Lipases and carboxylesterases affect moth sex pheromone compounds involved in interspecific mate recognition.

Moth sex pheromones are a classical model for studying sexual selection. Females typically produce a species-specific pheromone blend that attracts males. Revealing the enzymes involved in the interspecific variation in blend composition is key for understanding the evolution of these sexual communication systems. The nature of the enzymes involved in the variation of acetate esters, which are prominent compounds in moth pheromone blends, remains unclear. We identify enzymes involved in acetate degradation using two closely related moth species: Heliothis (Chloridea) subflexa and H. (C.) virescens, which have different quantities of acetate esters in their sex pheromone. Through comparative transcriptomic analyses and CRISPR/Cas9 knockouts, we show that two lipases and two esterases from H. virescens reduce the levels of pheromone acetate esters when expressed in H. subflexa females. Together, our results show that lipases and carboxylesterases are involved in tuning Lepidoptera pheromones composition.

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.

The Bellerophon pipeline, improving de novo transcriptomes and removing chimeras.

Transcriptome quality control is an important step in RNA-Seq experiments. However, the quality of de novo assembled transcriptomes is difficult to assess, due to the lack of reference genome to compare the assembly to. We developed a method to assess and improve the quality of de novo assembled transcriptomes by focusing on the removal of chimeric sequences. These chimeric sequences can be the result of faulty assembled contigs, merging two transcripts into one. The developed method is incorporated into a pipeline, which we named Bellerophon, that is broadly applicable and easy to use. Bellerophon first uses the quality assessment tool TransRate to indicate the quality, after which it uses a transcripts per million (TPM) filter to remove lowly expressed contigs and CD-HIT-EST to remove highly identical contigs. To validate the quality of this method, we performed three benchmark experiments: (1) a computational creation of chimeras, (2) identification of chimeric contigs in a transcriptome assembly, (3) a simulated RNA-Seq experiment using a known reference transcriptome. Overall, the Bellerophon pipeline was able to remove between 40% and 91.9% of the chimeras in transcriptome assemblies and removed more chimeric than nonchimeric contigs. Thus, the Bellerophon sequence of filtration steps is a broadly applicable solution to improve transcriptome assemblies.

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.

Behavioral Effect of Plant Volatiles Binding to Spodoptera littoralis Larval Odorant Receptors.

Phytophagous insects use volatile organic compounds (VOC) emitted by plants to orient towards their hosts. In lepidopteran pests, crop damages are caused by larval stages-the caterpillars-that feed extensively on leaves or other plant tissues. However, larval host plant choice has been poorly studied, and it is generally admitted that caterpillars feed on the plant where the female laid the eggs. The mobility of caterpillars has been generally overlooked even though several studies showed that they can orient towards odors and change host plant. Recently, a large number of odorant receptors (ORs) tuned to plant volatiles have been characterized in the model pest moth Spodoptera littoralis (Noctuidae). In the present work, we identified nine of these deorphanized ORs as expressed in S. littoralis caterpillars. In order to understand whether these ORs are involved in host searching, we tested the behavioral significance of their ligands using a larval two-choice assay. This OR-guided approach led to the identification of nine plant volatiles, namely 1-hexanol, benzyl alcohol, acetophenone, benzaldehyde, (Z)3-hexenol, (E)2-hexenol, indole, DMNT and (Z)3-hexenyl acetate, which are active on S. littoralis caterpillar behavior, increasing our knowledge on larval olfactory abilities. To further explore the link between OR activation and behavioral output induced by plant volatiles we used a modeling approach, thereby allowing identification of some ORs whose activation is related to caterpillar attraction. These ORs may be promising targets for future plant protection strategies.

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.

Advances in the identification and characterization of olfactory receptors in insects.

Olfactory receptors (ORs) are the key elements of the molecular machinery responsible for the detection of odors in insects. Since their initial discovery in Drosophila melanogaster at the beginning of the twenty-first century, insect ORs have been the focus of intense research, both for fundamental knowledge of sensory systems and for their potential as novel targets for the development of products that could impact harmful behaviors of crop pests and disease vectors. In recent years, studies on insect ORs have entered the genomic era, with an ever-increasing number of OR genes being characterized every year through the sequencing of genomes and transcriptomes. With the upcoming release of genomic sequences from hundreds of insect species, the insect OR family could very well become the largest multigene family known. This extremely rapid identification of ORs in many insects is driving the necessity for the development of high-throughput technologies that will allow the identification of ligands for this unprecedented number of receptors. Moreover, such technologies will also be important for the development of agonists or antagonists that could be used in the fight against pest insects.