Uridine diphosphate glycosyltransferases (UGTs) are multifunctional detoxification enzymes, which are involved in metabolizing various chemicals and contribute to the development of insecticide resistance. However, the possible roles of UGTs in chlorantraniliprole resistance in Chilo suppressalis have rarely been studied in detail. Based on genome data, 24 UGT genes in C. suppressalis belonging to 11 families were identified, which were designated by the UGT nomenclature committee. Synergism assay data suggested that UGTs are potentially involved in chlorantraniliprole resistance in C. suppressalis. CsUGT40AL1 and CsUGT33AG3 were significantly overexpressed in the chlorantraniliprole resistant strain (12.36- and 5.34-fold, respectively). The two UGTs were highly expressed in the larval Malpighian tubules, fat body, and midgut; however, expression was lowest in the head. Injection of individual dsRNAs reduced the expression of the two target genes (by 69.34% and 48.74%, respectively) and caused significant higher larval mortality (81.33% and 54.67%, respectively). Overexpression of CsUGT40AL1 and CsUGT33AG3 was potentially involved in chlorantraniliprole resistance in C. suppressalis, as confirmed by the RNAi assay. Our findings suggest that overexpression of UGTs may contribute to chlorantraniliprole resistance in C. suppressalis.

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.

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.

Chemosensory systems play an important role in insect behavior, and some key associated genes have potential as novel targets for pest control. Cacopsylla chinensis is an oligophagous pest and has become one of the main pests of pear trees, but little is known about the molecular-level means by which it locates its hosts. In this study, we assembled the head transcriptome of C. chinensis using Illumina sequencing, and 63,052 Unigenes were identified. A total of 36 candidate chemosensory genes were identified, including five different families: 12 odorant binding proteins (OBPs), 11 chemosensory proteins (CSPs), 7 odorant receptors (ORs), 4 ionotropic receptors (IRs), and 2 gustatory receptors (GRs). The number of chemosensory gene families is consistent with that found in other Hemipteran species, indicating that our approach successfully obtained the chemosensory genes of C. chinensis. The tissue expression of all genes using quantitative real-time PCR (qRT-PCR) found that some genes displayed male head, female head, or nymph-biased specific/expression. Our results enrich the gene inventory of C. chinensis and provide valuable resources for the analysis of the functions of some key genes. This will help in developing molecular targets for disrupting feeding behavior in C. chinensis.