Resistance monitoring of diamide insecticides and characterization of field-evolved chlorantraniliprole resistance among Chinese populations of the tomato pinworm Phthorimaea (=Tuta) absoluta (Lepidoptera: Gelechiidae).

The tomato pinworm, Phthorimaea (=Tuta) absoluta, is considered one of the most destructive and invasive insect pests worldwide, having developed significant resistance to many popular insecticides. In this study, we monitored the field resistance of P. absoluta populations from China to three diamide insecticides: flubendiamide, chlorantraniliprole, and cyantraniliprole. We found that one field population from Wuzhong City (WZ) exhibited high level of resistance to chlorantraniliprole. Using the WZ population and a susceptible reference strain (YN-S), we established a near-isogenic line (WZ-NIL) of P. absoluta with resistance to chlorantraniliprole. This strain also showed substantial cross-resistance to flubendiamide, and cyantraniliprole. Genetic analysis revealed that the inheritance of resistance to chlorantraniliprole in the WZ-NIL strain was autosomal and incompletely dominant. Additionally, the pesticide synergist piperonyl butoxide significantly inhibited chlorantraniliprole resistance by compromising P450 monooxygenase activity, which was significantly higher in the resistant strain. Furthermore, WZ-NIL had significantly prolonged developmental stages, lower pupation rates, reduced female fecundity, and lower egg hatchability than YN-S individuals. The fitness of WZ-NIL relative to YN-S was estimated to be 0.73, indicating significant fitness cost associated with chlorantraniliprole resistance. Rotating chlorantraniliprole with other insecticides that have different modes of action and degradation may be particularly useful for managing chlorantraniliprole resistance in P. absoluta.

Synthesis and Insecticidal Activity of Novel Anthranilic Diamide Insecticides Containing Indane and Its Analogs.

Diamide insecticides have always been a hot research topic in the field of pesticides. To further discover new compounds with high activity and safety, indane and its analogs were introduced into chlorantraniliprole, and a battery of chlorfenil derivatives, including indane and its analogs, were designed and prepared for biological testing. Their characterization and verification were carried out through nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Biological detection showed that all the compounds exhibited good insecticidal activity against Mythimna separata. At 0.8 mg/L, the insecticidal activity of compound 8q against Mythimna separata was 80%, which was slightly better than that of chlorantraniliprole. The results of the structure-activity relationship (SAR) analysis indicated that the indane moiety had a significant effect on insecticidal activity, especially in the R-configuration. The results indicated that chlorantraniliprole derivatives containing indane groups could serve as pilot compounds for the further development of new insecticides.

Diamide insecticides targeting insect ryanodine receptors: Mechanism and application prospect.

As a Lepidoptera pest, Spodoptera frugiperda has become one of the major migratory pests causing significant damage to crops. It should prevent and control Spodoptera frugiperda with strong reproductive ability, adaptability, and migration ability, and reduce economic losses as much as possible. Chemical insecticides are mainly used in the emergency control of Spodoptera frugiperda. Diamide insecticide is a kind of pesticide that specifically targets the ryanodine receptor of Lepidopteran pests, which makes it safe, effective, targeted, and low toxicity to mammals. So, it is one of the most concerned and fastest-growing pesticide products after neonicotinoid pesticides. Intracellular Ca2+ concentration can be regulated by ryanodine receptors, and the continuous release of Ca2+ eventually leads to the death of pests and achieve the insecticidal effect. This review introduces in detail diamide insecticides that mainly play roles in stomach toxicity, as well as its specific target-ryanodine receptor, and analyzes how the diamide insecticide acts on the ryanodine receptor and how its mechanism of action can provide a theoretical basis for the rational use of highly effective insecticides and solve the resistance problem. Moreover, we also propose several recommendations for reducing resistance to diamide insecticides, and provide a reference for chemical control and resistance studies of Spodoptera frugiperda, which has broad development prospects in today's increasingly concerned about the ecological environment and advocating green environmental protection.

Pyrrole-2 carboxamides - A novel class of insect ryanodine receptor activators.

The ryanodine receptor (RyR) is an intracellular calcium channel critical to the regulation of insect muscle contraction and the target site of diamide insecticides such as chlorantraniliprole, cyantraniliprole and flubendiamide. To-date, diamides are the only known class of synthetic molecules with high potency against insect RyRs. Target-based screening of an informer library led to discovery of a novel class of RyR activators, pyrrole-2-carboxamides. Efforts to optimize receptor activity resulted in analogs with potency comparable to that of commercial diamides when tested against RyR of the fruit fly, Drosophila melanogaster. Surprisingly, testing of pyrrole-2-carboxamides in whole-insect screens showed poor insecticidal activity, which is partially attributed to differential selectivity among insect receptors and rapid detoxification. Among various lepidopteran species field resistance to diamide insecticides has been well documented and in many cases has been attributed to a single point mutation, G4946E, of the RyR gene. As with diamide insecticides, the G4946E mutation confers greatly reduced sensitivity to pyrrole-2-carboxamides. This, coupled with findings from radioligand binding studies, indicates a shared binding domain between anthranilic diamides and pyrrole-2-carboxamides.

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

Mechanism of the different metabolome responses between Plutella xylostella and Pieris rapae treated with the diamide insecticides.

Diamide insecticides, such as chlorantraniliprole, cyantraniliprole, and tetrachlorantraniliprole, are a new class of insecticides that selectively target insects by affecting calcium homeostasis. While this class of insecticides are effective on a wide range of insect pests, the toxicities of diamide insecticides vary among species and life stages. In this study, we addressed the mechanism underlying the different responses of Plutella xylostella and Pieris rapae to diamide insecticides. The susceptibility to insecticides of P. xylostella and P. rapae larvae was assessed 2 and 4 days after exposure to chlorantraniliprole, cyantraniliprole, and tetrachlorantraniliprole. P. xylostella larvae treated with distilled water (Group A), chlorantraniliprole (Group B), cyantraniliprole (Group C), and tetrachlorantraniliprole (Group D) and P. rapae larvae treated with distilled water (Group E), chlorantraniliprole (Group F), cyantraniliprole (Group G) and tetrachlorantraniliprole (Group H) were subjected to metabolomics analysis. The differential metabolites in the B vs. F, C vs. G, and D vs. H groups were analyzed, followed by pathway enrichment analysis. Chlorantraniliprole, cyantraniliprole, and tetrachlorantraniliprole all showed high toxicities for P. xylostella and P. rapae larvae. P. rapae larvae were more sensitive to the diamide insecticides than P. xylostella larvae. There were 65 overlapped differential metabolites between P. xylostella and P. rapae larvae treated with these three diamide insecticides. Pathway analysis showed that the differential metabolites were closely related with fatty acid biosynthesis and metabolism-related pathways. The differential regulation of fatty acid biosynthesis and metabolism may contribute to the different response to diamide insecticides in P. xylostella and P. rapae.

Temperature-dependent variations in toxicity of diamide insecticides against three lepidopteran insects.

The effect of temperature on the toxicities of four diamide insecticides (chlorantraniliprole, cyantraniliprole, flubendiamide, tetraniliprole) against three lepidopteran insects (Helicoverpa armigera, Plutella xylostella, Athetis lepigone) were determined from 15 to 35 °C by exposing third-instar larvae to dip-treated cabbage leaf. The results indicated that increase in temperature led to an increase significantly and regularly in the toxicities of the four diamide insecticides against P. xylostella and H. armigera, but not for A. lepigone. The temperature coefficients (TCs) of the four diamide insecticides increased from 15 to 35 °C. Tetraniliprole for H. armigera (+825.83), chlorantraniliprole for P. xylostella (+315.65) and cyantraniliprole for H. armigera (+225.77) exhibited high positive TCs. For A. lepigone, temperature had a positively weak or no effect on the toxicities of most of the diamide insecticides from 20 to 30 °C, but a higher effect from 30 to 35 °C. In addition, the toxicities of chlorantraniliprole, cyantraniliprole and tetraniliprole all decreased from 15 to 20 °C. This study can guide pest managers in choosing suitable ambient field temperature when spraying diamide insecticides against lepidopteran insects.

Molecular cloning, mRNA expression and alternative splicing of a ryanodine receptor gene from the citrus whitefly, Dialeurodes citri (Ashmead).

Insect ryanodine receptors are the main targets of diamide insecticides that have highly selective insecticidal activity but are less toxic to mammals. Therefore, these insecticides are ideal for pest control. Ryanodine receptors (RyRs) play a critical role in Ca2+ signaling in muscle and non-muscle cells. In this study, we cloned the complete cDNA (DcRyR) of the RyR from the citrus whitefly, Dialeurodes citri, a serious pest of citrus orchards in China. The open reading frame of RyR is 15,378bp long and encodes a protein with 5126 amino acids with a computed molecular weight of 579.523kDa. DcRyR shows a high amino acid sequence identity to RyRs from other insects (76%-95%) and low identity to those from nematodes and mammals (44%-52%). DcRyR shares many features of insect and vertebrate RyRs, including a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeat domain, RIH-associated domain at the N-terminus, two consensus calcium-binding EF-hands and six transmembrane domains at the C-terminus. The expression of DcRyR mRNA was the highest in the nymphs and lowest in eggs; DcRyR mRNA was 1.85-fold higher in the nymphs than in the eggs. Among the tissues, DcRyR mRNA expression was 4.18- and 4.02-fold higher in the adult head and thorax than in the abdomen. DcRyR had three alternative splice sites and the splice variants showed body part-specific expression and were developmentally regulated. These results may help investigate target-based resistance to diamide insecticides in D. citri.

Discovery of Trisubstituted N-Phenylpyrazole Containing Diamides with Improved Insecticidal Activity.

To increase the structural diversity of insecticides and meet the needs of effective integrated insect management, the structure of chlorantraniliprole was modified based on a previously established three-dimensional quantitative structure-activity relationship (3D-QSAR) model. The pyridinyl moiety in the structure of chlorantraniliprole was replaced with a 4-fluorophenyl group. Further modifications of this 4-fluorophenyl group by introducing a halogen atom at position 2 and an electron-withdrawing group (e.g., iodine, cyano, and trifluoromethyl) at position 5 led to 34 compounds with good insecticidal efficacy against Mythimna separata, Plutella xylostella, and Spodoptera frugiperda. Among them, compound IV f against M. separata showed potency comparable to that of chlorantraniliprole. IV p against P. xylostella displayed a 4.5 times higher potency than chlorantraniliprole. In addition, IV d and chlorantraniliprole exhibited comparable potencies against S. frugiperda. Transcriptome analysis showed that the molecular target of compound IV f is the ryanodine receptor. Molecular docking was further performed to verify the mode of action and insecticidal activity against resistant P. xylostella.

Magnetic molecularly imprinted polymers integrated ionic liquids for targeted detecting diamide insecticides in environmental water by HPLC-UV following MSPE.

Efficiently detecting diamide insecticides in environmental water is challenging due to their low concentrations and complex matrix interferences. In this study, we developed ionic liquids (ILs)-incorporated magnetic molecularly imprinted polymers (IL-MMIPs) for the detection of diamide insecticides, capitalizing on the advantages of ILs and quick magnetic separation through surface imprinting. Tetrachlorantraniliprole was used as the template, and a specific IL, 1-vinyl-3-ethylimidazolium hexafluorophosphate ([VEIm][PF6]), was employed as the functional monomer. Various synthesis conditions were investigated to optimize adsorption efficiency. The prepared IL-MMIPs were successfully employed as adsorbents in magnetic solid-phase extraction (MSPE) to selectively extract, separate, and quantify three types of diamide insecticides from water samples using HPLC-UV detection. Under optimal conditions, the analytical method achieved low limits of detection (0.69 ng mL-1, 0.64 ng mL-1, 0.59 ng mL-1 for cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole, respectively). The method also displayed a wide linear range (0.003-10 µg mL-1 for cyantraniliprole and chlorantraniliprole, and 0.004-10 µg mL-1 for tetrachlorantraniliprole, respectively) with satisfactory coefficients (R2≥0.9996), and low relative standard deviation (RSD≤2.55%). Additionally, extraction recoveries fell within the range of 79.4%-109%. The results clearly demonstrate that IL-MMIPs exhibit exceptional recognition and rebinding capabilities. The developed IL-MMIPs-MSPE-HPLC-UV method is straightforward and rapid, making it suitable for the detection and analysis of three kinds of diamide insecticides in environmental water.

Synthesis of in-situ magnetized MOF-cellulose membranes for high-efficiency enrichment of diamide insecticides in vegetables and determination by LC-MS/MS.

This study presents a novel, eco-friendly composite adsorbent material designed for the magnetic solid-phase extraction of diamide insecticides from vegetable samples. The membrane, denoted as Fe-MMm, was incorporated with a cellulose framework embedded with Metal-Organic Frameworks (MOFs) and Multi-Walled Carbon Nanotubes (MWCNTs) magnetized with Fe3O4. This innovative material streamlined the conventional solid-phase extraction process, simplifying the sample pre-treatment. By combining it with liquid chromatography tandem mass spectrometry (LC-MS/MS), the method achieves significantly enhanced extraction efficiency through systematic optimization of experimental parameters, including adsorbent selection, pH, ionic strength, adsorption time, and elution time. The method had a wide linear range of 0.1-1000 ng/mL and an exceptionally low detection limit ranging from 0.023 to 0.035 ng/mL. The successful identification of diamide insecticides in vegetable samples underscores the potential of Fe-MMm as a robust material for sample pretreatment in analytical applications.

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase.

BACKGROUD: Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. RESULTS: The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 µM (Chlorantraniliprole), 0.21 µM (Flubendiamide), and 0.57 µM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 µM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. CONCLUSIONS: SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

Molecular cloning and mRNA expression of a ryanodine receptor gene in the cotton bollworm, Helicoverpa armigera.

Ryanodine receptors (RyRs) are the targets of novel diamide insecticides. The cotton bollworm, Helicoverpa armigera, is one of the most important cotton pests in the world. In this study, we report the full-length RyR cDNA sequence (named as HaRyR) of H. armigera. The 16,083-bp contiguous sequence encoded 5, 142 amino acid residues, which shares 80% and 78% overall identities with its homologues in Nilaparvata lugens (NlRyR) and Drosophila melanogaster (DmRyR), respectively. All hallmarks of RyR proteins are conserved in the HaRyR, including the GXRXGGGXGD motif conserved in the Ca(2+) release channels and four copies of RyR domain unique to RyR channels. The previously identified seven lepidopteran-specific RyR residues were also found in HaRyR (N(4977), N(4979), N(4990), L(5005), L(5036), N(5068) and T(5119)). An amino acid sequence alignment showed that the N-terminal region of HaRyR (residues 188-295) shared high sequence identity with NlRyR (94%) and DmRyR (92%), and moderate sequence identity (47-50%) with three rabbit RyR isoforms, while the short segment of the C-terminal transmembrane region of HaRyR (residues 4632-4676) exhibited moderate sequence identity with NlRyR (69%) and DmRyR (67%), and low sequence identity (19-28%) with three rabbit RyR isoforms. In addition, expression analysis of HaRyR revealed that the mRNA expression level in eggs was significantly lower than in third instar larvae, pupae and adults, and anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen. Our results lay a foundation for comprehensive structural and functional characterization of HaRyR and for understanding of the molecular mechanisms of toxicity selectivity of diamide insecticides among different species.

Comprehensive Overview of Diamide Derivatives Acting as Ryanodine Receptor Activators.

The world's hunger is continuously rising due to conflicts, climate change, pandemics (such as the recent COVID-19), and crop pests and diseases. It is widely accepted that zero hunger is impossible without using agrochemicals to control crop pests and diseases. Diamide insecticides are one of the widely used green insecticides developed in recent years and play important roles in controlling lepidopteran pests. Currently, eight diamine insecticides have been commercialized, which target the insect ryanodine receptors. This review summarizes the development and optimization processes of diamide derivatives acting as ryanodine receptor activators. The review also discusses pest resistance to diamide derivatives and possible solutions to overcome the limitations posed by the resistance. Thus, with reference to structural biology, this study provides an impetus for designing and developing diamide insecticides with improved insecticidal activities.

Neurotoxicity and behavioral disorders induced in mice by acute exposure to the diamide insecticide chlorantraniliprole.

Diamide insecticides activate ryanodine receptors expressed in lepidopteran skeletal muscle and promote Ca2+ release in the sarcoplasmic reticulum, causing abnormal contractions and paralysis, leading to death of the pest. Although they had been thought not to act on nontarget organisms, including mammals, adverse effects on vertebrates were recently reported, raising concerns about their safety in humans. We investigated the neurotoxicity of the acute no-observed-adverse-effect level of chlorantraniliprole (CAP), a diamide insecticide, in mice using clothianidin (CLO), a neonicotinoid insecticide, as a positive control. The CLO-administered group showed decreased locomotor activities, increased anxiety-like behaviors, and abnormal human-audible vocalizations, while the CAP-administered group showed anxiety-like behaviors but no change in locomotor activities. The CAP-administered group had greater numbers of c-fos-immunoreactive cells in the hippocampal dentate gyrus, and similar to the results in a CLO-administered group in our previous study. Blood corticosterone levels increased in the CLO-administered group but did not change in the CAP-administered group. Additionally, CAP was found to decreased 3-Methoxytyramine and histamine in mice at the time to maximum concentration. These results suggest that CAP-administered mice are less vulnerable to stress than CLO-administered mice, and the first evidence that CAP exposure increases neuronal activity and induces anxiety-like behavior as well as neurotransmitter disturbances in mammals.

Lack of Known Target-Site Mutations in Field Populations of Ostrinia furnacalis in China from 2019 to 2021.

The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera; Pyralidae), is one of the most destructive insect pests of corn, for which chemical insecticides have been the primary method of control, especially during outbreaks. Little information is currently available on the status of insecticide resistance and associated mechanisms in O. furnacalis field populations. Invasions and outbreaks of Spodoptera frugiperda in China in recent years have increased chemical application in corn fields, which adds to the selection pressure on O. furnacalis. This study was conducted to estimate the risk of insecticide resistance by investigating the frequency of insecticide resistant alleles associated with target site insensitivity in field populations of O. furnacalis. Using the individual-PCR genotype sequencing analysis, none of the six target-site insecticide resistant mutations were detected in O. furnacalis field populations collected from 2019 to 2021 in China. These investigated insecticide resistance alleles are common in resistant Lepidoptra pests and are responsible for resistance to pyrethroids, organophosphorus, carbamates, diamide, and Cry1Ab. Our results support the low insecticide resistance status in field O. furnacalis populations and betokens the unlikely development of high resistance mediated by the common target-site resistance alleles. Additionally, the findings would serve as references for further efforts toward the sustainable management of O. furnacalis.

Design, synthesis and biological activity of diamide compounds based on 3-substituent of the pyrazole ring†.

BACKGROUND: Diamide insecticides have attracted significant attention due to their high efficacy and low toxicity to non-target organisms since they were introduced to the market. In order to tackle the problems of insecticide resistance and ecological safety, 16 novel nitrobenzene substituted anthranilic diamides with ester, hydroxyl or sulfonyl at the 3-position of the pyrazole ring were designed and synthesized. RESULTS: All of these compounds possessed good activity against the ryanodine receptor (RyR) from Spodoptera frugiperda and relatively lower activity against mammalian RyR1, showing a better insect-selectivity compared to chlorantraniliprole in a cell-based assay. The molecular docking analysis predicted the binding conformations of these compounds, which showed a good correlation between the insecticidal activity and the binding scores. In vitro studies using a calcium imaging method demonstrated that the novel compounds could not only activate the RyR but may also target the dihydropyridine receptor on the plasma membrane of insect neurons, implicating a similar but not same mode of action. CONCLUSION: Substituted anthranilic diamides with an ester at the 3-position of the pyrazole ring exhibited a promising insecticidal activity and better insect-selectivity, which provided insight into the rational design of a new generation of effective diamide insecticides.

Diamide insecticide resistance in transgenic Drosophila and Sf9-cells expressing a full-length diamondback moth ryanodine receptor carrying an I4790M mutation.

BACKGROUND: Resistance to diamide insecticides in Lepidoptera is known to be caused primarily by amino acid changes on the ryanodine receptor (RyR). Recently, two new target site mutations, G4946V and I4790M, have emerged in populations of diamondback moth, Plutella xylostella, as well as in other lepidopteran species, and both mutations have been shown empirically to decrease diamide efficacy. Here, we quantify the impact of the I4790M mutation on diamide activation of the receptor, as compared to alterations at the G4946 locus. RESULTS: I4790M when introduced into P. xylostella RyR expressed in an insect-derived Sf9 cell line was found to mediate just a ten-fold reduction in chlorantraniliprole efficacy (compared to 104- and 146-fold reductions for the G4946E and G4946V variants, respectively), whilst in the field its presence is associated with a ≥150-fold reduction. I4790M-mediated resistance to flubendiamide was estimated to be >24-fold. When the entire coding sequence of P. xylostella RyR was integrated into Drosophila melanogaster, the I4790M variant conferred ~4.4-fold resistance to chlorantraniliprole and 22-fold resistance to flubendiamide in the 3rd instar larvae, confirming that it imparts only a moderate level of resistance to diamide insecticides. Although the I4790M substitution appears to bear no fitness costs in terms of the flies' reproductive capacity, when assessed in a noncompetitive environment, it does, however, have potentially major impacts on mobility at both the larval and adult stages. CONCLUSIONS: I4790M imparts only a moderate level of resistance to diamide insecticides and potentially confers significant fitness costs to the insect.

Enrichment of diamide insecticides from environmental water samples using metal-organic frameworks as adsorbents for determination by liquid chromatography tandem mass spectrometry.

A series of metal-organic frameworks composed of different metal ions and organic linkers were facilely synthesized and used as adsorbents for five diamide insecticides for the first time. Among them, MIL-101-NH2 performed much better than other materials due to extraordinarily high specific surface area, strong water stability, specific interaction with analytes. A sensitive method was developed with MIL-101-NH2 based dispersive solid phase extraction combining with liquid chromatography tandem mass spectrometry (dSPE-LC-MS/MS). Important parameters including adsorbent amount, enrichment time, elution solvent and volume, pH and salt effect were investigated to achieve the best enrichment efficiency. At selected conditions, the proposed method showed ultrahigh sensitivity with limits of detection low to 0.01-0.03 ng/mL, which was 2-3 orders of magnitude lower than reported methods. Wide linearity in the range of 0.03-1000 ng/mL (chlorantraniliprole, cyantraniliprole) and 0.1-2000 ng/mL (flubendiamide, cyclaniliprole, tetrachlorantraniliprole) were established with satisfactory coefficient of determination. The method was successfully used for analyzing of diamide insecticides in environmental water samples and flubendiamide was detected in several samples. This work demonstrated the first example of developing novel nanomaterials in trace amount diamide insecticide enrichment from practical samples, which opens a new perspective in establishing nanomaterial-based sample preparation method for diamide insecticide analysis.

Detection of ryanodine receptor target-site mutations in diamide insecticide-resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a widely distributed pest of corn. Since it invaded China in 2018, it has caused serious damage to local corn production. Chlorantraniliprole, an anthranilic diamide insecticide, has been widely used to control lepidopteran pests. Tetrachloropyramid is a new allosteric modulator insecticide developed based on chlorantraniliprole, so it has a similar mechanism and insecticidal effect. In this study, we investigated resistance levels to chlorantraniliprole and tetrachloropyramid in S. frugiperda from 13 populations in China. Among the populations tested, the relative highest resistance to chlorantraniliprole occurred in the Guangzhou population, and the most susceptible to chlorantraniliprole was found in the Wuhan population. The lethal dosage LD50 value of the Guangzhou population against chlorantraniliprole was 27.8-fold higher than that of the Wuhan population. Minimal differences were observed among S. frugiperda populations in terms of sensitivity to tetrachloropyramid. Heterozygous mutations at the I4734 site of the ryanodine receptor (RyR) were found, while no mutations were found in the G4891 site. The mutations were detected in only two of the 786 individuals analyzed, one from the Qinzhou population and other from the Anshun population (frequency below 2% in both cases). There were no significant differences in the expression levels of RyR between Guangzhou and Wuhan populations. In summary, our results indicate that: (i) S. frugiperda has low resistance levels to diamide insecticides in China; and (ii) the differences in relative resistance among the 13 populations analyzed are not caused by the mutations in RyR or the expression of RyR.