Gene cloning, protein expression, and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum.

RNA interference (RNAi) is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double-stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+ -binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species.

New insights into chlorantraniliprole metabolic resistance mechanisms mediated by the striped rice borer cytochrome P450 monooxygenases: A case study of metabolic differences.

The anthranilic diamide insecticide chlorantraniliprole has been extensively applied to control Lepidoptera pests. However, its overuse leads to the development of resistance and accumulation of residue in the environment. Four P450s (CYP6CV5, CYP9A68, CYP321F3, and CYP324A12) were first found to be constitutively overexpressed in an SSB CAP-resistant strain. It is imperative to further elucidate the molecular mechanisms underlying P450s-mediated CAP resistance for mitigating its environmental contamination. Here, we heterologously expressed these four P450s in insect cells and evaluated their abilities to metabolize CAP. Western blotting and reduced CO difference spectrum tests showed that these four P450 proteins had been successfully expressed in Sf9 cells, which are indicative of active functional enzymes. The recombinant proteins CYP6CV5, CYP9A68, CYP321F3, and CYP324A12 exhibited a preference for metabolizing the fluorescent P450 model probe substrates EC, BFC, EFC, and EC with enzyme activities of 0.54, 0.67, 0.57, and 0.46 pmol/min/pmol P450, respectively. In vitro metabolism revealed distinct CAP metabolic rates (0.97, 0.86, 0.75, and 0.55 pmol/min/pmol P450) and efficiencies (0.45, 0.37, 0.30, and 0.17) of the four recombinant P450 enzymes, thereby elucidating different protein catalytic activities. Furthermore, molecular model docking confirmed metabolic differences and efficiencies of these P450s and unveiled the hydroxylation reaction in generating N-demethylation and methylphenyl hydroxylation during CAP metabolism. Our findings not only first provide new insights into the mechanisms of P450s-mediated metabolic resistance to CAP at the protein level in SSB but also demonstrate significant differences in the capacities of multiple P450s for insecticide degradation and facilitate the evaluation and mitigation of toxic risks associated with CAP application in the environment.

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.

Adenosine Monophosphate-Activated Protein Kinase (AMPK) Phosphorylation Is Required for 20-Hydroxyecdysone Regulates Ecdysis in Apolygus lucorum.

The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect's molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status.

A Programmable, DNA-Exclusively-Guided Argonaute DNase and Its Higher Cleavage Specificity Achieved by 5'-Hydroxylated Guide.

Argonaute proteins exist widely in eukaryotes and prokaryotes, and they are of great potential for molecular cloning, nucleic acid detection, DNA assembly, and gene editing. However, their overall properties are not satisfactory and hinder their broad applications. Herein, we investigated a prokaryotic Argonaute nuclease from a mesophilic bacterium Clostridium disporicum (CdAgo) and explored its overall properties, especially with 5'-hydroxylated (5'-OH) guides. We found that CdAgo can exclusively use single-stranded DNA (ssDNA) as guide to cleave ssDNA and plasmid targets. Further, we found the length of the efficient guide is narrower for the 5'-OH guide (17-20 nt) than for the 5'-phosphorylated guide (5'-P, 14-21 nt). Furthermore, we discovered that the 5'-OH guides can generally offer stronger mismatch discrimination than the 5'-P ones. The 5'-OH guides offer the narrower length range, higher mismatch discrimination and more accurate cleavage than the 5'-P guides. Therefore, 5'-OH-guide-directed CdAgo has great potential in biological and biomedical applications.

Application of transcriptomic analysis to unveil the toxicity mechanisms of fall armyworm response after exposure to sublethal chlorantraniliprole.

The anthranilic diamide insecticide chlorantraniliprole is highly effective against Lepidoptera pests, but the underlying mechanisms of toxic effects of chlorantraniliprole exposures for adapting to the chemical environment are poorly known in fall armyworm (FAW), Spodoptera frugiperda (J.E.Smith). FAW being one of the most pests of maize in Latin America, suddenly appeared in China in 2019 and spread rapidly. In this study, using bioassay and transcriptomic and biochemical analyses, we comprehensively investigated gene expression changes of third instar larvae in response to different sublethal concentrations (LC10 and LC30) of chlorantraniliprole in this insect. Exposure to LC10 chlorantraniliprole (0.73 mg/L) causes 1266 differentially expressed genes (DEGs), of which 578 are up-regulated and 688 down-regulated. Exposure to LC30 (2.49 mg/L) causes differential expression of 3637 DEGs (1545 up-, 2092 down-regulated). Interestingly, the LC30 treatment led to a significant increase in the number of DEGs compared to that of the LC10, indicating a concentration effect manner. Moreover, enrichment analysis identified important DEGs belonging to specific categories, such as amino acid, carbohydrate, lipid, energy, xenobiotics metabolisms, signal transduction, and posttranslational modification pathways, and enzymes activities in enriched pathways were significantly altered at the LC10 and LC30, which matched transcriptome analysis to mediate toxic mechanisms. The DEGs encoding detoxification-related genes were identified and validated by quantitative real-time PCR (qRT-PCR), which correlated with the RNA-sequencing (RNA-seq) data. To our knowledge, these findings provide the first toxicity mechanisms for a better understanding of chlorantraniliprole action and detoxification in FAW and other insect pests at molecular level.

Constitutive overexpression of cytochrome P450 monooxygenase genes contributes to chlorantraniliprole resistance in Chilo suppressalis (Walker).

BACKGROUND: The rice striped stem borer (SSB), Chilo suppressalis (Walker), which is one of the most economically important phytophagous pests, has developed resistance to multiple insecticides. The resistance of SSB against chlorantraniliprole has been investigated in detail. However, the mechanism of its metabolic resistance has rarely been studied. RESULTS: A field population from Wuhu City, China was used to establish chlorantraniliprole resistant and susceptible strains (WHR and WHS) by laboratory continuous selection. Enzyme activities data suggested the potential involvement of cytochrome P450 monooxygenase in WHR. CYP6CV5, CYP9A68, CYP321F3, and CYP324A12 were significantly overexpressed in WHR (from 4.48 to 44.88-fold). These four P450 genes were expressed in the late developmental stages of WHR; however, they were almost absent during the egg stage. In addition, their expressions were much more sensitive to chlorantraniliprole induction in WHR than in WHS. Injection of individual and mixture dsRNAs reduced the expression of the four target genes (55.2-73.2% and 43.2-50.2%, respectively) and caused significant larvae mortality (55.1-65.1% and 88.2%, respectively). CONCLUSION: Multiple overexpressed P450 genes were potentially associated with chlorantraniliprole resistance, as confirmed by the RNA interference (RNAi) assay. Our findings suggested that metabolic resistance to chlorantraniliprole might be mediated by P450s. © 2018 Society of Chemical Industry.