Method validation and residue analysis of methoxyfenozide and metaflumizone in Chinese broccoli under field conditions by liquid chromatography with tandem mass spectrometry.

Methoxyfenozide and metaflumizone are insecticides used on Chinese broccoli to prevent insects and increase yield. However, the residues are potentially harmful to the environment and consumers. In this study, the quick, easy, cheap, effective, rugged, safe method with high-performance liquid chromatography with tandem mass spectrometry was modified and validated for determination of methoxyfenozide and metaflumizone in Chinese broccoli. The clean-up efficiency of different sorbents including C18 , primary secondary amine, graphitized carbon black, and carbon nanofiber was compared. Recoveries of the validated method were 71.8-94.6% with relative standard deviations of 1.5-3.2% and the limits of quantification were 0.01 and 0.005 mg/kg for methoxyfenozide and metaflumizone, respectively. A storage stability test showed almost no degradation of methoxyfenozide in Chinese broccoli, however, the degradation rate of metaflumizone was 22.9% after 10-wk storage at -20°C. In field trials in four producing regions, the dissipation of both methoxyfenozide and metaflumizone in Chinese broccoli was fast, with half-lives of only 1.0-5.1 and 0.7-2.5 days, respectively. Terminal residues after application of the two pesticides were all below 1.0 mg/kg after 5 days.

Molecular characterization, expression pattern and metabolic activity of flavin-dependent monooxygenases in Spodoptera exigua.

Enhanced detoxification is one of the important mechanisms for insecticide resistance. Most research in this field to date has focused on the role of cytochrome P450s. Our previous work revealed that flavin-dependent monooxygenases (FMOs) were involved in metabolic resistance of Spodoptera exigua. In the present study we investigated the molecular characteristics, expression patterns and oxidative activities of SeFMO on insecticides. Three FMO genes, which encode proteins with the typical FMO motifs, were cloned from S. exigua. The oxidative activities of eukaryotically expressed SeFMO enzymes were verified with the model substrate of FMO. Importantly, the SeFMOs had significantly higher oxidative activities on metaflumizone and lambda-cyhalothrin than on model substrates and other insecticides tested. The three SeFMOs were mainly expressed in the midgut, fat body and Malpighian tubules. The tissues responsible for xenobiotic metabolism and their expression characteristics were similar to those of P450s acting as detoxification genes. The study also revealed that the expression of SeFMOs could be induced by insecticide exposure, and that SeFMOs were over-expressed in a metaflumizone-resistant strain of S. exigua. These results suggest that SeFMOs are important insecticide detoxifying enzymes, and that over-expression of FMO genes may be one of the mechanisms for metabolic resistance in S. exigua.

The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides.

Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNav1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs.

Fitness and inheritance of metaflumizone resistance in Plutella xylostella.

The diamondback moth, Plutella xylostella (L.) has developed resistance to many types of insecticides in the field. To study inheritance and fitness cost of metaflumizone resistance, a susceptible strain of diamondback moth was continuously selected with metaflumizone during 37 generations under laboratory conditions. The resistance to metaflumizone was at a high level (resistance ratios from 250.37 to 1450.47-fold). We investigated a metaflumizone resistance strain (G27) and a susceptible strain of P. xylostella, using the age-stage, two-sex life table approach. Compared to the susceptible strain, egg duration, the developmental time of the first and second instar larvae, pupae duration, adult preoviposition period (APOP), total preoviposition period (TPOP), egg hatchability, the survival rate of second instar larva and the mean generation time (T) were significantly differences in the resistant strain. The resistant strain had a relative fitness of 0.78. The inheritance of metaflumizone resistance was also studied by crossing the metaflumizone resistant and susceptible populations. Results revealed an autosomal and incompletely recessive mode of inheritance for metaflumizone resistance in the resistant population of P. xylostella. The present study provided useful information for planning potential management strategies to delay development of metaflumizone resistance in P. xylostella.

Biochemical mechanisms for metaflumizone resistance in beet armyworm, Spodoptera exigua.

The metaflumizone, which belongs to the class of voltage-dependent sodium channel blockers, was registered to control Spodoptera exigua on vegetables in China in 2009. The present study revealed S. exigua has developed high resistance to this novel chemistry insecticide shortly after 2-3 years application in Guangdong Province of China. The metabolic mechanisms for metaflumizone resistance in this insect were analysed. The inhibitor of esterases greatly potentiates the toxicity of this chemical against the field resistant populations. The synergism ratio is 5.7 and 3.4-fold for S. exigua collected from Huizhou, Guangdong Province in 2011 and 2012, respectively. The activity of esterases in field populations (HZ12) is also significantly greater than that in the susceptible strain, and further significantly increased by challenge with metaflumizone for 3 generations. However, the inhibitor of P450s or GSTs only has slight synergism on metaflumizone toxicity against resistant populations, and there are no obvious differences in activities of P450s or GSTs between resistant populations and the susceptible strain. These results suggest that esterases might take pivotal role in conferring metabolic resistance to metaflumizone in the field populations of S. exigua, and P450s or GSTs are not involved in this resistance. Moreover, flavin-dependent monooxygenases (FMOs) are discovered to involve in metaflumizone resistance in the field populations of S. exigua. The FMO inhibitor, methimazole, potentiates metaflumizone toxicity in resistant larva of this species substantially. The synergism ratios for methimazole in resistant populations HZ11 and HZ12 were 3.1 and 1.9, respectively. Enzymatic assays also revealed higher FMO activities in resistant populations than in the susceptible strain, and successive selection with metaflumizone further increased the FMO activity in the field resistant population, but not significantly. The higher FMO activities in the older larval stages and in the larval midgut signify the importance of FMO in the detoxification of xenobiotic from food sources. The synergism assay and FMO activity analysis suggest that FMO contributes to metaflumizone detoxification in resistant populations of S. exigua and conferred metaflumizone resistance in S. exigua. A novel mechanism for insecticide resistance by insect was proposed.

Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels.

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.

Equivalent intensity but differential dominance of sodium channel blocker insecticide resistance conferred by F1845Y and V1848I mutations of the voltage-gated sodium channel in Plutella xylostella.

Two point mutations (F1845Y and V1848I) in the voltage-gated sodium channel gene of Plutella xylostella are involved in the target-site resistance to sodium channel blocker insecticides (SCBIs). The contribution of the individual mutations to the SCBI resistance and the associated inheritance modes is as yet unclear. Through 2 rounds of single-pair crossing and marker-assisted selection, 2 P. xylostella strains (1845Y and 1848I) bearing homozygous F1845Y or V1848I mutant alleles were successfully established from a field-collected population, and the contribution of each mutation to SCBI resistance, as well as associated inheritance patterns, was determined. When compared with the susceptible SZPS strain, each of the mutations individually conferred equally high-level resistance to indoxacarb (378 and 313 fold) and metaflumizone (734 and 674 fold), respectively. However, dominance levels of resistance to SCBIs were significantly different between the 2 resistant strains. Resistance of the 1845Y strain to indoxacarb and metaflumizone was inherited as an autosomal and incompletely dominant trait (D values ranged from 0.43 to 0.76). In contrast, that of the 1848I strain followed an autosomal but incompletely recessive to semidominant mode (D values: -0.24 to 0.09). Our findings enriched the current understanding of inheritance and mechanisms of SCBI resistance in P. xylostella, and will help develop resistance management programs for P. xylostella and other economic pests.

Control efficacy and joint toxicity of metaflumizone mixed with chlorantraniliprole or indoxacarb against the fall armyworm, Spodoptera frugiperda.

BACKGROUND: The fall armyworm (FAW), Spodoptera frugiperda is the main destructive pest of grain crops, and has led to substantial economic losses worldwide. Chemical pesticides are the most effective way to manage FAW. Here, a laboratory test using an artificial diet-incorporated assay was conducted to determine the toxicity of five insecticides and the joint effect of the binary combination insecticides to FAW larvae. A field plot test using foliar spray was carried out to assess the control efficacy of metaflumizone mixed with chlorantraniliprole or indoxacarb against FAW. RESULTS: The bioassay results showed that metaflumizone had a stronger insecticidal effect than indoxacarb toward FAW larvae. Furthermore, the mixture of metaflumizone and chlorantraniliprole in a volume ratio of 3:7 had the strongest synergistic effect against FAW, with a co-toxicity coefficient (CTC) of 317.18. The best synergistic effect for mixtures of metaflumizone and indoxacarb was observed at a 1:9 volume ratio, with a CTC of 185.98. However, there was an antagonistic effect of metaflumizone mixed with emamectin benzoate and with lufenuron, because the co-toxic factor was less than -20 at volume ratios of 8:2 and 9:1, respectively. According to the results of the field trial, metaflumizone mixed with chlorantraniliprole or indoxacarb at a 50% reduction of the application rate can effectively control FAW with efficacy ranging from 77.73% to 94.65% 1-7 days postapplication. CONCLUSION: Overall, our findings suggest that metaflumizone and its binary combination insecticides can be utilized in FAW integrated pest management programs. © 2022 Society of Chemical Industry.

Azobenzene-Semicarbazone Enables Optical Control of Insect Sodium Channels and Behavior.

Photopharmacology uses molecular photoswitches to establish control over the action of bioactive molecules. The application of photopharmacology in the research of invertebrate sodium channels has not been investigated. Here we report several photochromic ligands of metaflumizone. One ligand, termed ABM04, underwent reversible trans-cis isomerization under ultraviolet or blue light irradiation. cis-ABM04 had excellent larvicidal activity against mosquito larvae with an LC50 value of 4.39 µM and showed insecticidal activity against Mythimna separata with an LC50 value of 7.19 µM. However, trans-ABM04 was not found to have biological activity. ABM04 (10 µM) can induce depolarization of dorsal unpaired median neurons and enable the real-time photoregulation of mosquito larval behavior. The precise regulation of invertebrate sodium channels is realized for the first time, which provides a new strategy for the basic and accurate research of invertebrate sodium channels.

Baseline Response, Monitoring, and Cross-Resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Sodium Channel Blocker Insecticides in Brazil.

Spodoptera frugiperda (J.E. Smith) is one of the key cross-crop pests in Brazilian agroecosystems. Field-evolved resistance of S. frugiperda to some conventional insecticides and Bt proteins has already been reported. Thus, the use of insecticides with new mode of action such as sodium channel blockers (indoxacarb and metaflumizone) could be an important tool in insecticide resistance management (IRM) programs. To implement a proactive IRM, we conducted baseline response and monitoring to indoxacarb and metaflumizone in 87 field populations of S. frugiperda collected from major maize-growing regions of Brazil from 2017 to 2020, estimated the frequency of resistance alleles to indoxacarb, and evaluated cross-resistance of indoxacarb and metaflumizone to some selected insecticides and Bt proteins. Low variation in susceptibility to indoxacarb (4.6-fold) and metaflumizone (2.6-fold) was detected in populations of S. frugiperda in 2017. The frequency of the resistance allele to indoxacarb was 0.0452 (0.0382-0.0527 CI 95%), by using F2 screen method. The mean survival at diagnostic concentration, based on CL99, varied from 0.2 to 12.2% for indoxacarb and from 0.0 to 12.7% for metaflumizone, confirming high susceptibility of S. frugiperda to these insecticides in Brazil. No cross-resistance was detected between sodium channel blocker insecticides and other insecticides (organophosphate, pyrethroid, benzoylurea, spinosyn, and diamide) and Bt proteins. These findings showed that sodium channel blocker insecticides are important candidates to be exploited in IRM strategies of S. frugiperda in Brazil.

Biochemical Mechanisms, Cross-resistance and Stability of Resistance to Metaflumizone in Plutella xylostella.

The diamondback moth, Plutella xylostella (L.) is an important pest of cruciferous crops worldwide. It has developed resistance to many conventional and novel insecticide classes. Metaflumizone belongs to the new chemical class of semicarbazone insecticides. To delay the development of metaflumizone resistance in P. xylostella and to guide insecticide use in the field, the biochemical mechanisms, cross-resistance spectrum, and stability of resistance to metaflumizone were studied in a laboratory-selected resistant strain (metaflu-SEL). Synergism tests with the carboxylesterase inhibitor triphenyl phosphate (TPP), the glutathione S-transferase depletor diethyl maleate (DEM), and the P450 inhibitor piperonyl butoxide(PBO) had no obvious effect on metaflumizone in the metaflu-SEL strain and the susceptible strain (SS) of P. xylostella, with synergism ratios that ranged from 1.02 to 1.86. Biochemical studies revealed that the cytochrome P450-dependent monooxygenase increased only 1.13-fold in the metaflu-SEL strain compared with the UNSEL stain; meanwhile, carboxylesterase and glutathione S-transferase activity showed no difference. These results suggest that these detoxification enzymes may be not actively involved in metaflumizone resistance. Furthermore, the metaflu-SEL population showed a moderate level of cross-resistance to indoxacarb (11.63-fold), but only very low cross-resistance to spinosad (1.75-fold), spinetoram (3.52-fold), abamectin (2.81-fold), beta-cypermethrin (0.71-fold), diafenthiuron (0.79-fold), chlorantraniliprole (2.16-fold), BT (WG-001) (3.34-fold), chlorfenapyr (0.49-fold), and chlorfluazuron (0.97-fold). Moreover, metaflumizone resistance decreased from 1087.85- to 1.23-fold in the metaflu-SEL strain after 12 generations without exposure to metaflumizone. These results are useful for formulating insecticide resistance management strategies to control P. xylostella and to delay the development of metaflumizone resistance in the field.

Review of the existing maximum residue levels for metaflumizone according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance metaflumizone. To assess the occurrence of metaflumizone residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Commission Regulation (EU) No 188/2011, the MRLs established by the Codex Alimentarius Commission and the European authorisations reported by Member States (including the supporting residues data). Based on the assessment of the available data, MRL proposals were derived and a consumer risk assessment was carried out. Some information required by the regulatory framework was missing and a possible acute risk to consumers was identified. Hence, the consumer risk assessment is considered indicative only, some MRL proposals derived by EFSA still require further consideration by risk managers and measures for reduction of the consumer exposure should also be considered.

Resistance Risk Evaluated by Metaflumizone Selection and the Effects on Toxicities Over Other Insecticides in Spodoptera exigua (Lepidoptera: Noctuidae).

Metaflumizone is a novel semicarbazone insecticide. It functions as a sodium channel blocker insecticide (SCBI) with excellent insecticidal activity on most economically important lepidopterous pests. This study assessed the resistance risk of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) to metaflumizone in the laboratory and the effects of metaflumizone selection on toxicities to other insecticides. Spodoptera exigua collected from a field population at Huizhou in 2012 were successively challenged by metaflumizone to evaluate the risk of resistance evolution. Twelve generations of selection increased resistance to metaflumizone by 3.4-fold and threshold trait analysis revealed that the realized heritability (h2) of this resistance was 0.086. When h2 was equal to 0.086 and 90% of individuals were killed at each generation, LC50 to metaflumizone increased by 10-fold after 15 generations. The selection by metaflumizone did not increase the resistance to indoxacarb, chlorantraniliprole, spinosad, methomyl, or endosulfan, suggesting a lack of cross-resistance. However, metaflumizone challenge upheld the recession of resistance to emamectin benzoate, chlorfluazuron, and tebufenozide. The block of resistance drops by metaflumizone exposure implied a possible cross-resistance between metaflumizone and these three insecticides. These results contribute to integrated resistance management of S. exigua.

Functional validation of target-site resistance mutations against sodium channel blocker insecticides (SCBIs) via molecular modeling and genome engineering in Drosophila.

Sodium channel blocker insecticides (SCBIs) like indoxacarb and metaflumizone offer an alternative insecticide resistance management (IRM) strategy against several pests that are resistant to other compounds. However, resistance to SCBIs has been reported in several pests, in most cases implicating metabolic resistance mechanisms, although in certain indoxacarb resistant populations of Plutella xylostella and Tuta absoluta, two mutations in the domain IV S6 segment of the voltage-gated sodium channel, F1845Y and V1848I have been identified, and have been postulated through in vitro electrophysiological studies to contribute to target-site resistance. In order to functionally validate in vivo each mutation in the absence of confounding resistance mechanisms, we have employed a CRISPR/Cas9 strategy to generate strains of Drosophila melanogaster bearing homozygous F1845Y or V1848I mutations in the para (voltage-gated sodium channel) gene. We performed toxicity bioassays of these strains compared to wild-type controls of the same genetic background. Our results indicate both mutations confer moderate resistance to indoxacarb (RR: 6-10.2), and V1848I to metaflumizone (RR: 8.4). However, F1845Y confers very strong resistance to metaflumizone (RR: >3400). Our molecular modeling studies suggest a steric hindrance mechanism may account for the resistance of both V1848I and F1845Y mutations, whereby introducing larger side chains may inhibit metaflumizone binding.

Metaflumizone inhibits the honeybee NaV 1 channel by targeting recovery from slow inactivation.

Metaflumizone is the latest addition to the armamentarium of the Na+ channel inhibitor insecticide family. We used the Xenopus oocyte expression system and a Markovian model to assess the effect of metaflumizone on Apis mellifera Na+ channels (AmNaV 1). Our results reveal that metaflumizone inhibits AmNaV 1 channels by targeting the kinetics of recovery from slow inactivation. Multistate modeling of fast and slow inactivation of the AmNaV 1 channel made it possible to study the effects of metaflumizone on a set of rate constants underlying the transition between the open and inactivated conformations and provided insights into their specificity. We conclude that the methods we used could be extended to assessing the toxicity of other Na+ channel inhibitor insecticides.

Molecular Mechanism of Action and Selectivity of Sodium Ch annel Blocker Insecticides.

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides that are represented by two commercially registered compounds, indoxacarb and metaflumizone. SCBIs, like pyrethroids and DDT, target voltage-gated sodium channels (VGSCs) to intoxicate insects. In contrast to pyrethroids, however, SCBIs inhibit VGSCs at a distinct receptor site that overlaps those of therapeutic inhibitors of sodium channels, such as local anesthetics, anticonvulsants and antiarrhythmics. This review will recount the development of the SCBI insecticide class from its roots as chitin synthesis inhibitors, discuss the symptoms of poisoning and evidence supporting inhibition of VGSCs as their mechanism of action, describe the current model for SCBI-induced inhibition of VGSCs, present a model for the receptor for SCBIs on VGSCs, and highlight differences between data collected from mammalian and insect experimental models.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella.

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutations were never found to co-exist in the same allele of PxNav , suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.