An odorant receptor tuned to an attractive plant volatile vanillin in Spodoptera litura.

The insect olfaction plays crucial roles in many important behaviors, in which ORs are key determinants for signal transduction and the olfactory specificity. Spodoptera litura is a typical polyphagous pest, possessing a large repertoire of ORs tuning to broad range of plant odorants. However, the specific functions of those ORs remain mostly unknown. In this study, we functionally characterized one S. litura OR (OR51) that was highly expressed in the adult antennae. First, by using Xenopus oocyte expression and two-electrode voltage clamp recording system (XOE-TEVC), OR51 was found to be strongly and specifically responsive to vanillin (a volatile of S. litura host plants) among 77 tested odorants. Second, electroantennogram (EAG) and Y-tube behavioral experiment showed that vanillin elicited significant EAG response and attraction behavior especially of female adults. This female attraction was further confirmed by the oviposition experiment, in which the soybean plants treated with vanillin were significantly preferred by females for egg-laying. Third, 3D structural modelling and molecular docking were conducted to explore the interaction between OR51 and vanillin, which showed a high affinity (-4.46 kcal/mol) and three residues (Gln163, Phe164 and Ala305) forming hydrogen bonds with vanillin, supporting the specific binding of OR51 to vanillin. In addition, OR51 and its homologs from other seven noctuid species shared high amino acid identities (78-97%) and the same three hydrogen bond forming residues, suggesting a conserved function of the OR in these insects. Taken together, our study provides some new insights into the olfactory mechanisms of host plant finding and suggests potential applications of vanillin in S. litura control.

Transcriptome analysis and identification of chemosensory genes in the larvae of Plagiodera versicolora.

BACKGROUND: In insects, the chemosensory system is crucial in guiding their behaviors for survival. Plagiodera versicolora (Coleoptera: Chrysomelidae), is a worldwide leaf-eating forest pest in salicaceous trees. There is little known about the chemosensory genes in P. versicolora. Here, we conducted a transcriptome analysis of larvae heads in P. versicolora. RESULTS: In this study, 29 odorant binding proteins (OBPs), 6 chemosensory proteins (CSPs), 14 odorant receptors (ORs), 13 gustatory receptors (GRs), 8 ionotropic receptors (IRs) and 4 sensory neuron membrane proteins (SNMPs) were identified by transcriptome analysis. Compared to the previous antennae and foreleg transcriptome data in adults, 12 OBPs, 2 CSPs, 5 ORs, 4 IRs, and 7 GRs were newly identified in the larvae. Phylogenetic analyses were conducted and found a new candidate CO2 receptor (PverGR18) and a new sugar receptor (PverGR23) in the tree of GRs. Subsequently, the dynamic expression profiles of various genes were analyzed by quantitative real-time PCR. The results showed that PverOBP31, OBP34, OBP35, OBP38, and OBP40 were highly expressed in larvae, PverOBP33 and OBP37 were highly expressed in pupae, and PverCSP13 was highly expressed in eggs, respectively. CONCLUSIONS: We identified a total of 74 putative chemosensory genes based on a transcriptome analysis of larvae heads in P. versicolora. This work provides new information for functional studies on the chemoreception mechanism in P. versicolora.

Functional differentiation of three pheromone binding proteins in Orthaga achatina using mixed-type sex pheromones.

Pheromone-binding proteins (PBPs) play important roles in perception of insect sex pheromones, functioning to recognize and transport pheromone components onto the olfactory receptors of the odorant sensing neurons. Orthaga achatina, a serious pest of camphor trees, uses a mixture of three Type I (Z11-16:OAc, Z11-16:OH and Z11-16:Ald) and one Type II (Z3,Z6,Z9,Z12,Z15-23:H) sex pheromone components in its sex communication, in which Z11-16:OAc is the major component and others are minor components. In this study, we for the first time demonstrated that the three PBPs differentiated in recognition among pheromone components in a moth using mixed-type sex pheromones. First, tissue expression study showed that all three PBPs of O. achatina were expressed only in antennae and highly male-biased, suggesting their involvement in perception of the sex pheromones. Second, the three PBPs were expressed in Escherichia coli and the binding affinities of PBPs to four sex pheromone components and some pheromone analogs were determined by the fluorescence competition binding assays. The results showed that OachPBP1 bound all four sex pheromone components with high binding affinity, while OachPBP2 had high or moderate binding affinity only to three Type I components, and OachPBP3 had high binding affinity only to three minor pheromone components. Furthermore, key amino acid residues that bind to sex pheromone components were identified in three PBPs by 3-D structure modeling and ligand molecular docking, predicting the interactions between PBPs and pheromone components. Our study provides a fundamental insight into the olfactory mechanism in moths that use mixed-type sex pheromones.

Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

BACKGROUND: Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. RESULTS: First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. CONCLUSION: Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

Ionotropic Receptor IR75q.2 Mediates Avoidance Reaction to Nonanoic Acid in the Fall Armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae).

Ionotropic receptors (IRs) play an important role in olfaction, but little is known in nondrosophila insects. Here, we report in vitro and in vivo functional characterization of IR75q.2 in the invasive moth pest Spodoptera frugiperda. First, 13 IRs (including four coreceptor IRs) were found specifically or highly expressed in adult antennae. Second, these IRs were tested for responding profiles to 59 odorants using the Xenopus oocyte expression system, showing that only SfruIR75q.2 responded to 8-10C fatty acids and their corresponding aldehydes, with SfruIR8a as the only coreceptor. Third, the three acids (especially nonanoic acid) showed repellent effects on moth's behavior and oviposition, but the repellence significantly reduced to the insects with IR75q.2 knockout by CRISPR/Cas9. Taken together, our study reveals the function of SfruIR75q.2 in perception of acid and aldehyde odorants and provides the first in vivo evidence for olfactory function of an odor-specific IR in Lepidoptera.

Foreleg Transcriptomic Analysis of the Chemosensory Gene Families in Plagiodera versicolora (Coleoptera: Chrysomelidae).

Plagiodera versicolora (Coleoptera: Chrysomelidae) is a worldwide leaf-eating forest pest in salicaceous trees. The forelegs play important roles in the chemoreception of insects. In this study, we conducted a transcriptome analysis of adult forelegs in P. versicolora and identified a total of 53 candidate chemosensory genes encoding 4 chemosensory proteins (CSPs), 19 odorant binding proteins (OBPs), 10 odorant receptors (ORs), 10 gustatory receptors (GRs), 6 ionotropic receptors (IRs), and 4 sensory neuron membrane proteins (SNMPs). Compared with the previous antennae transcriptome data, 1 CSP, 4 OBPs, 1 OR, 3 IRs, and 4 GRs were newly identified in the forelegs. Subsequently, the tissue expression profiles of 10 P. versicolora chemosensory genes were performed by real-time quantitative PCR. The results showed that PverOBP25, PverOBP27, and PverCSP6 were highly expressed in the antennae of both sexes. PverCSP11 and PverIR9 are predominately expressed in the forelegs than in the antennae. In addition, the expression levels of PverGR15 in female antennae and forelegs were significantly higher than those in the male antennae, implying that it may be involved in some female-specific behaviors such as oviposition site seeking. This work would greatly further the understanding of the chemoreception mechanism in P. versicolora.

Functional Characterization of Sex Pheromone Receptors in the Fall Armyworm (Spodoptera frugiperda).

Pheromone receptors (PRs) found in the antennae of male moths play a vital role in the recognition of sex pheromones released by females. The fall armyworm (FAW), Spodoptera frugiperda, is a notorious invasive pest, but its PRs have not been reported. In this report, six candidate PRs (SfruOR6, 11, 13, 16, 56 and 62) suggested by phylogenetic analysis were cloned, and their tissue-sex expression profiles were determined by quantitative real-time PCR (qPCR). All six genes except for SfruOR6 were highly and specifically expressed in the antennae, with SfruOR6, 13 and 62 being male-specific, while the other three (SfruOR11, 16 and 56) were male biased, suggesting their roles in sex pheromone perception. A functional analysis by the Xenopus oocyte system further demonstrated that SfruOR13 was highly sensitive to the major sex pheromone component Z9-14:OAc and the pheromone analog Z9,E12-14:OAc, but less sensitive to the minor pheromone component Z9-12:OAc; SfruOR16 responded weakly to pheromone component Z9-14:OAc, but strongly to pheromone analog Z9-14:OH; the other four candidate PRs did not respond to any of the four pheromone components and four pheromone analogs. This study contributes to clarifying the pheromone perception in the FAW, and provides potential gene targets for developing OR-based pest control techniques.

The Molecular Basis of Host Selection in a Crucifer-Specialized Moth.

Glucosinolates (GSs) are sulfur-containing secondary metabolites characteristic of cruciferous plants [1, 2]. Their breakdown products, isothiocyanates (ITCs), are released following tissue disruption by insect feeding or other mechanical damages [3, 4]. ITCs repel and are toxic to generalist herbivores, while specialist herbivores utilize the volatile ITCs as key signals for localizing host plants [5, 6]. However, the molecular mechanisms underlying detection of ITCs remain open. Here, we report that in the diamondback moth Plutella xylostella, a crucifer specialist, ITCs indeed drive the host preference for Arabidopsis thaliana, and the two olfactory receptors Or35 and Or49 are essential for this behavior. By performing gene expression analyses, we identified 12 (out of 59 in total) female-biased Ors, suggesting their possible involvement in oviposition choice. By ectopically expressing these Ors in Xenopus oocytes and screening their responses with 49 odors (including 13 ITCs, 25 general plant volatiles, and 11 sex pheromone components), we found that Or35 and Or49 responded specifically to three ITCs (iberverin, 4-pentenyl ITC, and phenylethyl ITC). The same ITCs also exhibited highest activity in electroantennogram recordings with female antennae and were the strongest oviposition stimulants. Knocking out either Or35 or Or49 via CRISPR-Cas9 resulted in a reduced oviposition preference for the ITCs, while double Or knockout females lost their ITC preference completely and were unable to choose between wild-type A. thaliana and a conspecific ITC knockout plant. We hence conclude that the ITC-based oviposition preference of the diamondback moth for its host A. thaliana is governed by the cooperation of two highly specific olfactory receptors.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.