection, even after long delays. Here, we address the question of the neural mechanisms underlying such long-term modulation. As previous work has shown increased responses to pheromone in central olfactory neurons, we asked whether brief exposure to the pheromone increases input activity from olfactory receptor neurons. Males pre-exposed to sex pheromone exhibited increased peripheral sensitivity to the main pheromone component. Among nine antennal genes targeted as putatively involved in pheromone reception, one encoding a pheromone-binding protein showed significant upregulation upon exposure. In the primary olfactory centre (antennal lobe), the neural compartment processing the main pheromone component was enlarged after a brief pheromone exposure, thus suggesting enduring structural changes. We hypothesise that higher peripheral sensitivity following pre-exposure leads to increased input to the antennal lobe, thus contributing to the structural and functional reorganization underlying a stable change in behaviour.

Functional evolution of Lepidoptera olfactory receptors revealed by deorphanization of a moth repertoire

Insects detect their hosts or mates primarily through olfaction, and olfactory receptors (ORs) are at the core of odorant detection. Each species has evolved a unique repertoire of ORs whose functional properties are expected to meet its ecological needs, though little is known about the molecular basis of olfaction outside Diptera. Here we report a pioneer functional analysis of a large array of ORs in a lepidopteran, the herbivorous pest Spodoptera littoralis. We demonstrate that most ORs are narrowly tuned to ubiquitous plant volatiles at low, relevant odorant titres. Our phylogenetic analysis highlights a basic conservation of function within the receptor repertoire of Lepidoptera, across the expansive evolutionary radiation of different major clades. Our study provides a reference for further studies of olfactory mechanisms in Lepidoptera, a historically crucial insect order in olfactory research.