Molecular Mechanism of Action of Cycloxaprid, An Oxabridged cis-Nitromethylene Neonicotinoid.

Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity in Hemipteran insect pests. In this study, the action of cycloxaprid was characterized by recombinant receptor Nlα1/rß2 and cockroach neurons. On Nlα1/ß2 in Xenopus oocytes, cycloxaprid acted as a full agonist. The imidacloprid resistance-associated mutation Y151S reduced the Imax of cycloxaprid by 37.0% and increased EC50 values by 1.9-fold, while the Imax of imidacloprid was reduced by 72.0%, and EC50 values increased by 2.3-fold. On cockroach neurons, the maximum currents elicited by cycloxaprid were only 55% of that of acetylcholine, a full agonist, but with close EC50 values of that of trans-neonicotinoids. In addition, cycloxaprid inhibited acetylcholine-evoked currents on insect neurons in a concentration-dependent manner when co-applied with acetylcholine. Cycloxaprid at low concentrations significantly inhibited the activation of nAChRs by acetylcholine, and its inhibition potency at 1 µM was higher than its activation potency on insect neurons. Two action potencies, activation, and inhibition, by cycloxaprid on insect neurons provided an explanation for its high toxicity to insect pests. In summary, as a cis-nitromethylene neonicotinoid, cycloxaprid showed high potency on both recombinant nAChR Nlα1/ß2 and cockroach neurons, which guaranteed its high control effects on a variety of insect pests.

Photostability study of cis-configuration neonicotinoid insecticide cycloxaprid in water.

Cycloxaprid (CYC) is a new cis-configuration neonicotinoid insecticide, which is currently under development in China for agricultural pest control. Considering the photodegradation of CYC is important for the application of CYC in the future, the photochemical behavior of CYC in aqueous solution was herein investigated in a "merry-go-round" reactor under a 300 W high-pressure mercury lamp. Twenty-five photodegradation products were identified via UPLC-TOF-ESI-MS/MS. The results suggested that NTN32692, the precursor of CYC was the predominant photodegradation product. CYC photodegrades via a more complex mechanism than imidacloprid and four potential photodegradation pathways were proposed.

Cycloxaprid: A novel cis-nitromethylene neonicotinoid insecticide to control imidacloprid-resistant cotton aphid (Aphis gossypii).

Imidacloprid is a nicotinic acetylcholine receptor (nAChR) agonist with potent insecticidal activity. However, resistance to imidacloprid is a significant threat and has been identified in several pest species. Cycloxaprid with cis-configuration is a novel neonicotinoid insecticide, which shows high activity against imidacloprid-resistant pests. The LC50 of imidacloprid against the resistant Aphis gossypii was 14.33mgL(-1) while it was only 0.70mgL(-1) for the susceptible population, giving a resistance ratio of 20.47. In this imidacloprid-resistant population, a point mutation (R81T) located in the loop D region of the nAChR ß1 subunit was found out. But this point mutation did not decrease the activity of cycloxaprid against A. gossypii. The LC50 of cycloxaprid was 1.05 and 1.36mgL(-1) for the imidacloprid-susceptible and imidacloprid-resistant populations, respectively. In addition, cycloxaprid provided better efficacies against resistant A. gossypii than imidacloprid in the fields. Although cycloxaprid was highly toxic to A. gossypii, it showed high selective activity between A. gossypii and its predominant natural enemies, Harmonia axyridis and Chrysoperla sinica. These results demonstrate that cycloxaprid is a promising insecticide against imidacloprid-resistant A. gossypii and suitable for the integrated pest management.

The Toxicity and Detoxifying Mechanism of Cycloxaprid and Buprofezin in Controlling Sogatella furcifera (Homoptera: Delphacidae).

The effects of cycloxaprid (a modified neonicotinoid insecticide) and buprofezin (a thiadiazine insecticide) on mortality of the white-backed planthopper (WBPH), Sogatella furcifera, were determined in laboratory assays. Cycloxaprid killed WBPH nymphs and adults but buprofezin killed only nymphs, and cycloxaprid acted faster than buprofezin. One day after infestation, mortality of third-instar nymphs was >65% with cycloxaprid at 125 mg liter(-1) but was <38% with buprofezin at 148 mg liter(-1). By the 4th day after infestation, however, control of nymphs by the two insecticides was similar, and cycloxaprid at 125 mg liter(-1) caused ≥ 80% mortality of adults but buprofezin at 148 mg liter(-1) (the highest rate tested) caused almost no adult mortality. LC50 values for cycloxaprid were lowest with nymphs, intermediate with adult males, and highest with adult females. Although buprofezin was slower acting than cycloxaprid, its LC50 for nymphs 5 d after infestation was 3.79-fold lower than that of cycloxaprid. Mean carboxylesterase (CarE) specific activity of nymphal WBPH treated with cycloxaprid and buprofezin was higher than that of control, but there was no significant difference between cycloxaprid and control (no insecticide), and it was significantly higher for buprofezin than those of cycloxaprid and control. For glutathione S-transferase and mixed function oxygenase, the specific activity of nymphal WBPH treated with buprofezin was significantly higher than those of cycloxaprid and control, too.

Simultaneous determination of twelve neonicotinoids and six metabolites in human urine with isotope-dilution UPLC-Q-Orbitrap HRMS.

The extensive global use of neonicotinoid insecticides (NNIs) has led to widespread human exposure, necessitating the development of effective methods for large-scale biomonitoring. However, current methods are inadequate in simultaneously and accurately detecting various NNIs or their metabolites (m-NNIs). In this study, we aimed to establish a robust method using solid-phase extraction (SPE)-ultra high performance liquid chromatography tandem Q-Orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) for the simultaneous determination of 12 NNIs and 6 m-NNIs in human urine. Samples were prepared using Oasis HLB 96 well plate with Isopropanol: methanol (7:3, v/v) as the elution solvent. The target compounds were separated using the Accucore RP-MS column and subsequently analyzed under parallel reaction monitoring mode. NTN32692 (m/z = 255.06433) was confirmed to be the specific metabolite of cycloxaprid for the further detection. Satisfactory recoveries (81.6-122.4 %) of the NNIs and m-NNIs were observed, with intra- (n = 3) and inter-day (n = 9) relative standard deviation (RSD) ranging from 0.8 % to 13.7 % and from 1.1 % to 18.6 %, respectively. Good linearity (R2 > 0.99) was achieved for all analytes. The limits of detection (LODs) for all target analytes ranged from 0.01 ng/mL to 0.65 ng/mL. This method was applied to urine samples collected from 10 children recruited from an agricultural area in China. Our study provides an effective method to identify and assess human exposure to NNIs and their metabolites.

Fabrication of nanogels to improve the toxicity and persistence of cycloxaprid against Diaphorina citri, the vector of citrus huanglongbing.

INTRODUCTION: Diaphorina citri is the most serious pest of citrus worldwide because it is the natural insect vector of huanglongbing. Cycloxaprid (Cyc) was highly toxic to D. citri. However, the poor solubility and stability had limited its development. OBJECTIVES: In order to improve the insecticidal effect and stability to harsh climatic conditions of Cyc. METHODS: Cyc was chosen as the representative pesticide, 4,4'-methylenebis (phenyl isocyanate), PEG-600 and n-butanol were used to prepare sustained-release nano-gelation particles (Cyc@NGs). RESULTS: Cyc@NGs enhance the toxicity of Cyc more than 3 folds. Furthermore, Cyc@NGs showed excellent anti-rain and anti-UV capacity. After being exposed to ultraviolet light for 12 h, Cyc decreased by 100 %, while the insecticide content of Cyc@NGs only decreased by 25 %. Additionally, Cyc@NGs possessed better wettability on citrus leaves, mainly benefitting from its lower contact angle on citrus leaves. Moreover, FITC-labeled nano-gelation particles (FITC-NGs) exhibited high capability to penetrate and enrich in citrus leaf tissue and D. citri midgut. Consequently, NGs promoted the translocation and durability of insecticides, thereby, increasing the insecticidal activity. The results suggested that nano-gelation particle is a promising platform to deliver insecticides and Cyc@NGs would be the suitable candidate for the effective management of D. citri.

Enantioselective uptake and translocation of a novel chiral neonicotinoid insecticide cycloxaprid in Youdonger (Brassica campestris subsp. Chinensis).

For a novel potential commercial chiral pesticide, an independent study on the fate characteristics and residues of each stereoisomer is essential if the application rates for the pesticide and human exposure are to be reduced. The absorption and translocation behavior of a chiral insecticide, cycloxaprid, in plants treated by root immersion and blade smearing was studied using (14)C-labeling tracer techniques. With the root treatment, total absorption of (1R;8S)-cycloxaprid (RS) (12.39%) was much greater than that of (1S;8R)-cycloxaprid (SR) (3.31%) at 192 h after treatment (HAT). The mass concentrations (RS/SR) of cycloxaprid in the roots, cotyledons, leaf 1, leaf 2, and leaf 3 were 37.0/16.8, 8.3/2.8, 11.7/6.5, 5.1/4.8, and 8.0/4.7 mg kg(-1) (fresh weight), respectively, at 192 HAT at an initial concentration 1.6 mg kg(-1). With the foliar application treatment, no significant difference was observed between the total absorption of RS (3.11%) and SR (4.03%) at the end of the treatment. Both acropetal and basipetal transport of absorbed (14)C occurred and more than 71.83% of absorbed RS and 82.42% of SR remained in the treated leaf. Stereoselective absorption was observed during root uptake but not during foliar absorption.

Metabolite of chiral cycloxaprid in solvent and in the raw of Puer tea.

Cycloxaprid (CYC) with a chiral oxabridged cis- structure contains a pair of enantiomers. Enantioselective degradation, transformation and metabolite of CYC was performed in different solvents under light and raw Puer tea processing. The results showed that cycloxaprid enantiomers in acetonitrile and acetone was stable over 17 day, however the transformation of 1S, 2R-(-)-cycloxaprid or 1R, 2S-(-)-cycloxaprid was founded in methanol. The fastest degradation of cycloxaprid occurred in acetone under light, the metabolites were founded with retention times (TR) at 34.83, 15.78 min, which mainly was via the reduce reaction of NO2 to NO, and rearrange reaction to tetrahydropyran. Degradation pathways were via the cleavage of the oxabridge seven member ring and the whole C ring. However, the degradation pathway under raw Puer tea processing was via the cleavage of whole C ring and the cleavage of oxabridge seven member ring and reducing NO2, then it underwent an elimination of nitromethylene and rearrange reaction. This pathway of Puer tea processing was firstly founded.

Computer-aided toxicity prediction and potential risk assessment of two novel neonicotinoids, paichongding and cycloxaprid, to hydrobionts.

Paichongding (IPP) and cycloxaprid (CYC) have been effectively used as the alternative products of imidacloprid (IMI) against IMI-resistant insects and exhibit a great market potential. However, risk assessment of IPP and CYC for non-target organisms, especially ecological risk assessment for non-target aquatic organisms, is still lacking. Here, we predicted the toxicity and potential risks of IPP, CYC, and their transformation products (TPs) to hydrobionts. The results indicated that IPP and CYC could generate 428 and 113 TPs, respectively, via aerobic microbial transformation. Nearly half of the IPP TPs and nearly 41 % of the CYC TPs exhibited high or moderate toxicity to Daphnia or fish. Moreover, we found that IPP, CYC, and 80 TPs of them posed potential risks to aquatic ecosystems. Almost all harmful TPs contained a 6-chloropyridine ring structure, suggesting that this structure may be associated with the strong toxicity of these TPs to aquatic organisms, and these TPs (IPP-TP2 or CYC-TP2, IPP-TP197 or CYC-TP71, IPP-TP198 or CYC-TP72, and IPP-TP212 or CYC-TP80) may appear in aquatic environments as final products. The risks posed by these TPs to aquatic ecosystems require more attention. This study provides insights into the toxicity and ecological risks of IPP and CYC.

Transcriptome and metabolome comprehensive analysis reveal the molecular basis of slow-action and non-repellency of cycloxaprid against an eusocial pest, Solenopsis invicta.

The eusocial pest, red imported fire ant (Solenopsis invicta), is a highly invasive species that poses significant threats to public safety, agriculture, and the ecological environment. Cycloxaprid, a newly identified effective, slow-acting, and non-repellent insecticide against S. invicta, allows contaminated individuals to transfer the insecticide among nestmates through body contact. However, the molecular-level changes occurring in S. invicta post cycloxaprid exposure and any molecular alterations contributing to the slow demise or decreased sensitivity remain unclear. In this study, transcriptomic and metabolomic techniques were used to investigate the molecular mechanisms of S. invicta exposed to cycloxaprid. Differential analysis results revealed 275, 323, and 536 differentially expressed genes at 12, 24, and 48 h, respectively. Genes involved in lipid and energy metabolism, DNA integration, and hormone synthesis were largely upregulated at 12 h, suggesting S. invicta might actively resist cycloxaprid impacts, and predominantly downregulated at 48 h, indicating further functional impairment and impending death. Also, we observed an imbalance in olfactory perception pathways at 12 h, which may indicate a disruption in the olfactory system of S. invicta. Metabolomic results showed that the regulation of most differential metabolites (DMs) was consistent with the expression changes of their related DEGs at different time points. Our study provides insights into the mechanism underlying slow-acting and non-repellent properties of cycloxaprid against S. invicta.

Molecular Evolutionary Mechanisms of CYP6ER1vA-Type Variant Associated with Resistance to Neonicotinoid Insecticides in Field Populations of Nilaparvata lugens.

The evolution of insecticide resistance has threatened the control of Nilaparvata lugens. Research on mechanisms behind neonicotinoid resistance in N. lugens remains incomplete. This study examined P450-mediated resistance to neonicotinoids in a resistant N. lugens strain (XA-2017-3G). The overexpression of CYP6ER1 in the XA-2017-3G strain plays a role in neonicotinoid resistance, as confirmed by RNA interference. Phenotypic analyses of CYP6ER1-mediated resistance in strains, including laboratory-susceptible, field-collected, and imidacloprid-laboratory further-selected strains, revealed that the vA-type/vL-type genotype exhibited greater resistance to neonicotinoids compared to the vA-type/vA-type genotype. The mRNA expression levels of CYP6ER1vA-type were closely correlated with the levels of neonicotinoid resistance in N. lugens strains, in which CYP6ER1vA-type overexpression is in part attributed to increased copy numbers of CYP6ER1. CYP6ER1vA-type-mediated neonicotinoid resistance was further confirmed by a CYP6ER1vA-type transgenic Drosophila melanogaster line. Taken together, our findings strongly suggest that the overexpression of CYP6ER1vA-type, which can be partially attributed to copy number variations, plays a crucial role in N. lugens resistance to neonicotinoids.

Translocation and metabolism of the chiral neonicotinoid cycloxaprid in oilseed rape (Brassica napus L.).

Neonicotinoids have been banned in some countries because of increased nontarget resistance and ecological toxicity. Cycloxaprid is a potentially promising substitute, but its metabolism in plants is still poorly understood. The study aims to clarify the translocation of cycloxaprid, identify its metabolites, propose possible metabolic pathways and compare differences between enantiomers in oilseed rape via 14C tracing technology and HPLC-QTOF-MS. The results showed that most cycloxaprid remained in the treated leaves, and only a small amount translocated to the anthers. Seven metabolites were identified, and the possible metabolic pathway was divided into two phases. Phase Ⅰ metabolism included two metabolites obtained via cleavage of the oxa-bridged seven-membered ring. Phase II metabolism was responsible for glucose conjugate formation. The possible metabolic pathways revealed that the proportion of phase I metabolites gradually decreased over time, and the phase II metabolites transformed from monosaccharide and disaccharide conjugates to trisaccharide and tetrasaccharide conjugates. The levels of metabolites were significantly different between the enantiomers. In particular, the main metabolite was M4, which has confirmed biological toxicity. M2 was the only metabolite detected in rapeseed. The results will promote the scientific application of cycloxaprid in agriculture and could have implications for assessing environmental risk.

Fate of the neonicotinoid insecticide cycloxaprid in different soils under oxic conditions.

Neonicotinoids are the most widely used pesticides worldwide due to their high toxicity to invertebrates. However, these compounds also increase the probability of environmental contamination. Cycloxaprid (CYC) is a promising neonicotinoid due to its insecticidal effectiveness and low cross resistance, but little is known about its fate in soils. Using radioisotope tracing techniques, the fate of 14C-labeled CYC enantiomers and racemic mixtures in aerobic soil was investigated in this research. After 100 d of incubation, the extractable residue (ER) of CYC decreased from 89.6% to 36.4% in red clay soil, from 46.1% to 10.1% in yellow loam soil, and from 93.2% to 12.2% in coastal saline soil. The radioactivity was substantially lower in methanol than in the other two solvents, but the distribution of CYC ER in various solvents across the three soils dramatically differed. The fraction of radioactive CYC that diffused into bound residue (BR) in the three soils increased over time to 56.8-83.0%. The variability in BR was influenced by soil properties such as organic matter concentration, pH, and residual microbial activity. Among the soils, yellow loam soil had the greatest tendency (53.0-83.0%) to form BR, while red clay soil showed the lowest capacity (7.5-61.2%). Cumulative mineralization (MI) to 14CO2 accounted for 0.12-0.23%, 6.69-7.31% and 14.82-20.06% in acidic soil, neutral soil and alkaline soil, respectively, which suggests that the environmental fate of chiral pesticides may be influenced by soil pH. No stereoselective behavior was detected in this study. These findings provide a framework to assess the environmental impact and ecological safety of CYC application.

Cross-Resistance and Fitness Costs of the cis-Nitromethylene Neonicotinoid Cycloxaprid Resistance in Melon Aphid, Aphis gossypii (Hemiptera: Aphididae).

The melon aphid, Aphis gossypii Glover, is an important pest on various vegetables around the world and has developed resistance to neonicotinoids in fields. Cycloxaprid is a novel cis-nitromethylene configuration neonicotinoid insecticide that is different from trans-configuration neonicotinoids like imidacloprid and thiamethoxam. Herein, the cross-resistance to the other seven insecticides and fitness costs were investigated in the cycloxaprid-resistant A. gossypii strain (Cpd-R), which has developed 69.5-fold resistance to cycloxaprid. The results showed that the Cpd-R strain had very low levels of cross-resistance to imidacloprid (4.3-fold), acetamiprid (2.9-fold), thiamethoxam (3.7-fold), nitenpyram (6.1-fold), flupyradifurone (2.2-fold), and sulfoxaflor (4.5-fold), while it exhibited a cross-resistance to dinotefuran (10.6-fold). The fitness of the Cpd-R strain by life table analysis was only 0.799 compared to the susceptible strain (Cpd-S). This Cpd-R strain exhibited significantly reduction in fecundity, oviposition days, and developmental time of nymph stage compared to the Cpd-S strain. Moreover, the expression levels of some genes related to the development and reproduction, including EcR, USP, JHAMT, and JHEH were significantly up-regulated, while Vg was down-regulated in the Cpd-R strain. This study indicates that the Cpd-R strain possessed a certain fitness cost. The above research results are useful for rational application of cycloxaprid and implementing the appropriate resistance management strategy for A. gossypii.

Enantioselectivity and residue analysis of cycloxaprid and its metabolite in the pile and fermentation processing of Puer tea by ultraperformance liquid chromatography-high-resolution mass spectrometry.

The residues of cycloxaprid enantiomers and metabolites are investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) during raw and ripen Puer tea processing. A Chiralpak AG column with chiral stationary phase of amylose tris (3-chloro-5-methylphenylcarbamate) is succeed to separate the 1R, 2S-cycloxaprid, 1S, 2R-cycloxaprid, and their metabolite, which is identified as nitrylene-imidazolidine. It is not conversed 1R, 2S -cycloxaprid into 1S,2R-cycloxaprid during Puer tea processing. The estimated half-lives of the 1R,2S-cycloxaprid and 1S,2R-cycloxaprid are 0.97 and 1.1 h, respectively, and 1R,2S-cycloxaprid decreases more quickly than the 1S,2R-cycloxaprid. During raw Puer tea processing, the half-lives of 1R, 2S-cycloxaprid and 1S, 2R-cycloxaprid are 1.68 h and 1.77 h, but the residue is still detected even if it is over 730 day. However, the half-lives of 1R,2S -cycloxaprid and 1S,2R-cycloxaprid are 0.60 day and 0.63 day during ripen tea processing. The amounts of metabolite are more in raw tea than in ripen tea; the terminal residues are still detected until 730 days during raw tea. A significant enantioselectivity of 1R, 2S-cycloxaprid and 1S, 2R-cycloxaprid is observed during raw tea or ripen tea processing. The degration result shows the enantioselectivity of cycloxaprid in raw or ripen Puer tea processing.

Sublethal toxicity, transgenerational effects, and transcriptome expression of the neonicotinoid pesticide cycloxaprid on demographic fitness of Coccinella septempunctata.

Evaluating side effects of new neonicotinoids in terms of sublethal doses and transcriptome expression is a crucial but challenging part of integrated pest management (IPM) approaches. To this end, a study of lethal and sublethal effects on Coccinella septempunctata larvae was conducted, and an age-stage, two-sex life table procedure was performed to investigate life-table parameters. Cycloxaprid (CYC) was shown to have adverse effects on survival, development, total longevity, reproductive capacity, and predation ability in C. septempunctata. In addition, demographic growth parameters of the F1 generation such as net reproductive rate, and the intrinsic and finite rates of increase were significantly decreased under sublethal dosage LR30 (1.91 g ai/hm2). These results demonstrated that the population growth of C. septempunctata was impacted by a sublethal dosage of CYC. For transcriptome expression, 544 up- and 338 down-regulated significantly differentially expressed genes (DEGs), were observed between LR30 treatment and control groups. Moreover, pathways related to metabolism of retinol, carcinogenesis, biosynthesis of steroid hormone, P450 metabolism, and metabolism of xenobiotics were identified in KEGG pathway analysis. Ten DEGs were chosen and confirmed with quantitative real-time PCR analysis. Based on these findings, CYC should be considered as a component of IPM strategies in the field.

Toxicological, Behavioral, and Horizontal Transfer Effects of Cycloxaprid Against Formosan Subterranean Termites (Blattodea: Rhinotermitidae).

Cycloxaprid, 9-((6-chloropyrid-3-yl)methyl)-4-nitro-8-oxa-10,11-dihydroimidazo-[2,3-a]-bicyclo-[3,2,1]-oct-3-ene, is a cis-configuration neonicotinoid insecticide. In the present study, the lethal and sublethal effect of cycloxaprid against Formosan subterranean termites, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae), was evaluated and compared with fipronil. Toxicity bioassays showed that cycloxaprid had slightly lower toxicity than fipronil. The minimum cycloxaprid concentration in sand and soil that causes 100% termite mortality was 100 ppm. Similar to fipronil, cycloxaprid significantly reduced wood consumption and tunneling activities of termites. In the tunneling-choice tests, termite tunneling activity measured in both length and area was significantly lower in sand treated with cycloxaprid (10 or 100 ppm) than that in untreated sand. In the aggregation-choice tests, cycloxaprid exhibited inhibition to termite aggregation starting from 100 ppm. In addition, cycloxaprid exhibited significant horizontal transfer effect at 10 ppm. In conclusion, our study showed that cycloxaprid is slightly less toxic than fipronil and more repellent to C. formosanus than fipronil. Future studies are needed to evaluate the effectiveness of cycloxaprid against subterranean termites in the field.

A comprehensive review on the pretreatment and detection methods of neonicotinoid insecticides in food and environmental samples.

In recent years, the residues of neonicotinoid insecticide in food and environmental samples have attracted extensive attention. Neonicotinoids have many adverse effects on human health, such as cancer, chronic disease, birth defects, and infertility. They have substantial toxicity to some non-target organisms (especially bees). Hence, monitoring the residues of neonicotinoid insecticides in foodstuffs is necessary to guarantee public health and ecological stability. This review aims to summarize and assess the metabolic features, residue status, sample pretreatment methods (solid-phase extraction (SPE), Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS), and some novel pretreatment methods), and detection methods (instrument detection, immunoassay, and some innovative detection methods) for neonicotinoid insecticide residues in food and environmental samples. This review provides detailed references and discussion for the analysis of neonicotinoid insecticide residues, which can effectively promote the establishment of innovative detection methods for neonicotinoid insecticide residues.

Combination of high specific activity carbon-14 labeling and high resolution mass spectrometry to study pesticide metabolism in crops: Metabolism of cycloxaprid in rice.

The study of pesticide metabolism in crops is critical for assessing the mode of action and environmental risks of pesticides. However, the study of pesticide metabolism in crops is usually complicated and it is often a daunting challenge to accurately screen the metabolites of novel pesticides in complex matrices. This study demonstrated a combined use of high-specific activity carbon-14 labeling and high-resolution mass spectrometry (HSA-14C-HRMS) for metabolism profiling of a novel neonicotinoid cycloxaprid in rice. By generating the characteristic radioactive peaks on the liquid chromatogram, the use of 14C can eliminate the severe interference of complex matrices and quickly probe target compounds; by producing ion pairs with unique abundance ratios on HRMS, high-specific activity labeling can effectively exclude false matrix positives and promote the elucidation of metabolite structure. The structures of 15 metabolites were identified, three of which were further confirmed by authentic standards. Based on these metabolites, a metabolic profile of cycloxaprid was established, which includes denitrification, demethylation, imidazolidine hydroxylation and ring cleavage olefin formation, oxidation and carboxylation reactions. The strategy of combining high-specific activity 14C labeling and HRMS offers unique advantages and provides a powerful solution for profiling unknown metabolites of novel pesticides in complex matrices, especially when traditional non-labeling methods are not feasible.

Changes in Transcriptome and Gene Expression in Sogatella furcifera (Hemiptera: Delphacidae) in Response to Cycloxaprid.

The white-backed planthopper, Sogatella furcifera (Horváth), causes substantial damage to crops by direct feeding or virus transmission, especially southern rice black-streaked dwarf virus, which poses a serious threat to rice production. Cycloxaprid, a novel cis-nitromethylene neonicotinoid insecticide, has high efficacy against rice planthoppers, including imidacloprid-resistant populations. However, information about the influence of cycloxaprid on S. furcifera (Hemiptera: Delphacidae) at the molecular level is limited. Here, by de novo transcriptome sequencing and assembly, we constructed two transcriptomes of S. furcifera and profiled the changes in gene expression in response to cycloxaprid at the transcription level. We identified 157,906,456 nucleotides and 131,601 unigenes using the Illumina technology from cycloxaprid-treated and untreated S. furcifera. In total, 38,534 unigenes matched known proteins in at least one database, accounting for 29.28% of the total unigenes. The number of coding DNA sequences was 28,546 and that of amino acid sequences in the coding region was 22,299. In total, 15,868 simple sequence repeats (SSRs) were identified. The trinucleotide repeats accounted for 45.1% (7,157) of the total SSRs and (AAG/CTT)n were the most frequent motif. There were 359 differentially expressed genes that might have been induced by cycloxaprid. There were 131 upregulated and 228 downregulated genes. Twenty-two unigenes might be involved in resistance against cycloxaprid, such as cytochrome P450, glutathione S-transferase (GST), acid phosphatase (ACP), and cadherin. Our study provides vital information on cycloxaprid-induced resistance mechanisms, which will be useful to analyze the molecular mechanisms of cycloxaprid resistance and may lead to the development of novel strategies to manage S. furcifera.