Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura.

Insect gustatory receptors (GRs) aid in the precise identification of deterrent or stimulant compounds associated with food, mating, and egg-laying. Thus, they are promising targets for developing efficient insecticides. Here, 61 GRs in the chemosensory organs of Spodoptera litura larvae and adults were identified. Among them, SlitGR206 exhibited larval labium (LL)-specific expression characteristics. To explore the role of SlitGR206, a bacterial expression system was established to produce high-quality double-stranded RNA (dsRNA) and suppress SlitGR206 expression in LL. Subsequent behavioral assessments revealed that SlitGR206 silencing influenced larval feeding preferences and absorption. Moreover, it was found to reduce the ability of larvae to forage the five crucial host odorants. These findings demonstrate that SlitGR206 likely plays an indirect regulatory role in host recognition, consequently affecting foraging behavior. This provides a crucial foundation for the analysis of functional diversity among insect GRs and the precise development of nucleic acid pesticides in the future.

Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones.

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

Screening of behaviorally active compounds based on the interaction between two chemosensory proteins and mung bean volatiles in Callosobruchus chinensis.

Chemosensory proteins (CSPs) are involved in the earliest steps of the olfactory process by binding and transporting odorants and play a crucial role in the insect's search for food and egg-laying sites. In the present study, the tissue expression profiles showed that both CchiCSP3 and CchiCSP5 of Callosobruchus chinensis were highly expressed in the adult antennae. Subsequently, the recombinant CchiCSP3 and CchiCSP5 proteins were analysed using fluorescence competitive binding assays, and both showed binding affinities for the three mung bean volatiles. Molecular docking and site-directed mutagenesis revealed four key amino acid residues in CchiCSP3 (L47, W80, Y81, and L84) and CchiCSP5 (Y28, K46, L49, and I72). Electroantennogram (EAG) and dual-choice biobehavioral assays showed that the antennae of adult C. chinensis were electrophysiologically active in response to stimulation with all three behaviorally active compounds and that octyl 4-methoxycinnamate and ß-ionone had a significant luring effect on adult C. chinensis, whereas vanillin had a significant avoidance effect. Our study screened three effective behaviorally active compounds based on the involvement of two CchiCSPs in the recognition of mung bean volatiles, providing an opportunity to develop an alternative control strategy using behavioral disruptors to limit the impact of pests.

Binding properties of chemosensory protein 12 in Riptortus pedestris to aggregation pheromone (E)-2-hexenyl (Z)-3-hexenoate.

Riptortus pedestris (bean bug), a common soybean pest, has a highly developed olfactory system to find hosts for feeding and oviposition. Chemosensory proteins (CSPs) have been identified in many insect species; however, their functions in R. pedestris remain unknown. In this study, quantitative real time-polymerase chain reaction (qRT-PCR) revealed that the expression of RpedCSP12 in the adult antennae of R. pedestris increased with age. Moreover, a significant difference in the expression levels of RpedCSP12 was observed between male and female antennae at one and three days of age. We also investigated the binding ability of RpedCSP12 to different ligands using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP12 only bound to one aggregation pheromone, (E)-2-hexenyl (Z)-3-hexenoate, and its binding decreased with increasing pH. Furthermore, homology modelling, molecular docking, and site-directed mutagenesis revealed that the Y27A, L74A, and L85A mutants lost their binding ability to (E)-2-hexenyl (Z)-3-hexenoate. Our findings highlight the olfactory roles of RpedCSP12, providing insights into the mechanism by which RpedCSPs bind to aggregation pheromones. Therefore, our study can be used as a theoretical basis for the population control of R. pedestris in the future.

Ligand-binding properties of chemosensory protein 1 in Callosobruchus chinensis to mung bean volatiles.

Callosobruchus chinensis (Coleoptera: Fabaceae) is a worldwide pest that feeds exclusively on legumes, and is the most serious pest affecting mung beans. Usually, the insect olfactory system plays a predominant role in searching for host plants and egg-laying locations. Chemosensory proteins (CSPs), are mainly responsible for transporting specific odour molecules from the environment. In this study, we found that the CSP1 gene of adult C. chinensis displayed antennae-biased expression using quantitative real-time PCR (qRT-PCR) analysis. The binding properties of 23 mung bean volatiles were then determined through several analyses of in vitro recombinant CSP1 protein, including fluorescence competitive binding assay, homology modelling, molecular docking, and site-directed mutagenesis. Fluorescence competitive binding assays showed that CchiCSP1 protein could bind to four mung bean volatiles and was most stable at pH 7.4. After site-directed mutation of three key amino acid bases (L39, V25, and Y35), their binding affinities to each ligand were significantly decreased or lost. This indicated that these three amino acid residues may be involved in the binding of CchiCSP1 to different ligands. We further used Y-tube behavioural bioassays to find that the four mung bean volatiles had a significant attraction or repulsion response in adult C. chinensis. The above findings confirm that the CchiCSP1 protein may be involved in the response of C. chinensis to mung bean volatiles and plays an important role in olfactory-related behaviours. The four active volatiles are expected to develop into new behavioural attractants or repellents in the future.

Functional differentiation of two general odorant-binding proteins to sex pheromones in Spodoptera frugiperda.

A precise chemosensory system can help insects complete various important behavioral responses by accurately identifying different external odorants. Therefore, deeply understanding the mechanism of insect recognition of important odorants will help us develop efficient and environmentally-friendly behavioral inhibitors. Spodoptera frugiperda is a polyphagous pest that feeds on >350 different host plants worldwide and also harms maize production in China. However, the molecular mechanism of the first step for males to use odorant-binding proteins (OBPs) to recognize sex pheromones remains unclear. Here, we obtained 50 OBPs from the S. frugiperda genome, and the expression level of SfruGOBP1 in females was significantly higher than that in males, whereas SfruGOBP2 displayed male-biased expression. Fluorescence competitive binding assays showed that only SfruGOBP2 showed binding affinities for the four sex pheromones of female S. frugiperda. Subsequently, we identified some key amino acid residues that can participate in the interaction between SfruGOBP2 and sex pheromones using molecular docking and site-directed mutagenesis methods. These findings will help us explore the interaction mechanism between GOBPs and sex pheromones in moths, and provide important target genes for developing new mating inhibitors of S. frugiperda in the future.

Two Odorant-Binding Proteins Involved in the Recognition of Sex Pheromones in Spodoptera litura Larvae.

Usually, the recognition of sex pheromone signals is restricted to adult moths. Here, our behavioral assay showed that fourth-instar Spodoptera litura larvae are attracted to cabbage laced with minor sex pheromones Z9,E12-tetradecadienyl acetate (Z9,E12-14:Ac) or Z9-tetradecenyl acetate (Z9-14:Ac). Seven odorant-binding proteins (OBPs) were upregulated after exposure to Z9,E12-14:Ac, and one OBP was upregulated after exposure to Z9-14:Ac. Fluorescence competitive binding assays showed that GOBP2 and OBP7 bound to sex pheromones. RNAi treatment significantly downregulated GOBP2 and OBP7 mRNA expression by 70.37 and 63.27%, respectively. The siOBP-treated larvae were not attracted to Z9,E12-14:Ac or Z9-14:Ac, and the corresponding preference indices were significantly lower than those in siGFP-treated larvae. Therefore, we concluded that GOBP2 and OBP7 are involved in the attraction of S. litura larvae to food containing Z9,E12-14:Ac and Z9-14:Ac. These results provide an important basis for exploring the olfactory mechanisms underlying sex pheromone attraction in moth larvae.

Genome-Wide Analysis of Odorant-Binding Proteins and Chemosensory Proteins in the Bean bug Riptortus pedestris.

Insects have sensitive olfactory systems to interact with environment and respond to the change in host plant conditions. Key genes in the system can be potential targets for developing new and efficient pest behaviour control methods. Riptortus pedestris is an important soybean pest in East Asia and has caused serious damage to the soybean plants in Huang-Huai-Hai region of China. However, the current treatment of pests is dominated by chemical insecticides and lacks efficient sustainable prevention and control technologies. In this study, we identified 49 putative odorant-binding proteins (OBPs) (43 were new genes) and 25 chemosensory proteins (CSPs) (17 were new genes) in R. pedestris genome. These OBP and CSP genes are clustered in highly conserved groups from other hemipteran species in phylogenetic trees. Most RpedOBPs displayed antennal-biased expression. Among the 49 RpedOBPs, 33 were significantly highly expressed in the antennae, including three male-biased and nine female-biased. While many RpedCSPs were detected both in the antennae and in non-antennal tissues, only 11 RpedCSPs displayed antennal-biased expression, in which four RpedCSPs were male-biased and five RpedCSPs were female-biased. Some OBP and CSP genes showed sex-biased expression profiles. Our results not only provide a foundation for future exploration of the functions of RpedOBPs and RpedCSPs but also aid in developing environmentally friendly insecticides in the future.

Binding properties of odorant-binding protein 4 from bean bug Riptortus pedestris to soybean volatiles.

The bean bug Riptortus pedestris is a notorious insect pest that can damage various crops, especially soybean, in East Asia. In insects, the olfactory system plays a crucial role in host finding and feeding behaviour in which the odorant-binding proteins (OBPs) are believed to be involved in initial step in this system. In this study, we produced the R. pedestris adult antennae-expressed RpedOBP4 protein using a recombinant expression system in E. coli. Fluorescence competitive binding confirmed that RpedOBP4 has binding affinities to 7 of 20 soybean volatiles (ligands), and that a neutral condition is the best environment for it. The binding property of RpedOBP4 to these ligands was further revealed by integrating data from molecular docking, site-directed mutagenesis and ligand binding assays. This demonstrated that five amino acid residues (I30, L33, Y47, I57 and Y121) are involved in the binding process of RpedOBP4 to corresponding ligands. These findings will not only help us to more thoroughly explore the olfactory mechanism of R. pedestris during feeding on soybean, but also lead to the identification of key candidate targets for developing environmental and efficient behaviour inhibitors to prevent population expansion of R. pedestris in the future.