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Sex pheromone biosynthesis in moths relies on the activity of multiple enzymes, including Δ9 desaturase, which plays an important role in catalyzing desaturation at the Δ9 position of the carbon chain. However, the physiological function of moth Δ9 desaturase has not been elucidated in vivo. In this study, we used the CRISPR/Cas9 system to knockout the Δ9 desaturase gene (SlitDes11) of Spodoptera litura to analyze its role in sex pheromone biosynthesis. First, through the direct injection of SlitDes11 -single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, gene editing was induced in around 30% of eggs 24 h after injection and was induced in 20.8% of the resulting adult moths. Second, using a sibling-crossing strategy, insects with mutant SlitDes11 (bearing a premature stop codon) were selected, and homozygous mutants were obtained in the G5 generation. Third, pheromone gland extracts of adult female homozygous SlitDes11 mutants were analyzed using Gas chromatography (GC). The results showed that titers of all three ester sex pheromone components; Z9, E11-14:Ac, Z9,E12-14:Ac, and Z9-14:Ac; were reduced by 62.40%, 78.50%, and 72.50%, respectively. This study provides the first direct evidence for the role of SlitDes11 in sex pheromone biosynthesis in S. litura , and indicates the gene could be as potential target to disrupt sexual communication in S. litura for developing a new pollution-free insecticide. Unlabelled Image • We obtained SlitDes11 homozygous knockout mutants by CRISPR/Cas9 system in S. litura. • The SlitDes11 play a key role in the ester sex pheromone biosynthesis of S. litura. • SlitDes11 could be as potential target for developing a new pollution-free insecticide. [ABSTRACT FROM AUTHOR]

Odorant-degrading enzymes (ODEs) in sensillar lymph are proposed to play important roles in the maintenance of the sensitivity of the olfactory sensilla, by timely degrading the odorants that have already fulfilled the activation of the odorant receptor (OR). Here we reported the cloning and characterization of an ODE gene ( SexiCXE10 ) from the polyphagous insect pest Spodoptera exigua . SexiCXE10 is a carboxylesterase (CXE) gene, encoding a protein with 538 amino acid residues, and bearing typical characteristics of Carboxyl/cholinesterase (CCE, EC 3.1.1.1.) gene family. Tissue-temporal expression pattern by qPCR revealed that the SexiCXE10 mRNA was highly antenna biased, and maintained at high level throughout the adult stage. Further fluorescence in situ hybridization demonstrated that SexiCXE10 mRNA signal was detected under sensilla basiconica and short and long sensilla trichodea. Finally, enzymatic study using purified recombinant enzyme showed that SexiCXE10 had high activity specifically for ester plant volatiles with 7–10 carbon atoms, while no activity was found with S. exigua sex pheromone components and plant volatiles with more carbon atoms. In addition, SexiCXE10 displayed lower activity at acidic pH (pH 5.0), while higher activity was found at neutral and alkaline conditions (pH 6.5–9.0). Our results suggest that SexiCXE10 may play an important role in the degradation of the host plant volatiles, and thus contributes to the high sensitivity of the olfactory system in S. exigua . Meanwhile, the CXE would be a potential target for developing behavioral antagonists and pesticides against S. exigua . [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The expression of RpedCSP4 in the adult antennae increased with age. • We acquired a high-quality recombinant RpedCSP4 protein. • RpedCSP4 had binding affinity to E2Z3, E2E2, and E2HH and most stable at pH 5. • L29A mutant should participate in the interaction between RpedCSP4 and ligands. [ABSTRACT FROM AUTHOR]

Athetis lepigone (Alep) is a polyphagous pest native to Europe and Asia that has experienced major outbreaks in the summer maize area of China since 2011 and has shown evidence of resistance to some insecticides. Insect olfaction is crucial for recognition of sex pheromones, host plant volatiles and even insecticides, in which two general-odorant binding proteins (GOBPs) play important roles. To elucidate the functions of GOBPs in A. lepigone, we first expressed the two AlepGOBP proteins in the E. coli expression system. Then, the results of fluorescence competitive binding assays demonstrated that the high binding affinity of AlepGOBP2 with sex pheromones [(Z)-7-dodecenyl acetate (Z7–12:Ac), Ki = 0.65 μM; (Z)-9-tetradecenyl acetate (Z9–14:Ac), Ki = 0.83 μM], two maize plant volatiles [Ocimene, Ki = 9.63 μM; (E)-β-Farnesene, Ki = 4.76 μM] and two insecticides (Chlorpyrifos Ki =5.61 μM; Phoxim, Ki = 4.38 μM). However, AlepGOBP1 could only bind Ocimene (Ki = 13.0 μM) and two insecticides (Chlorpyrifos Ki =4.46 μM; Phoxim, Ki = 3.27 μM). These results clearly suggest that AlepGOBP1 and AlepGOBP2 differentiate among odorants and other ligands. The molecular docking results further revealed different key residues involved in the ligand binding of AlepGOBPs. In summary, this study provides a foundation for exploring the olfactory mechanism of A. lepigone and identified two potential target genes for the development of highly effective insecticides in the future. Unlabelled Image • We first expressed the two AlepGOBPs of Athetis lepigone by E. coli system. • AlepGOBP2 has high binding to sex pheromones, two maize plant volatiles and two insecticides. • AlepGOBP1 could only bind one plant volatile and two insecticides. • Molecular docking revealed the different key residues involved in the ligand binding of AlepGOBPs. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The expression of RpedCSP12 in the adult antennae increased with age. • The expression levels of RpedCSP12 increased consistently over time. • We acquired a high-quality recombinant RpedCSP12 protein. • RpedCSP12 had binding affinity to (E)-2-hexenyl (Z)-3-hexenoate. • Three residues participate in the interaction between RpedCSP12 and pheromone. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • We acquired a high-quality recombinant CchiCSP1 protein. • CchiCSP1 had binding affinity to four different mung bean volatiles. • The binding affinity of CchiCSP1 to different volatiles was most stable at pH 7.4. • Three key residues participate in the interaction between CchiCSP1 and four volatiles. • The four mung bean volatiles had a significant behavioural response to C. chinensis. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The expression level of SfruGOBP1 in females was significantly higher than that in males. • SfruGOBP2 displayed male-biased expression. • Only SfruGOBP2 showed binding affinities for the four sex pheromones of female S. frugiperda. • Some key amino acid residues that can participate in the interaction between SfruGOBP2 and sex pheromones. [ABSTRACT FROM AUTHOR]

Boric acid, a well-established chemical insecticide, has a good control effect on various types of cockroaches. In this study, we investigated the oral virulence effect of boric acid on German cockroach (Blattella germanica) of various instars and characterized its effect on the gut microbiota by high-throughput sequencing technology. The results of an oral toxicity test showed that the toxicity of boric acid was positively correlated with its concentration and negatively correlated with the instar of cockroach nymphs. The 1–3 instar nymphs showed the strongest sensitivity to boric acid, which exhibited a median lethal time of only 3.16 d, while the 6–7 instar nymphs showed the weakest sensitivity, and exhibited a median lethal time of 10.15 d. There was no significant difference between male and female insects regarding their sensitivity to boric acid. Oral treatment of boric acid resulted in severe dysbiosis in cockroaches, the relative abundances of Bacteroides , which can degrade a variety of complex macromolecules, and Enterococcus , which can inhibit pathogenic microorganisms, were significantly reduced, while the relative abundance of the opportunistic pathogenic bacterium Weissella was significantly increased. It was speculated that dysbiosis of gut microbiota might accelerate the toxicity of boric acid on German cockroaches. Unlabelled Image • Different instars of Blattella germanica differed in sensitivity to boric acid. • Oral treatment of boric acid altered the composition of gut microbes of Blattella germanica. • Dysbiosis of gut microbiota might accelerate the toxicity of boric acid on Blattella germanica. [ABSTRACT FROM AUTHOR]