Trial and error: New insights into recombinant expression of membrane-bound insect cytochromes P450 in Escherichia coli systems.

Insect cytochromes P450 (CYP450s) are key enzymes responsible for a wide array of oxidative transformations of both endogenous and exogenous substrates. However, there is currently no a universal guideline established for heterologous expression of membrane-bound CYP450s, which hampers their downstream biochemical and structural studies. In this study, we conducted large-scale screening of protein overexpression in Escherichia coli using 71 insect CYP450 sequences and optimized the expression of a difficult-to-express CYP450 (CYP6HX3) using eight different optimizations, including selection of host strains and expression vectors, alternative of leader signal peptides, and N-terminal modifications. We confirmed that 1) Only insect CYP450s belonging to the CYP347 family could be expressed with N-terminal fusion of ompA2+ signal peptide in E. coli expression system. 2) E. coli Lemo 21 (DE3) effectively improved the expression of CYP6HX3 in the plasma membrane. 3) A brick-red appearance occurred frequently in the expressed thallus or membrane proteins, but this phenomenon could not necessarily indicate successful overexpression of target CYP450s. These findings provide new insights into the recombinant expression of insect CYP450s in E. coli systems and will facilitate the theoretical approaches for functional expression and production of eukaryotic CYP450s.

Heat stress-induced oviposition behavioral change correlates with sperm damage in the pine sawyer beetle, Monochamus alternatus.

BACKGROUND: Global climate change is causing an increase in extreme high temperatures (EHTs), which subject insects to unprecedented stress. Behavior plasticity in response to EHTs, particularly oviposition behavior, is important for the persistence and outbreak of insect populations. Investigating the plasticity of oviposition behavior and its underlying mechanisms has theoretical importance to pest management, but knowledge gaps still remain. RESULTS: Herein, we characterized the reproductive traits of Monochamus alternatus, a dominant insect vector of the destructive pine wilt disease, including oviposition behavioral patterns, fecundity, offspring fitness and sperm viability, under simulated heatwave conditions in the laboratory. The results showed that (i) EHTs induced a novel oviposition behavior, whereby females deposited multiple eggs into a single groove rather than laying one egg per groove under normal condition; (ii) EHTs exerted stage- and sex-specific effects on fecundity, offspring fitness and sperm viability; and (iii) there was a significant correlation between frequency of the novel oviposition strategy and sperm viability. CONCLUSION: We hypothesized that this beetle pest has the ability to flexibly shift towards a low-cost oviposition strategy to counteract the fitness costs caused by heat stress. Taken together, these findings provide a theoretical foundation for personalized pest management strategies in the context of climate change. © 2024 Society of Chemical Industry.

Biology of Euwallacea interjectus, an emerging poplar pest, reared on an ambrosia beetle artificial diet and medium of fungal symbiont.

Euwallacea interjectus, a recently discovered pest in poplar plantations, poses a significant economic threat due to its role in causing widespread tree mortality. This pest's cryptic behaviour has hindered research and control efforts, making laboratory rearing a valuable tool for studying its development and biology. We investigated the development period and biological characteristics of E. interjectus using artificial diets and fungal medium. Our findings revealed that the development time for eggs, larvae, and pupae averages approximately 6, 18, and 6 days, respectively. Notably, first and second instar larvae displayed peak moulting periods at 3.45 ± 0.64 SD and 7.92 ± 1.77 SD days, respectively. Furthermore, we measured head capsule widths of postmolt larvae, yielding values of 318.02 ± 7.38 SD µm for first-instar larvae, 403.01 ± 11.08 SD µm for second-instar larvae, and 549.54 ± 20.74 SD µm for third-instar larvae. Our research also uncovered a positive correlation between the number of progeny (eggs, larvae, pupae, and adults) and the mean length of the gallery system. Interestingly, the haplodiploid reproductive strategy did not significantly affect the number of offspring produced by the foundress. Additionally, we observed that foundresses displayed higher fecundity when subjected to nutrient-rich diets as compared to nutrient-poor diets. Our results will deepen our understanding of the biology of E. interjectus and provide criteria for larval instar classification. Additionally, managing nutrient availability within the colony could be considered a viable approach to regulating population size.

Microbial changes and associated metabolic responses modify host plant adaptation in Stephanitis nashi.

Symbiotic microorganisms are essential for the physiological processes of herbivorous pests, including the pear lace bug Stephanitis nashi, which is known for causing extensive damage to garden plants and fruit trees due to its exceptional adaptability to diverse host plants. However, the specific functional effects of the microbiome on the adaptation of S. nashi to its host plants remains unclear. Here, we identified significant microbial changes in S. nashi on 2 different host plants, crabapple and cherry blossom, characterized by the differences in fungal diversity as well as bacterial and fungal community structures, with abundant correlations between bacteria or fungi. Consistent with the microbiome changes, S. nashi that fed on cherry blossom demonstrated decreased metabolites and downregulated key metabolic pathways, such as the arginine and mitogen-activated protein kinase signaling pathway, which were crucial for host plant adaptation. Furthermore, correlation analysis unveiled numerous correlations between differential microorganisms and differential metabolites, which were influenced by the interactions between bacteria or fungi. These differential bacteria, fungi, and associated metabolites may modify the key metabolic pathways in S. nashi, aiding its adaptation to different host plants. These results provide valuable insights into the alteration in microbiome and function of S. nashi adapted to different host plants, contributing to a better understanding of pest invasion and dispersal from a microbial perspective.

Identification and Functional Analysis of an Epsilon Class Glutathione S-Transferase Gene Associated with α-Pinene Adaptation in Monochamus alternatus.

Alpha-pinene is one of the main defensive components in conifers. Monochamus alternatus (Coleoptera: Cerambycidae), a wood borer feeding on Pinaceae plants, relies on its detoxifying enzymes to resist the defensive terpenoids. Here, we assayed the peroxide level and GST activity of M. alternatus larvae treated with different concentrations of α-pinene. Meanwhile, a gst gene (MaGSTe3) was isolated and analyzed. We determined its expression level and verified its function. The results showed that α-pinene treatment led to membrane lipid peroxidation and thus increased the GST activity. Expression of MaGSTe3 was significantly upregulated in guts following exposure to α-pinene, which has a similar pattern with the malonaldehyde level. In vitro expression and disk diffusion assay showed that the MaGSTe3 protein had high antioxidant capacity. However, RNAi treatment of MaGSTe3 did not reduce the hydrogen peroxide and malonaldehyde levels, while GST activity was significantly reduced. These results suggested MaGSTe3 takes part in α-pinene adaptation, but it does not play a great role in the resistance of M. alternatus larvae to α-pinene.

Spatiotemporal Patterns of Five Small Heat Shock Protein Genes in Hyphantria cunea in Response to Thermal Stress.

Hyphantria cunea (Drury), a destructive polyphagous pest, has been spreading southward after invading northern China, which indicates that this insect species is facing a huge thermal challenge. Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones that protect insects from heat stress damage. In order to explore the role of sHSPs in the thermotolerance of H. cunea, five novel sHSP genes of H. cunea were cloned, including an orthologous gene (HcHSP21.4) and four species-specific sHSP genes (HcHSP18.9, HcHSP20.1, HcHSP21.5, and HcHSP29.8). Bioinformatics analysis showed that the proteins encoded by these five HcHSPs contained typical α-crystallin domains. Quantitative real-time PCR analysis revealed the ubiquitous expression of all HcHSPs across all developmental stages of H. cunea, with the highest expression levels in pupae and adults. Four species-specific HcHSPs were sensitive to high temperatures. The expression levels of HcHSPs were significantly up-regulated under heat stress and increased with increasing temperature. The expression levels of HcHSPs in eggs exhibited an initial up-regulation in response to a temperature of 40 °C. In other developmental stages, the transcription of HcHSPs was immediately up-regulated at 30 °C or 35 °C. HcHSPs transcripts were abundant in the cuticle before and after heat shock. The expression of HcHSP21.4 showed weak responses to heat stress and constitutive expression in the tissues tested. These results suggest that most of the HcHSPs are involved in high-temperature response and may also have functions in the normal development and reproduction of H. cunea.

An oral dsRNA delivery system based on chitosan induces G protein-coupled receptor kinase 2 gene silencing for Apolygus lucorum control.

RNA interference (RNAi) is recognized as a new and environmentally friendly pest control strategy due to its high specificity. However, the RNAi efficiency is relatively low in many sucking insect pests, such as Apolygus lucorum. Therefore, there is an urgent need to develop new and effective ways of dsRNA delivery. Bacterially expressed or T7 synthesized dsRNA targeting a G Protein-Coupled Receptor Kinase 2 gene was mixed with chitosan in a 1:2 ratio by mass. The size of the chitosan/dsRNA nanoparticles was 69 ± 12 nm, and the TEM and AFM images showed typical spherical or ellipsoidal structures. The chitosan nanoparticles protected the dsRNA from nuclease activity, and pH and temperature-dependent degradation, and the fluorescently-tagged nanoparticles were found to be stable on the surface of green bean plants (48 h) (Phaseolus vulgaris) and were absorbed by midgut epithelial cells and transported to hemolymph. Once fed to the A. lucorum nymph, chitosan/dsRNA could effectively inhibit the expression of the G protein-coupled receptor kinase 2 gene (70%), and led to significantly increase mortality (50%), reduced weight (26.54%) and a prolonged developmental period (8.04%). The feeding-based and chitosan-mediated dsRNA delivery method could be a new strategy for A. lucorum management, providing an effective tool for gene silencing of piercing-sucking insects.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

BACKGROUND: Commensal microorganisms are widely distributed in insect gut tissues and play important roles in host nutrition, metabolism, reproductive regulation, and especially immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. RESULTS: We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that re-introduction of the HcM7 strain to germfree larvae preactivated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. CONCLUSION: These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest. © 2023 Society of Chemical Industry.

Isolation and characterization of gut bacteria associated with the degradation of host-specific terpenoids in Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae.

Insect intestinal bacteria play an important role in resisting defensive substances of host plants. Pagiophloeus tsushimanus (Coleoptera: Curculionidae) feeds exclusively on camphor trees (Cinnamomum camphora, Laurales: Lauraceae) in China, causing substantial economic and ecological losses. It is unclear how the larvae of P. tsushimanus outcome the main secondary metabolites of C. camphora such as D-camphor, eucalyptol, and linalool. In this study, we isolated terpenoid-degrading bacteria from the gut of P. tsushimanus larvae by using selective culture medium. Maximum likelihood phylogenetic analyses were performed with 16S rDNA sequences to identify the bacteria, and results showed ten strains belonged to four genera, including Pseudomonas, Enterobacter, Serratia, and Corynebacterium. Then, gas chromatography was employed to determine the degradability of D-camphor, eucalyptol, and linalool by the isolated strains, results showed that Z5 strain (i.e., Corynebacterium variabile, Actinomycetales: Corynebacteriaceae), F1 strain (i.e., Pseudomonas aeruginosa, Pseudomonadales: Pseudomonaceae), and A3 strain (i.e., Serratia marcescens, Enterobacterales: Enterobacteriaceae) had the highest degradation rates of D-camphor, linalool, and eucalyptol, respectively. The intestinal bacteria were capable of terpenoid degradation in vitro, which suggested that these gut bacteria associated with P. tsushimanus play an important role in overcoming host plant secondary metabolite defense, thereby facilitating the host specialization of this pest.

Effects of midgut bacteria in Hyphantria cunea (Lepidoptera: Erebidae) on nuclear polyhedrosis virus and Bacillus thuringiensis (Bacillales: Bacillaceae).

Hyphantria cunea Drury (Lepidoptera: Erebidae) is a quarantine pest in China that can cause damage to hundreds of plants. As biological control agents, Nuclear Polyhedrosis Virus (NPV) and Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt) are commonly used to inhibit the prevalence of H. cunea. To investigate the role of midgut bacteria in the infection of NPV and Bt in H. cunea, we performed a series of tests, including isolating the dominant culturable bacteria in the midgut, eliminating intestinal bacteria, and respectively inoculating the dominant strains with NPV and Bt for bioassay. Two dominant bacteria, Klebsiella oxytoca Lautrop (Enterobacterales: Enterobacteriaceae) and Enterococcus mundtii Collins (Lactobacillales: Enterococcaceae), in the midgut of H. cunea were identified, and a strain of H. cunea larvae without intestinal bacteria was successfully established. In the bioassays of entomopathogen infection, K. oxytoca showed significant synergistic effects with both NPV and Bt on the death of H. cunea. In contrast, E. mundtii played antagonistic effects. This phenomenon may be attributed to the differences in the physico-chemical properties of the two gut bacteria and the alkaline environment required for NPV and Bt to infect the host. It is worth noting that the enhanced insecticidal activity of K. oxytoca on NPV and Bt provides a reference for future biological control of H. cunea by intestinal bacteria.

Tolerance to dietary linalool primarily involves co-expression of cytochrome P450s and cuticular proteins in Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae using SMRT sequencing and RNA-seq.

BACKGROUND: Pagiophloeus tsushimanus (Coleoptera: Curculionidae), an emerging forest pest exclusively infesting camphor trees, has recently caused severe ecological and economic damage in localized areas in China. Its population outbreak depends largely on the capacity to overcome the pressure of terpenoid-derived metabolites (e.g. linalool) from camphor trees. At present, the molecular basis of physiological adaptation of P. tsushimanus to dietary linalool is poorly understood, and there is no available reference genome or transcriptome. RESULTS: Herein, we constructed the transcriptome profiling of P. tsushimanus larvae reared on linalool-infused diets using RNA sequencing and single-molecule real-time sequencing. A total of 20,325 high-quality full-length transcripts were identified as a reference transcriptome, of which 14,492 protein-coding transcripts including 130 transcription factors (TFs), and 5561 long non-coding RNAs (lncRNAs) were detected. Also, 30 alternative splicing events and 8049 simple sequence repeats were captured. Gene ontology enrichment of differential expressed transcripts revealed that overall up-regulation of both cytochrome P450s (CYP450s) and cuticular proteins (CPs), was the primary response characteristic against dietary linalool. Other physiological effects possibly caused by linalool exposure, such as increase in Reactive Oxygen Species (ROS) and hormetic stimulation, were compensated by a handful of induced genes encoding antioxidases, heat shock proteins (HSPs), juvenile hormone (JH) epoxide hydrolases, and digestive enzymes. Additionally, based on co-expression networks analysis, a diverse array of hub lncRNAs and TFs co-expressed with CYP450s and CPs were screened as the potential gene regulators. Temporal expression of candidate transcripts determined by quantitative real-time PCR also indicated a cooperative relationship between the inductions of CYP450s and CPs upon exposure to linalool. CONCLUSIONS: Our present study provides an important transcriptome resource of P. tsushimanus, and lays a valuable foundation for understanding how this specialist pest copes with chemical challenges in its specific host environments.

An alkaline protease from Bacillus cereus NJSZ-13 can act as a pathogenicity factor in infection of pinewood nematode.

Endophytic bacteria are an important biological control for nematodes. We isolated the nematicidal Bacillus cereus NJSZ-13 from healthy Pinus elliottii trunks. Bioassay experiments showed killing of all tested nematodes by proteins from the NJSZ-13 culture filtrate within 72 h. Degradation of the nematode cuticles was observed, suggesting the action of extracellular bacterial enzymes. The responsible protease was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography, and SDS-PAGE. The protease had a molecular weight of 28 kDa and optimal activity at 55 °C and pH 9, indicating an alkaline protease. The study suggests the potential for using this B. cereus NJSZ-13 strain protease to prevent pinewood nematode infection.

Comparing the Performance of Hyphantria cunea (Lepidoptera: Arctiidae) on Artificial and Natural Diets: Feasibility of Mass-Rearing on Artificial Diets.

In China, Hyphantria cunea (Drury) is an invasive phytophagous pest; it attacks nearly all species of defoliated trees. To develop integrated pest management programs (IPM) for H. cunea, we need to ensure the availability of insects by mass-rearing them on artificial diets under laboratory conditions. This study compared the growth characteristics, nutritional indices, growth indices, and digestive enzyme activity of insects reared on Pterocarya stenoptera C.DC (Fagales: Juglandaceae), the Chinese wingnut, and an artificial diet. We also investigated the correlation between diet components and growth indices using principal components analysis and Pearson correlation analysis. We found that mass-rearing of H. cunea on an artificial diet was feasible. It led to a shorter developmental period, with heavier larvae and pupae than natural diets. The principal components analysis indicated that the growth indices and α-Amylase were significantly positively associated with PC1, which explained 82.45% of the total data variability. Pearson correlation analysis showed a significant correlation between digestion, absorption parameters, and growth. Developing a mass-rearing program to produce H. cunea on an artificial diet will be valuable for improving IPM strategies. Understanding the mechanism of the responses of phytophagous insect populations to anthropogenic diet regulation can provide new ideas and methods for pest control.

A heat shock 70kDa protein MaltHSP70-2 contributes to thermal resistance in Monochamus alternatus (Coleoptera: Cerambycidae): quantification, localization, and functional analysis.

BACKGROUND: Heat Shock Proteins 70 (HSP70s) in insects act on a diverse range of substrates to assist with overcoming extreme high temperatures. MaltHSP70-2, a member of HSP70s, has been characterized to involve in the thermotolerance of Monochamus alternatus in vitro, while quantification and localization of MaltHSP70-2 in various tissues and its functional analysis in vivo remain unclear. RESULTS: In this study, temporal expression of MaltHSP70-2 indicated a long-last inductive effect on MaltHSP70-2 expression maintained 48 hours after heat shock. MaltHSP70-2 showed a global response to heat exposure which occurring in various tissues of both males and females. Particularly in the reproductive tissues, we further performed the quantification and localization of MaltHSP70-2 protein using Western Blot and Immunohistochemistry, suggesting that enriched MaltHSP70-2 in the testis (specifically in the primary spermatocyte) must be indispensable to protect the reproductive activities (e.g., spermatogenesis) against high temperatures. Furthermore, silencing MaltHSP70-2 markedly influenced the expression of other HSP genes and thermotolerance of adults in bioassays, which implied a possible interaction of MaltHSP70-2 with other HSP genes and its role in thermal resistance of M. alternatus adults. CONCLUSIONS: These findings shed new insights into thermo-resistant mechanism of M. alternatus to cope with global warming from the perspective of HSP70s functions.

Comparative Transcriptome Analysis Reveals Molecular Insights in Overwintering Monochamus alternatus (Coleoptera: Cerambycidae).

Monochamus alternatus, the dominant vector of Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), has caused immense damage to forest resources. In China, this vector was native to the southern regions but has spread northward recently. To adapt to more challenging environments in the northern winter, M. alternatus has evolved an intricate strategy for overwintering, which remains largely unknown. Herein, we compared the transcriptome data of the overwintering and non-overwintering larvae of M. alternatus larvae to investigate the molecular mechanisms in overwintering. A total of 53.10 GB clean bases and 28, 245 unigenes were obtained by RNA-seq. Analysis of 2597 upregulated and 2429 downregulated unigenes, as well as the enrichment of DEGs showed that many genes and pathways were jointly involved in the overwintering period. Besides, the accuracy of the RNA-seq data was tested by using qPCR experiment involving 13 selected genes. The results revealed that the overwintering process relied largely on the energy allocation trade-off. Specifically, overwintering M. alternatus inhibited energy-intensive activities, such as growth and molting, detoxification, and trehalose transport, and the reserved energy was skewed towards the synthesis of antifreeze compounds and immune response to cope with the deleterious effects of winter.

iTRAQ Proteomic Analysis of Interactions Between 20E and Phospholipase C in Apolygus lucorum (Meyer-Dür).

Polyphagous Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. Hormone 20-hydroxyecdysone (20E) regulates multiple insect development and physiology events. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes and related proteins that can be affected by PLC still remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblotting techniques were used to compare differentially expressed proteins (DEPs) in A. lucorum in response to the treatment of 20E and the PLC inhibitor U73122 as well as their combination. A total of 1,624 non-redundant proteins and 97, 248, 266 DEPs were identified in the 20E/control, U73122/control, and 20E + U73122/control groups, respectively. Only 8 DEPs, including pathogenesis-related protein 5-like, cuticle protein 19.8, trans-sialidase, larval cuticle protein A2B-like, cathepsin L1, hemolymph juvenile hormone-binding protein, ATP-dependent RNA helicase p62-like, and myosin-9 isoform X1, were detected in all three groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEPs were involved in diverse signaling pathways. The results were validated by immunoblotting, which highlighted the reliability of proteomics analysis. These findings provided novel insights into the function of PLC in 20E signaling pathway in A. lucorum.

Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury).

The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, ß-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

Insights into chemosensory genes of Pagiophloeus tsushimanus adults using transcriptome and qRT-PCR analysis.

Pagiophloeus tsushimanus is a new, destructive, and monophagous weevil pest that thrives on Cinnamomum camphora, found in Shanghai. The functions of chemosensory genes involved in the host location and intraspecific communication of P. tsushimanus remain unknown. The male-female transcriptomes of P. tsushimanus adults were assembled using Illumina sequencing, and we focused on all chemosensory genes in transcriptomes. In general, 58,088 unigenes with a mean length of 1018.19 bp were obtained. In total, 39 odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 22 olfactory receptors (ORs), 16 gustatory receptors (GRs), eight ionotropic receptors (IRs), and five sensory neuron membrane proteins (SNMPs) were identified. PtsuOBPs comprised four subfamilies (20 Minus-C, one Plus-C, two Dimer, and 15 Classic). Both PtsuOBPs and PtsuCSPs contained a highly conserved sequence motif of cysteine residues. PtsuORs including one olfactory receptor co-receptors (Ptsu/Orco) comprised seven predicted transmembrane domains. Phylogenetic analysis revealed that PtsuOBPs, PtsuCSPs, and PtsuORs in P. tsushimanus exhibited low homology compared to other insect species. The results of tissue- and sex-specific expression patterns indicated that PtsuOBPs and PtsuORs were highly abundant in the antennae; whereas, PtsuCSPs were not only highly abundant in antennae, but also abdominal apexes, wings, and legs. In conclusion, these results enrich the gene database of P. tsushimanus, which may serve as a basis for identifying novel targets to disrupt olfactory key genes and may provide a reverse validation method to identify attractants for formulating potential eco-friendly control strategies for this pest.

Phospholipase C gamma (PLCγ) regulates soluble trehalase in the 20E-induced fecundity of Apolygus lucorum.

Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis (Bt) cotton in China. Additionally, 20-hydroxyecdysone (20E) has important functions in many biological processes, including insect reproduction. Phospholipase C (PLC), which is an essential enzyme for phosphoinositide metabolism, is involved in 20E signal transduction, but its function in 20E-mediated reproduction in A. lucorum remains unclear. In this study, 20E increased AlPLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis. The 20E treatment also induced the considerable accumulation of two second messengers, inositol triphosphate and diacylglycerol. The expression levels of genes encoding vitellogenin (AlVg) and soluble trehalase (AlTre-1) were similar to those of AlPLCγ, and were upregulated in response to 20E. The silencing of AlPLCγ resulted in downregulated expression of AlTre-1 and AlVg. However, the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression. Moreover, the silencing of AlVg did not alter AlTre-1 expression. Furthermore, an examination of the insect specimens indicated that AlPLCγ is required for female adult reproduction, and that downregulated expression of this gene is associated with decreases in fecundity, adult longevity, and egg hatching rate as well as delayed oocyte maturation. We propose that 20E regulates AlTre-1 expression via AlPLCγ and affects Vg expression as well as ovary development to facilitate the reproductive activities of A. lucorum females.