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.

Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis.

General odorant-binding proteins (GOBPs) are long considered responsible for the perception of plant odorants. In this study with the important noctuid pest Spodoptera litura, we functionally characterized that GOBP2 is also involved in the perception of sex pheromone components using in vivo CRISPR/Cas9 technique. First, the GOBP2 sgRNA and Cas9 protein were injected into the newly laid insect eggs, resulting in a 35.6% target mutagenesis in G0 moths. Then, the homozygous GOBP2 knockout strain (GOBP2-/-) was obtained after the screening of three generations. The knockout male and female moths displayed a significant reduction in EAG responses to the sex pheromone components, and the knockout females also displayed a significant reduction to plant odorants. In the behavioral assay of food choice, GOBP2-/- larvae lost the preference to artificial diet added with the major sex pheromone component Z9, E11-tetradecadienyl acetate (Z9, E11-14:Ac), whereas the WT larvae highly preferred the pheromone diet. Y-tube olfactometer assay and direct pheromone stimulation assay showed that GOBP2-/- male adults reduced significantly than WT males in percentages of choice, hair pencil displaying and mating attempt to Z9, E11-14:Ac. In the oviposition test, GOBP2-/- females showed significantly reduced preference for the soybean plants compared to the WT females. Our study demonstrated that GOBP2 plays an important role in perceiving sex pheromones in adult and larval stages, providing new insight into sex pheromone perception and a potential target for sex pheromone-based behavioral regulation in the pest.

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.