Analysis of chemosensory genes in Semiothisa cinerearia reveals sex-specific contributions for type-II sex pheromone chemosensation.

Insects employ a sensitive chemosensory system to accurately recognize external odorants, which help them to make a behavioral response quickly. Semiothisa cinerearia has caused serious damages to Sophora japonica L. in recent years, and there is still a lack of effective strategy to control the pest. Although the two type-II sex pheromones of S. cinerearia, 6Z,9Z-cis-3,4-epoxy-17:H and 3Z,6Z,9Z-17:H, have been identified for 30 years, the molecular mechanisms underlying the chemosensation of the two sex pheromones are still unknown. Here, we found that there are differences in the types of antennae sensilla between sexes, and revealed 146 putative chemosensory genes in the antennal transcriptome. Among these genes, 11 and 40 of them displayed male-biased and female-biased expression, respectively. Our findings greatly improve the chemosensory gene resources for S. cinerearia and provide a foundation for functional studies of these sex-biased genes on the chemosensation of sex pheromones and on other sex-related behaviors.

Molecular characterization and functional analysis of a novel candidate of cuticle carboxylesterase in Spodoptera exigua degradating sex pheromones and plant volatile esters.

Odorant-degrading enzymes (ODEs) are considered to play key roles in odorant inactivation to maintain the odorant receptor sensitivity of insects. Some members of carboxylesterase (CXE) is a major sub-family of ODEs. However, only a few CXEs have been functionally characterized so far. In the present study, we cloned the antennal esterase SexiCXE11 cDNA full-length sequences from the male antennae of a notorious crop pest, Spodoptera exigua, and its encoded 538 amino acids. It was similar to other insect esterases and had the characteristics of a carboxylesterase. We expressed recombinant enzyme in High-Five insect cells and obtained the high level purified recombinant protein by affinity column. Furthermore we test enzyme activity toward its two acetate sex pheromone components (Z9,E12-Tetradecadienyl acetate, Z9E12-14:Ac and Z9-Tetradecenyl acetate, Z9-14:Ac) and other 18 ester plant volatiles. Our results demonstrated that SexiCXE11 degraded acetate sex pheromone components with similar degradation activities (about 15.75% with Z9E12-14:Ac and 19.28% with Z9-14:Ac) and plant volatiles with a relatively high activity such as pentyl acetate and (Z)-3-hexenyl caproate. SexiCXE11 had high hydrolytic activity with these two ester odorants (>50% degradation), which is characterized that although a ubiquitous expression esterase SexiCXE11 may be partly involved with olfaction. This study may facilitate a better understanding of moth ODE differentiation and suggest strategies for the development of new pest behavior inhibitors.

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.

Functional Disparity of Three Pheromone-Binding Proteins to Different Sex Pheromone Components in Hyphantria cunea (Drury).

Hyphantria cunea (Drury) is a destructive invasive pest species in China that uses type II sex pheromone components. To date, however, the binding mechanisms of its sex pheromone components to their respective pheromone-binding proteins (HcunPBPs 1/2/3) have not been explored. In the current study, all three HcunPBPs were expressed in the antennae of both sexes. The prokaryotic expression and ligand binding assays were employed to study the binding of the moth's four sex pheromone components, including two aldehydes and two epoxides, and 24 plant volatiles to the HcunPBPs. Our results showed that the abilities of these HcunPBPs to bind to the aldehydes were significantly different from binding to the epoxides. These three HcunPBPs also selectively bind to some of the plant volatiles tested. Our molecular docking results indicated that some crucial hydrophobic residues might play a role in the binding of HcunPBPs to their sex pheromone components. Three HcunPBPs have different selectivities for pheromone components with both major and minor structural differences. Our study provides a fundamental insight into the olfactory mechanism of moths at the molecular level, especially for moth species that use various type II pheromone components.

Identification and Expression Profile of Olfactory Receptor Genes Based on Apriona germari (Hope) Antennal Transcriptome.

Insects' olfactory receptor plays a central role in detecting chemosensory information from the environment. Odorant receptors (ORs) and ionotropic receptors (IRs) are two types of olfactory receptors, and they are essential for the recognition of ligands at peripheral neurons. Apriona germari (Hope) (Coleoptera: Cerambycidae) is one of the most serious insect pests that cause damage to economic trees and landscaping trees, resulting in massive environmental damages and economic losses. Olfactory-based management strategy has been suggested as a promising strategy to control this wood-boring beetle. However, the olfactory perception mechanism in A. germari is now almost unknown. In the present study, RNA sequencing analysis was used to determine the transcriptomes of adult A. germari antennae. Among 36,834 unigenes derived from the antennal assembly, we identified 42 AgerORs and three AgerIRs. Based on the tissue expression pattern analysis, 27 AgerORs displayed a female-biased expression. Notably, AgerOR3, 5, 13, 33, and 40 showed a significant female-biased expression and were clustered with the pheromone receptors of Megacyllene caryae in the phylogenetic tree, suggesting that these AgerORs could be potential pheromone receptors for sensing male-produced sex pheromones in A. germari. The AgerIRs expression profile demonstrated that AgerIR2 had high expression levels in male labial palps, suggesting that this receptor may function to detect female-deposited trail-sex pheromone blend of A. germari. In addition, the phylogenetic tree showed that the Orco gene of five cerambycidae species was highly conservative. These results provide a foundation for further studies on the molecular mechanisms of olfactory chemoreception in A. germari apart from suggesting novel targets for the control of this pest in the future.

Organophosphorus insecticide interacts with the pheromone-binding proteins of Athetis lepigone: Implication for olfactory dysfunction.

Athetis lepigone is one of the most severe polyphagous pests, and it has developed resistance to different chemical insecticides. Insects primarily rely on the olfactory system to recognize various environmental chemicals, including xenobiotics such as insecticides. Here, we expressed two A. lepigone pheromone-binding proteins (AlepPBP2 and AlepPBP3), and observed they had higher binding affinities to phoxim than other insecticides, with Ki was 3.30 ±â€¯0.38 µM and 3.27 ±â€¯0.10 µM, respectively. Molecular dynamics simulation, binding mode analysis, and computational alanine scanning showed that six residues (Phe15, Phe39, Ile55, Leu65, Ile97, and Phe122) of AlepPBP2 and three residues (Phe12, Ile52, and Ile134) of AlepPBP3 maybe as potential residues that can change protein ability to bind an organophosphorus insecticide phoxim. Then, we used site-directed mutagenesis assay to mutate these residues into alanine, respectively. Subsequently, the binding assays displayed that Phe15, Phe39, and Ile97 of AlepPBP2, Phe12 and Ile134 of AlepPBP3 caused a significant decrease of AlepPBPs binding ability to phoxim, suggesting they should play crucial roles in the AlepPBPs/phoxim interactions. Our findings could further advance in using PBPs as unique targets to design and develop precise and environmentally-friendly pest control agents with high insecticidal potential using a computer-aided drug design (CADD) approach.

Key Amino Acid Residues Influencing Binding Affinities of Pheromone-Binding Protein from Athetis lepigone to Two Sex Pheromones.

Athetis lepigone is a polyphagous pest found around the world that feeds on maize, wheat, and various other important crops. Although it exhibits a degree of resistance to various chemical insecticides, an effective pest-control method has not yet been developed. The sex pheromone communication system plays an essential role in the mating and reproduction of moths, in which pheromone-binding proteins (PBPs) are crucial genes. In this study, we cloned and purified the protein AlepPBP1 using an E. coli expression system and found it had a higher binding affinity to two sex pheromones of A. lepigone, namely, Z7-12:Ac and Z9-14:Ac (with Ki 0.77 ± 0.10 and 1.10 ± 0.20 µM, respectively), than to other plant volatiles. The binding-mode analysis of protein conformation with equilibrium stabilization was obtained using molecular dynamics (MD) simulation and indicated that hydrophobic interactions involving several nonpolar residues were the main driving force for the binding affinity of AlepPBP1 with sex pheromones. Computational alanine scanning (CAS) was performed to further identify key amino acid residues and validate their binding contributions. Each key residue, including Phe36, Trp37, Val52, and Phe118, was subsequently mutated into alanine using site-directed mutagenesis. Binding assays showed that the efficient binding abilities to Z7-12:Ac (F36A, W37A, and F118A) and Z9-14:Ac (F36A, W37A, V52A, and F118A) were almost lost in the mutated proteins. Our results demonstrated that these key amino acid residues are crucial for determining the binding ability of AlepPBP1 to sex pheromones. These findings provide a basis for the use of AlepPBP1 in the studies as a specific target for the development of novel behavioral antagonists with marked inhibition or mating-disruption abilities using computer-aided drug design (CADD).