Substrate Report for: Indoxacarb

Insecticides tolerance in herbivorous arthropods is associated with preadaptation to host plant allelochemicals. However, how plant secondary metabolites activate detoxifying metabolic genes to develop tolerance remains unclear. Herein, the tolerance of Spodoptera litura larvae to cyantraniliprole was increased after nicotine exposure. An S. litura alpha esterase, SlCOE030, was predominantly expressed in the midgut and induced after exposure to cyantraniliprole, nicotine, and cyantraniliprole plus nicotine. Drosophila melanogaster with ectopically overexpressed SlCOE030 enhanced cyantraniliprole and nicotine tolerance by 4.91- and 2.12-fold, respectively. Compared to UAS-SlCOE030 and Esg-GAL4 lines, the Esg > SlCOE030 line laid more eggs after nicotine exposure. SlCOE030 knockdown decreased the sensitivity of nicotine-treated S. litura larvae to cyantraniliprole. Metabolism assays indicated that recombinant SlCOE030 protein metabolizes cyantraniliprole. Homology modeling and molecular docking analysis demonstrated that SlCOE030 exhibits effective affinities for cyantraniliprole and nicotine. Thus, insect CarEs may result in the development of cross-tolerance between synthetic insecticides and plant secondary metabolites.

BACKGROUND: Indoxacarb is an important active ingredient extensively used for the control of Tuta absoluta, a major tomato pest, playing a particular role in insecticide resistance management schemes.
RESULTS: Reduced susceptibility to indoxacarb was identified (1794-fold resistance) through toxicological bioassays in a field population from Greece and evolved rapidly to resistance after short laboratory selection. Combined bioassays with synergists and biochemical analysis suggested only a partial involvement of detoxification enzymes in the resistant phenotype. To investigate the role of target-site resistance, segment 6 of domain IV of the sodium channel in T. absoluta was cloned and the sequences compared between susceptible and indoxacarb-resistant T. absoluta insects. The presence of the F1845Y and the V1848I indoxacarb resistance mutations was detected and was strongly associated with the phenotype. These amino acid substitutions correspond to recently characterised indoxacarb resistance mutations in diamondback moth (Plutella xylostella). Robust and accurate PCR-RFLP assays were subsequently developed and successfully validated for detecting both indoxacarb resistance mutations in field T. absoluta populations. CONCLUSION: The identification of indoxacarb resistance mutations and the development of diagnostic tools will allow early detection of indoxacarb resistance, facilitating implementation of appropriate resistance management strategies, thus delaying the spread of resistance. (c) 2016 Society of Chemical Industry.

The toxicity of the oxadiazine insecticide indoxacarb to the European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae), was evaluated in the presence and absence of S,S,S-tributyl phosphorotrithioate (DEF), an inhibitor of hydrolytic metabolism. Bioassays involving topical application of different concentrations of indoxacarb to third instars of a susceptible O. nubilalis laboratory strain were performed, and in vitro metabolism experiments involving [14C] indoxacarb were examined to determine the role of hydrolytic metabolism in indoxacarb activation. Indoxacarb toxicity decreased in the presence of DEF indicating antagonism of toxicity. Results of in vivo and in vitro inhibition experiments indicated a reduction of indoxacarb activation and formation of the hydrolytic metabolite. These results are consistent with the proposed mechanism of hydrolytic activation for this compound.

Insecticides tolerance in herbivorous arthropods is associated with preadaptation to host plant allelochemicals. However, how plant secondary metabolites activate detoxifying metabolic genes to develop tolerance remains unclear. Herein, the tolerance of Spodoptera litura larvae to cyantraniliprole was increased after nicotine exposure. An S. litura alpha esterase, SlCOE030, was predominantly expressed in the midgut and induced after exposure to cyantraniliprole, nicotine, and cyantraniliprole plus nicotine. Drosophila melanogaster with ectopically overexpressed SlCOE030 enhanced cyantraniliprole and nicotine tolerance by 4.91- and 2.12-fold, respectively. Compared to UAS-SlCOE030 and Esg-GAL4 lines, the Esg > SlCOE030 line laid more eggs after nicotine exposure. SlCOE030 knockdown decreased the sensitivity of nicotine-treated S. litura larvae to cyantraniliprole. Metabolism assays indicated that recombinant SlCOE030 protein metabolizes cyantraniliprole. Homology modeling and molecular docking analysis demonstrated that SlCOE030 exhibits effective affinities for cyantraniliprole and nicotine. Thus, insect CarEs may result in the development of cross-tolerance between synthetic insecticides and plant secondary metabolites.

BACKGROUND: As an important family of detoxification enzymes, carboxylesterases (CarEs) play important roles in the development of insecticides resistance in almost all agricultural pests. Previous studies suggested that the enhancement of CarE activity was an important mechanism mediating indoxacarb resistance in Spodoptera litura, and several CarE genes were found to be overexpressed in the indoxacarb-resistant strains. However, the functions of these CarE genes in indoxacarb resistance needs to be further investigated. RESULTS: The synergist triphenyl phosphate (TPP) effectively reduced the resistance of S. litura to indoxacarb, suggesting an involvement of CarEs in indoxacarb resistance. Among seven identified S. litura CarE genes (SlituCOE hereinafter), six were overexpressed in two indoxacarb-resistant strains, but there were no significant differences in gene copy number. Knockdown of SlituCOE009 and SlituCOE050 enhanced indoxacarb sensitivity in both susceptible and resistant strains, whereas knockdown of SlituCOE090, SlituCOE093 and SlituCOE074 enhanced indoxacarb sensitivity only in the resistant strain. Knockdown of the sixth gene SlituCOE073 did not have any effect. Furthermore, knockdown of the five SlituCOE genes simultaneously had a greater effect on increasing indoxacarb sensitivity than silencing them individually. By contrast, overexpression of the five SlituCOE genes individually in Drosophila melanogaster significantly decreased the toxicity of indoxacarb to transgenic fruit flies. Furthermore, modeling and docking analysis indicated that the catalytic pockets of SlituCOE009 and SlituCOE074 were ideally shaped for indoxacarb and DCJW, but the binding affinity for DCJW was stronger than indoxacarb. CONCLUSION: This study reveals that multiple overexpressed CarE genes are involved in indoxacarb resistance in S. litura.

The red imported fire ant (Solenopsis invicta) is one of the deadliest invasive ant species that threatens the world by disrupting biodiversity, important functions within a natural ecosystem, and community structure. They are responsible for huge economic losses in the infested countries every year. Synthetic insecticides, especially indoxacarb, have been broadly used to control S. invicta for many years. However, the biochemical response of S. invicta to indoxacarb remains largely undiscovered. Here, we used the sublethal doses of indoxacarb on the S. invicta collected from the eight different cities of Southern China. The alteration in the transcriptome profile of S. invicta following sublethal dosages of indoxacarb was characterized using high-throughput RNA-seq technology. We created 2 libraries, with 50.93 million and 47.44 million clean reads for indoxacarb treatment and control, respectively. A total of 2018 unigenes were regulated after insecticide treatment. Results indicated that a total of 158 differentially expressed genes (DEGs) were identified in the indoxacarb-treated group, of which 100 were significantly upregulated and 58 were downregulated, mostly belonging to the detoxification enzymes, such as AChE, CarE, and GSTs. Furthermore, results showed that most of these DEGs were found in several KEGG pathways, including steroid biosynthesis, other drug metabolizing enzymes, glycerolipid metabolism, chemical carcinogenesis, drug-metabolizing cytochrome P450, glutathione metabolism, glycerophospholipid metabolism, glycolysis/gluconeogenesis, and metabolism of xenobiotics. Together, these findings indicated that indoxacarb causes significant alteration in the transcriptome profile and signaling pathways of S. invicta, providing a foundation for further molecular inquiry.

Indoxacarb is an important insecticide for the selective control of Helicoverpa armigera. It can be bioactivated to the more effective N-decarbomethoxylated indoxacarb (DCJW) by esterases in pests. It was observed that both field and laboratory selected populations of H. armigera showed negative cross-resistance between indoxacarb and methoxyfenozide. The Handan population exhibited moderate resistance to indoxacarb, but was susceptible to methoxyfenozide; the Baoding and Yishui populations exhibited moderate resistance to methoxyfenozide, but they were susceptible to indoxacarb. Moreover, the toxicity of indoxacarb was enhanced 1.83-fold in the laboratory methoxyfenozide-resistant H. armigera, and susceptibility to methoxyfenozide was increased 2.81-fold in the laboratory indoxacarb-resistant H. armigera. In vivo, DCJW concentrations in the susceptible and methoxyfenozide-selected (laboratory methoxyfenozide-resistant) populations were 4.59- and 4.31-fold greater than in the indoxacarb-resistant Handan population 1 h after dosing. After 2 h, the highest concentrations of DCJW and indoxacarb appeared in the methoxyfenozide-selected population. Meanwhile, increased carboxyl esterase (CarE) and decreased glutathione S-transferase (GST) activities were observed in the methoxyfenozide-selected population. However, the indoxacarb-selected (laboratory indoxacarb-resistant) and Handan populations showed a higher disappearance of indoxacarb and DCJW, and the activity of cytochrome P450 mono-oxygenase in these populations were significantly increased. This study showed that the improved toxicity of indoxacarb, as observed in the methoxyfenozide-selected H. armigera, was correlated with increased CarE activity, decreased GST activity, and the in vivo accumulation of indoxacarb and DCJW. The significantly increased cytochrome P450 activity and higher disappearance of indoxacarb and DCJW in indoxacarb-resistant H. armigera resulted in the decreased toxicity of indoxacarb.

The tobacco cutworm, Spodoptera litura, is an important pest of crop and vegetable plants worldwide, and its resistance to insecticides have quickly developed. However, the resistance mechanisms of this pest are still unclear. In this study, the change in mRNA and miRNA profiles in the susceptible, indoxacarb-resistant and field indoxacarb-resistant strains of S. litura were characterized. Nine hundred and ten co-up-regulated and 737 co-down-regulated genes were identified in the resistant strains. Further analysis showed that 126 co-differentially expressed genes (co-DEGs) (cytochrome P450, carboxy/cholinesterase, glutathione S-transferase, ATP-binding cassette transporter, UDP-glucuronosyl transferase, aminopeptidase N, sialin, serine protease and cuticle protein) may play important roles in indoxacarb resistance in S. litura. In addition, a total of 91 known and 52 novel miRNAs were identified, and 10 miRNAs were co-differentially expressed in the resistant strains of S. litura. Furthermore, 10 co-differentially expressed miRNAs (co-DEmiRNAs) had predicted co-DEGs according to the expected miRNA-mRNA negative regulation pattern and 37 indoxacarb resistance-related co-DEGs were predicted to be the target genes. These results not only broadened our understanding of molecular mechanisms of insecticide resistance by revealing complicated profiles, but also provide important clues for further study on the mechanisms of miRNAs involved in indoxacarb resistance in S. litura.

An intracellular esterase (BCE) from Bacillus cereus WZZ006 was purified to homogeneity with an 89.5-fold purification, specific activity of 1.79U/mg, and 26.7% recovery. The estimated molecular weight of BCE was 96kDa which was analyzed by SDS-PAGE and MALDI-TOF-MS. Activity staining denotes that BCE has an unexplored new carboxyl esterase characteristic. BCE enzyme activity was maximum at pH8.5 and also at 50 degrees C with pNP-caproate as a substrate. This indicates that the studied BCE as a thermoalkaliphilic esterase. The kinetic properties like Km, Vmax, kcat and kcat/Km value for BCE was found to be 0.98mM, 0.03mM/min, 69.47min(-1) and 70.89mM(-1)min(-1), respectively. Synthesis of (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indole-2-carboxylic acid methyl ester ((S)-CODHCM) by BCE can be shortened to 3h compared to 36h with whole-cell catalysis. The e.e.s achieved was 93.83%, and conversion around 52.78% with E being 39.95. These features render BCE as a promising biocatalyst for the synthesis of a key chiral intermediate for indoxacarb.

BACKGROUND: Indoxacarb is an important active ingredient extensively used for the control of Tuta absoluta, a major tomato pest, playing a particular role in insecticide resistance management schemes.
RESULTS: Reduced susceptibility to indoxacarb was identified (1794-fold resistance) through toxicological bioassays in a field population from Greece and evolved rapidly to resistance after short laboratory selection. Combined bioassays with synergists and biochemical analysis suggested only a partial involvement of detoxification enzymes in the resistant phenotype. To investigate the role of target-site resistance, segment 6 of domain IV of the sodium channel in T. absoluta was cloned and the sequences compared between susceptible and indoxacarb-resistant T. absoluta insects. The presence of the F1845Y and the V1848I indoxacarb resistance mutations was detected and was strongly associated with the phenotype. These amino acid substitutions correspond to recently characterised indoxacarb resistance mutations in diamondback moth (Plutella xylostella). Robust and accurate PCR-RFLP assays were subsequently developed and successfully validated for detecting both indoxacarb resistance mutations in field T. absoluta populations. CONCLUSION: The identification of indoxacarb resistance mutations and the development of diagnostic tools will allow early detection of indoxacarb resistance, facilitating implementation of appropriate resistance management strategies, thus delaying the spread of resistance. (c) 2016 Society of Chemical Industry.

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32mumol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26mumol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

The control of the most important pest of stored maize, the weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), is mainly achieved with the use of pyrethroid insecticides. However, the intensive use of these compounds has led to the selection of resistant populations and has compromised the control efficacy of this insect pest. Here, the toxicity of indoxacarb for a potential use in the control of S. zeamais was assessed on 13 Brazilian populations. Concentration-mortality bioassays, in the presence of synergists (piperonyl butoxide, triphenyl phosphate, and diethyl maleate), were used to assess potential metabolic-based indoxacarb resistance mechanisms. We also assessed the behavioral (locomotory) responses of these populations to indoxacarb exposure. The results showed significant differences between the populations (LD50 values ranged from 0.06 to 13.99 mg a.i/kg of grains), resulting in resistance ratios of >200-fold between the least (Canarana-MT) and the most (Espirito Santo do Pinhal-SP) susceptible populations. The results obtained with synergized indoxacarb suggest the involvement of esterases and glutathione-S-transferases on indoxacarb action, and also suggest the involvement of cytochrome P450-dependent monooxygenases as a potential indoxacarb resistance mechanism in Brazilian populations of S. zeamais. Although indoxacarb-induced behavioral avoidance varied among populations, some resistant populations (e.g., Canarana-MT) were able to reduce exposure to indoxacarb by spending more time in the nontreated areas. Collectively, our findings indicate that the behavioral (locomotory) and physiological responses of these insects may compromise the control efficacy of oxadiazine insecticides (e.g., indoxacarb) in Brazilian populations of S. zeamais.

BACKGROUND: Tuta absoluta(Meyrick) is one of the most serious pests of tomato recently introduced in the Mediterranean region. A novel bioassay method designed for the accurate determination of insecticide toxicity on T. absoluta (IRAC method No. 022) was validated by three different laboratories [Greece (NAGREF), Italy (UC) and Spain (UPCT)] on European populations.
RESULTS: The insecticides indoxacarb and chlorantraniliprole were used as reference products. The IRAC leaf dip method is easy to perform, producing repeatable, homogeneous responses. LC(50) values for indoxacarb ranged between 1.8 and 17.9 mg L(-1) (NAGREF), 0.93 and 10.8 mg L(-1) (UC) and 0.20 and 0.70 mg L(-1) (UPCT), resulting in a tenfold, 12-fold and fourfold difference between the least and most susceptible populations at each laboratory respectively. For chlorantraniliprole, LC(50) values ranged between 0.10 and 0.56 mg L(-1) (NAGREF), 0.23 and 1.34 mg L(-1) (UC) and 0.04 and 0.24 mg L(-1) (UPCT), resulting in a sixfold difference in all three cases. Overall, UPCT reported lower mean LC(50) to indoxacarb, while UC reported higher LC(50) to chlorantraniliprole.
CONCLUSIONS: The new bioassay is reliable, providing a useful tool in the design of IRM strategies. Within each country/lab, the variability observed in the results for both indoxacarb and chlorantraniliprole can be attributed to natural variation. Future research is necessary to determine the extent to which it is possible to compare results among laboratories.

Indoxacarb was treated to Plutella xylostella for 10 generations to develop a resistant strain and biochemical analysis of indoxacarb resistance in different tissues of P. xylostella was carried out. Biochemical analysis found maximum esterase activity in gut homogenates of indoxacarb resistant strains followed by whole body and cuticle homogenates. In gut homogenates of indoxacarb resistant strains, maximum increase in esterases was found as compared to the unselected strain. Acetylcholineesterase activity was higher in head homogenates of the resistant strain than in the unselected strain. Glutathione-S-transferase activity was highest in whole body homogenates. However, maximum increase was found in gut homogenates of indoxacarb resistant strains over the unselected. Induced resistance was suppressed using known synergists. Maximum synergism occurred using diethyl-maleate (DEM), followed by triphenyl phosphate (TPP).

The toxicity of the oxadiazine insecticide indoxacarb to the European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae), was evaluated in the presence and absence of S,S,S-tributyl phosphorotrithioate (DEF), an inhibitor of hydrolytic metabolism. Bioassays involving topical application of different concentrations of indoxacarb to third instars of a susceptible O. nubilalis laboratory strain were performed, and in vitro metabolism experiments involving [14C] indoxacarb were examined to determine the role of hydrolytic metabolism in indoxacarb activation. Indoxacarb toxicity decreased in the presence of DEF indicating antagonism of toxicity. Results of in vivo and in vitro inhibition experiments indicated a reduction of indoxacarb activation and formation of the hydrolytic metabolite. These results are consistent with the proposed mechanism of hydrolytic activation for this compound.

Owing to the spread of pyrethroid resistance in Anopheles gambiae s.s. (Diptera: Culicidae) and other vector mosquitoes, there is an urgent need to develop alternative insecticides to supplement the pyrethroids for malaria control. Indoxacarb is an oxadiazine insecticide initially commercialized by DuPont for control of agricultural pests. Performance against An. gambiae bearing kdr (pyrethroid and DDT resistance) or Ace-1R insensitive acetylcholinesterase (organophosphate and carbamate resistance) mechanisms was studied using larval and adult bioassays and a simulated experimental hut system (tunnel tests) that allows fuller expression of the behavioral responses to insecticide. Larval and adult bioassays (topical application and cone tests on treated netting) showed a standard probit dosage-mortality response and no evidence of cross-resistance to the kdr and Ace-1R resistance mechanisms. Toxic activity was slow compared with standard insecticides and additional mortality was observed. Indoxacarb induced no excitorepellency in adults. In tunnel tests, indoxacarb induced no inhibition of mosquito penetration or blood feeding through the holed netting, but it induced delayed mortality over 24-96 h. There was > 90% mortality of the kdr strain on netting treated with the 500 mg/m2 dosage, whereas permethin at 500 mg/nm2 only killed 30% of this strain. A mixture of indoxacarb and pyrethroid showed neither synergism nor antagonism. The absence of cross-resistance to current insecticides indicates that indoxacarb has malaria vector control potential as larvicide or adulticide where mosquitoes are pyrethroid resistant.

A laboratory study assessed the contact toxicity of indoxacarb, abamectin, endosulfan, insecticidal soap, S-kinoprene and dimethoate to Amblyseius fallacis (Garman), Phytoseiulus persimilis Athias-Henriot and nymphs of Orius insidiosus (Say). Amblyseius fallacis is a predacious phytoseiid mite and an integral part of integrated pest management (IPM) programmes in North American apple orchards. The other two beneficials are widely used in greenhouses to manage various arthropod pests infesting vegetable and ornamental crops. Indoxacarb is a slow-acting insecticide, so toxicity data were recorded 7 days post-treatment when the data had stabilised. It showed no toxicity to O. insidiosus nymphs or to A. fallacis or P. persimilis adults. The LC50 values for O. insidiosus nymphs and P. persimilis could not be estimated with their associated confidence limits, because the g values were greater than 0.5 and under such circumstances the lethal concentration would lie outside the limits. The LC50 for A. fallacis was 7.6x the label rate. The fecundity of P. persimilis was reduced by 26.7%. The eclosion of treated eggs from both species of beneficial mites was not affected adversely. Among the other pest control products, S-kinoprene and endosulfan affected adversely at least one species of the predators, whereas dimethoate, abamectin and insecticidal soap were very toxic to all three beneficials. Indoxacarb should be evaluated as a pest control product in IPM programmes.

The contact toxicity of indoxacarb, abamectin, endosulfan, insecticide soap, S-kinoprene and dimethoate to Orius insidiosus (Say) and Aphidius colemani Viereck were studied in the laboratory. These beneficials are often used in the greenhouses to manage various insect pests. Indoxacarb is slow acting and therefore, to estimate lethal dosages, observations should be continued for several days until data stabilize. Seven days after treatment, the LC50 was 0.119 g AI litre(-1) for O insidiosus adults and 0.019 g AI litre(-1) for A colemani. At that time, the recommended field concentration was 0.479 times the LC50 for O insidiosus adults and three times the LC50 for A colemani. In contrast, indoxacarb had no adverse effect on the reproductive capacity of wasps surviving a treatment or the developing wasps in the aphid mummy. Among the other insecticides S-kinoprene was the most innocuous while dimethoate was the most toxic to the two beneficials. The other insecticides had overlapping toxicities.

1. Decarbomethoxyllated JW062 (DCJW), the active component of a new oxadiazine insecticide DPX-JW062 (Indoxacarb), was tested on action potentials and the inward sodium current recorded from short-term cultured dorsal unpaired median neurones of the cockroach Periplaneta americana. 2. Under whole-cell current-clamp conditions, 100 nM DCJW reduced the amplitude of action potentials and induced a large hyperpolarization of the resting membrane potential associated with a 41% increase in input resistance. 3. In voltage-clamp, DCJW resulted in a dose-dependent inhibition (IC(50) 28 nM) of the peak sodium current. Based on IC(50) values, the effect of DCJW was about 10 fold less potent than tetrodotoxin (TTX) but 1000 fold more potent than the local anaesthetic lidocaine. DCJW (100 nM) was without effect on activation properties of the sodium current, reversal potential, voltage dependence of sodium conductance and on both fast and slow steady-state inactivations. 4. TTX (2 nM) resulted in 48% inhibition of the peak inward sodium current. Co-application of TTX (2 nM) with various concentrations of DCJW produced an additional inhibition of the peak inward current, indicating that the blocking actions of DCJW and TTX were distinct. Co-application of lidocaine (IC(50) 30 microM) with various concentrations of DCJW produced a reduction of the apparent potency of DCJW, suggesting that DCJW and lidocaine acted at the same site. 5. DCJW (100 nM) did not affect inward calcium or outward potassium currents. 6. This study describes, for the first time, the action on insect neuronal voltage-dependent sodium channels of Indoxacarb, a new class of insecticides.

The evolution of the insecticidal pyrazoline moiety that was originally discovered in 1972 has led to the discovery of a new crop insecticide, indoxacarb, which is the first commercialized pyrazoline-type sodium-channel blocker. Both monocyclic and fused-tricyclic pyrazolines and pyridazines, as well as structurally related semicarbazones were examined prior to the discovery of analogous tricyclic oxadiazines which had similarly high activity as well as favorable environmental dissipation rates and low toxicity to non-target organisms. The eventual leading candidate, DPX-JW062, was originally obtained as a racemic molecule, but a chiral synthesis was developed which produces material that is 50% ee in the insecticidal (+)-S-enantiomer (DPX-MP062, indoxacarb).