Stage-specific expression of an odorant receptor underlies olfactory behavioral plasticity in Spodoptera littoralis larvae.

BACKGROUND: The detection of environmental cues and signals via the sensory system directs behavioral choices in diverse organisms. Insect larvae rely on input from the chemosensory system, mainly olfaction, for locating food sources. In several lepidopteran species, foraging behavior and food preferences change across larval instars; however, the molecular mechanisms underlying such behavioral plasticity during larval development are not fully understood. Here, we hypothesize that expression patterns of odorant receptors (ORs) change during development, as a possible mechanism influencing instar-specific olfactory-guided behavior and food preferences. RESULTS: We investigated the expression patterns of ORs in larvae of the cotton leafworm Spodoptera littoralis between the first and fourth instar and revealed that some of the ORs show instar-specific expression. We functionally characterized one OR expressed in the first instar, SlitOR40, as responding to the plant volatile, ß-caryophyllene and its isomer α-humulene. In agreement with the proposed hypothesis, we showed that first but not fourth instar larvae responded behaviorally to ß-caryophyllene and α-humulene. Moreover, knocking out this odorant receptor via CRISPR-Cas9, we confirmed that instar-specific responses towards its cognate ligands rely on the expression of SlitOR40. CONCLUSION: Our results provide evidence that larvae of S. littoralis change their peripheral olfactory system during development. Furthermore, our data demonstrate an unprecedented instar-specific behavioral plasticity mediated by an OR, and knocking out this OR disrupts larval behavioral plasticity. The ecological relevance of such behavioral plasticity for S. littoralis remains to be elucidated, but our results demonstrate an olfactory mechanism underlying this plasticity in foraging behavior during larval development.

Genetic mapping of male pheromone response in the European corn borer identifies candidate genes regulating neurogenesis.

The sexual pheromone communication system of moths is a model system for studies of the evolution of reproductive isolation. Females emit a blend of volatile components that males detect at a distance. Species differences in female pheromone composition and male response directly reinforce reproductive isolation in nature, because even slight variations in the species-specific pheromone blend are usually rejected by the male. The mechanisms by which a new pheromone signal-response system could evolve are enigmatic, because any deviation from the optimally attractive blend should be selected against. Here we investigate the genetic mechanisms enabling a switch in male response. We used a quantitative trait locus-mapping approach to identify the genetic basis of male response in the two pheromone races of the European corn borer, Ostrinia nubilalis Male response to a 99:1 vs. a 3:97 ratio of the E and Z isomers of the female pheromone is governed by a single, sex-linked locus. We found that the chromosomal region most tightly linked to this locus contains genes involved in neurogenesis but, in accordance with an earlier study, does not contain the odorant receptors expressed in the male antenna that detect the pheromone. This finding implies that differences in the development of neuronal pathways conveying information from the antenna, not differences in pheromone detection by the odorant receptors, are primarily responsible for the behavioral response differences among the males in this system. Comparison with other moth species reveals a previously unexplored mechanism by which male pheromone response can change in evolution.

Pheromone Receptor Knock-Out Affects Pheromone Detection and Brain Structure in a Moth.

Sex pheromone receptors are crucial in insects for mate finding and contribute to species premating isolation. Many pheromone receptors have been functionally characterized, especially in moths, but loss of function studies are rare. Notably, the potential role of pheromone receptors in the development of the macroglomeruli in the antennal lobe (the brain structures processing pheromone signals) is not known. Here, we used CRISPR-Cas9 to knock-out the receptor for the major component of the sex pheromone of the noctuid moth Spodoptera littoralis, and investigated the resulting effects on electrophysiological responses of peripheral pheromone-sensitive neurons and on the structure of the macroglomeruli. We show that the inactivation of the receptor specifically affected the responses of the corresponding antennal neurons did not impact the number of macroglomeruli in the antennal lobe but reduced the size of the macroglomerulus processing input from neurons tuned to the main pheromone component. We suggest that this mutant neuroanatomical phenotype results from a lack of neuronal activity due to the absence of the pheromone receptor and potentially reduced neural connectivity between peripheral and antennal lobe neurons. This is the first evidence of the role of a moth pheromone receptor in macroglomerulus development and extends our knowledge of the different functions odorant receptors can have in insect neurodevelopment.

Spodoptera littoralis genome mining brings insights on the dynamic of expansion of gustatory receptors in polyphagous noctuidae.

The bitter taste, triggered via gustatory receptors, serves as an important natural defense against the ingestion of poisonous foods in animals, and the increased host breadth is usually linked to an increase in the number of gustatory receptor genes. This has been especially observed in polyphagous insect species, such as noctuid species from the Spodoptera genus. However, the dynamic and physical mechanisms leading to these gene expansions and the evolutionary pressures behind them remain elusive. Among major drivers of genome dynamics are the transposable elements but, surprisingly, their potential role in insect gustatory receptor expansion has not been considered yet. In this work, we hypothesized that transposable elements and possibly positive selection would be involved in the highly dynamic evolution of gustatory receptor in Spodoptera spp. We first sequenced de novo the full 465 Mb genome of S. littoralis, and manually annotated the main chemosensory genes, including a large repertoire of 373 gustatory receptor genes (including 19 pseudogenes). We also improved the completeness of S. frugiperda and S. litura gustatory receptor gene repertoires. Then, we annotated transposable elements and revealed that a particular category of class I retrotransposons, the SINE transposons, was significantly enriched in the vicinity of gustatory receptor gene clusters, suggesting a transposon-mediated mechanism for the formation of these clusters. Selection pressure analyses indicated that positive selection within the gustatory receptor gene family is cryptic, only 7 receptors being identified as positively selected. Altogether, our data provide a new good quality Spodoptera genome, pinpoint interesting gustatory receptor candidates for further functional studies and bring valuable genomic information on the mechanisms of gustatory receptor expansions in polyphagous insect species.

bric à brac controls sex pheromone choice by male European corn borer moths.

The sex pheromone system of ~160,000 moth species acts as a powerful form of assortative mating whereby females attract conspecific males with a species-specific blend of volatile compounds. Understanding how female pheromone production and male preference coevolve to produce this diversity requires knowledge of the genes underlying change in both traits. In the European corn borer moth, pheromone blend variation is controlled by two alleles of an autosomal fatty-acyl reductase gene expressed in the female pheromone gland (pgFAR). Here we show that asymmetric male preference is controlled by cis-acting variation in a sex-linked transcription factor expressed in the developing male antenna, bric à brac (bab). A genome-wide association study of preference using pheromone-trapped males implicates variation in the 293 kb bab intron 1, rather than the coding sequence. Linkage disequilibrium between bab intron 1 and pgFAR further validates bab as the preference locus, and demonstrates that the two genes interact to contribute to assortative mating. Thus, lack of physical linkage is not a constraint for coevolutionary divergence of female pheromone production and male behavioral response genes, in contrast to what is often predicted by evolutionary theory.

A novel lineage of candidate pheromone receptors for sex communication in moths.

Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication.

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.

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.

Sex in the night: fatty acid-derived sex pheromones and corresponding membrane pheromone receptors in insects.

The moth sex pheromone communication is one of the most striking examples of chemical communication in the animal kingdom. Investigating the molecular mechanisms of pheromone biosynthesis in the female pheromone gland and of pheromone reception in the male antennae not only defines new concepts in signalling research but also opens new perspectives for insect control. In this mini-review, we use the cotton leafworm Spodoptera littoralis as a guideline to illustrate the recent advances gained in the understanding of moth sex pheromone communication.