Substrate Report for: Chlorantraniliprole

Predicted climate change could impact the effects that various chemicals have on organisms. Increased temperature or change in precipitation regime could either enhance or lower toxicity of pesticides. The aim of this study is to assess how change in temperature and soil moisture affect biochemical biomarkers in Eisenia fetida earthworm and microbial activity in their excrements after exposure to a fungicide - propiconazole (PCZ) and an insecticide - chlorantraniliprole (CAP). For seven days, earthworms were exposed to the pesticides under four environmental conditions comprising combinations of two different temperatures (20 degrees C and 25 degrees C) and two different soil water holding capacities (30% and 50%). After exposure, in the collected earthworm casts the microbial activity was measured through dehydrogenase activity (DHA) and biofilm forming ability (BFA), and in the postmitochondrial fraction of earthworms the activities of acetylcholinesterase (AChE), catalase (CAT) and glutathione-S-transferase (GST) respectively. The temperature and the soil moisture affected enzyme activities and organism's response to pesticides. It was determined that a three-way interaction (pesticide concentration, temperature and moisture) is statistically significant for the CAT and GST after the CAP exposure, and for the AChE and CAT after the PCZ exposure. Interestingly, the AChE activity was induced by both pesticides at a higher temperature tested. The most important two-way interaction that was determined occurred between the concentration and temperature applied. DHA and BFA, as markers of microbial activity, were unevenly affected by PCZ, CAP and environmental conditions. The results of this experiment demonstrate that experiments with at least two different environmental conditions can give a very good insight into some possible effects that the climate change could have on the toxicity of pesticides. The interaction of environmental factors should play a more important role in the risk assessments for pesticides.

Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance.

The anthranilic and phthalic diamides, chlorantraniliprole (CAP) and flubendiamide (FLU), respectively, represent a new class of very effective insecticides that activate the ryanodine-sensitive intracellular calcium release channel (ryanodine receptor). This paper reports an analytical method for the simultaneous determination of the two insecticides on fruits and vegetables by liquid chromatography-electrospray tandem mass spectrometry operated in the positive and negative ionization switching mode. The two diamides were extracted with acetonitrile and separated on a Zorbax Column Eclipse XDB C8 (4.6 mm x 150 mm i.d., 3 microm) by isocratic elution with a mobile phase consisting of acetonitrile and water with 0.1% formic acid pumped at a flow rate of 0.4 mL/min. The diamides were selectively detected by multiple reaction monitoring for transitions of proton adduct precursor ions simultaneously: positive m/z 484.3-->285 for CAP, m/z 445.5-->169 for internal standard, and negative m/z 681.4-->253 for FLU. For CAP calibration in the positive mode was linear over a working range of 2 to 1000 microg/L with r > 0.992. The limit of detection (LOD) and limit of quantification (LOQ) for CAP were 0.8 and 1.6 microg/kg, respectively. For FLU in the negative mode the corresponding values were 1-1000 microg/L for linear working range, with r > 0.996 and 0.4 and 0.8 microg/L for LOD and LOQ, respectively. Moreover, the presence of interfering compounds in the fruit and vegetable extracts was found to be minimal. Due to the linear behavior of the MS detector response for the two analytes, it was concluded that the multiple reaction transitions of molecular ions in the ion-switching mode can be used for analytical purposes, that is, for identification and quantification of diamides in fruit and vegetable extracts at trace levels.

Chlorantraniliprole is an anthranilic diamide insecticide that binds to the insect ryanodine receptor (RyR) and induces an uncontrolled release of Ca(2+) , resulting in paralysis and ultimately death of the target insects. Recently, it was reported that chlorantraniliprole-resistant diamondback moths, Plutella xylostella Linnaeus, have mutations in their RyR. In this study, we developed two different chlorantraniliprole-resistant Drosophila melanogaster strain. The resistance ratio (RR) of the low-concentration chlorantraniliprole-treated resistant (Low-Res) strain was 2.3, while that of the high-concentration chlorantraniliprole-treated resistant (High-Res) strain was 21.3. The LC 50 of the untreated control (Con) strain was 23.8~25.9 ppm, which was significantly higher than that reported for the susceptible diamondback moth (0.03~0.51 ppm). The high LC 50 of the Con may be because the helix S2 amino acid sequence of D. melanogaster RyR ( DmRyR) is identical to the I4790M mutation of the chlorantraniliprole-resistant diamondback moths, resulting in a lower binding affinity of DmRyR for chlorantraniliprole. Among the tested detoxification enzymes, the activity of esterase was significantly increased in the two Res strains, but glutathione S-transferases and acetylcholinesterase were significantly decreased in the two Res strains. The cross-resistance of the High-Res strain to other insecticides with different modes of actions (MoAs) revealed that the RRs of the neuronal acetylcholine receptor allosteric and competitive modulators were significantly increased, while those of the Na (2+) channel modulators were significantly reduced. Our studies showed that RRs against the same insecticide vary with the treatment concentration, and that RRs against other insecticides with different MoAs can be altered.

The oriental armyworm Mythimna separata is a serious polyphagous pest in China and there are major efforts to control this pest. In the present study, an RNA-Seq method was used to explore transcriptome data of M. separata and identify the responses of genes to chlorantraniliprole. Sequencing and de novo assembly yielded 134,533 transcripts that were further assembled into 77,628 unigenes with an N50 length of 2165bp. A total of 76 unigenes encoding insecticide targets were identified. Furthermore, 62 cytochrome P450s, 34 glutathione S-transferase (GSTs)and 64 carboxylesterase (CCEs) were curated to construct phylogenetic trees. In addition, we identified 647 the differentially expressed genes following treatment with chlorantraniliprole. The pathways of calcium signaling was identified as response to the pesticide The transcriptome data we generated represents a comprehensive genomic resource for further studies focused on control of M. separata. The response of genes to chlorantraniliprole treatment will elucidate the molecular mechanisms of insecticide resistance and allow for the development of new chemical pesticides to control this pest.

Predicted climate change could impact the effects that various chemicals have on organisms. Increased temperature or change in precipitation regime could either enhance or lower toxicity of pesticides. The aim of this study is to assess how change in temperature and soil moisture affect biochemical biomarkers in Eisenia fetida earthworm and microbial activity in their excrements after exposure to a fungicide - propiconazole (PCZ) and an insecticide - chlorantraniliprole (CAP). For seven days, earthworms were exposed to the pesticides under four environmental conditions comprising combinations of two different temperatures (20 degrees C and 25 degrees C) and two different soil water holding capacities (30% and 50%). After exposure, in the collected earthworm casts the microbial activity was measured through dehydrogenase activity (DHA) and biofilm forming ability (BFA), and in the postmitochondrial fraction of earthworms the activities of acetylcholinesterase (AChE), catalase (CAT) and glutathione-S-transferase (GST) respectively. The temperature and the soil moisture affected enzyme activities and organism's response to pesticides. It was determined that a three-way interaction (pesticide concentration, temperature and moisture) is statistically significant for the CAT and GST after the CAP exposure, and for the AChE and CAT after the PCZ exposure. Interestingly, the AChE activity was induced by both pesticides at a higher temperature tested. The most important two-way interaction that was determined occurred between the concentration and temperature applied. DHA and BFA, as markers of microbial activity, were unevenly affected by PCZ, CAP and environmental conditions. The results of this experiment demonstrate that experiments with at least two different environmental conditions can give a very good insight into some possible effects that the climate change could have on the toxicity of pesticides. The interaction of environmental factors should play a more important role in the risk assessments for pesticides.

Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance.

We challenged Locusta migratoria (Meyen) grasshoppers with simultaneous doses of both the insecticide chlorantraniliprole and the fungal pathogen, Metarhizium anisopliae. Our results showed synergistic and antagonistic effects on host mortality and enzyme activities. To elucidate the biochemical mechanisms that underlie detoxification and pathogen-immune responses in insects, we monitored the activities of 10 enzymes. After administration of insecticide and fungus, activities of glutathione-S-transferase (GST), general esterases (ESTs) and phenol oxidase (PO) decreased in the insect during the initial time period, whereas those of aryl acylamidase (AA) and chitinase (CHI) increased during the initial period and that of acetylcholinesterase (AChE) increased during a later time period. Activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) decreased at a later time period post treatment. Interestingly, treatment with chlorantraniliprole and M. anisopliae relieved the convulsions that normally accompany M. anisopliae infection. We speculate that locust mortality increased as a result of synergism via a mechanism related to Ca(2+) disruption in the host. Our study illuminates the biochemical mechanisms involved in insect immunity to xenobiotics and pathogens as well as the mechanisms by which these factors disrupt host homeostasis and induce death. We expect this knowledge to lead to more effective pest control.

The need to overcome pesticide resistance has led to the development of novel insecticides such as chlorantraniliprole (CAP), an anthranilic diamide. CAP disrupts calcium homeostasis in nerve and muscle cells and is used in several agricultural crops due to its potency and selectivity. However, chronic toxicity data for aquatic invertebrates are limited. Our objective was to evaluate the toxicity of CAP at different levels of biological organization using Chironomus riparius. Organismal endpoints (survival, larval growth and emergence), and 5 biomarkers associated with important physiological functions (acetylcholinesterase - AChE; catalase - CAT; glutathione-S-transferase - GST; total glutathione - TG; and lipid peroxidation - LPO) were investigated. Effects of CAP on cellular energy allocation (CEA) were also assessed. Acute tests revealed a 48h LC50 for C. riparius of 77.5mug/L and life-cycle tests revealed a chronic LOEC of 3.1mug/L based on effects on C. riparius larval growth and emergence. C. riparius females exposed as larvae to low concentrations of CAP emerged at a smaller size which might also translate into effects on reproduction. Chironomid larvae were not under oxidative stress, since short exposures to CAP did not affect LPO levels, despite the significant inhibition of GST (0.6-9.6mug/L) and CAT (9.6mug/L). It seems that detoxification of reactive intermediates and ROS is still achieved due to glutathione consumption, since TG levels were significantly decreased in organisms exposed to CAP (0.6-9.6mug/L). Moreover, it was observed that CEA was disturbed due to increased activity of the electron transport system (ETS), suggesting extra energy expenditure in larvae. These results show that environmental concentrations of CAP can impair the fitness of C. riparius natural populations and at the same time that chironomids, as for most insecticides, are suitable test organisms to evaluate the organismal and biochemical effects of anthranilic diamides.

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.

Neonate larvae of obliquebanded leafroller, Choristoneura rosaceana, from a laboratory colony were exposed to two reduced-risk insecticides, chlorantraniliprole and spinetoram. After nine generations of selection, significant levels of resistance to each insecticide were observed. Biochemical assays were performed on third instars to determine potential resistance mechanisms. Enzyme assays indicated that esterase activity was significantly increased in the chlorantraniliprole-selected colony, whereas mixed-function oxidase levels were elevated in the spinetoram-selected colony as compared to the unselected colony. No difference in glutathione-S-transferase activity was seen in either of the insecticide-selected colonies. These results indicate the potential involvement of esterases and mixed-function oxidases as detoxification mechanisms responsible for resistance to chlorantraniliprole and spinetoram, respectively. Furthermore, the results of this study suggest that chlorantraniliprole and spinetoram are not detoxified by similar mechanisms and could therefore be incorporated into resistance management programs in tree fruit leading to sustainable management of C. rosaceana.

BACKGROUND: Chlorantraniliprole is a novel anthranilic diamide insecticide, efficacious for control of lepidopteran insect pests, as well as some species in the orders Coleoptera, Diptera and Hemiptera. In this study, the speed of action of chlorantraniliprole was compared with that of seven commercial insecticides by means of ingestion bioassays against larvae of Plutella xylostella L., Trichoplusia ni (Hbner), Spodoptera exigua (Hbner) and Helicoverpa zea (Boddie). RESULTS: Based on feeding cessation and reduction in feeding damage, chlorantraniliprole is among the fastest-acting insecticides available for control of lepidopteran pests, comparable in speed of action with methomyl, lambda-cyhalothrin and esfenvalerate, and faster than emamectin benzoate, indoxacarb, methoxyfenozide and metaflumizone. CONCLUSION: The speed of action of chlorantraniliprole against target pest species, based on time for feeding cessation and reduction in feeding damage, is significantly greater than that of most recently developed insecticides and comparable only with the speed of action of the fast-acting carbamates and pyrethroids. In addition, chlorantraniliprole has a favorable toxicological and ecotoxicological profile. It belongs to a new chemical class with a novel mode of action and is effective against insect populations that have developed resistance to other insecticide groups, thus representing an attractive new tool for integrated pest management programs.

The anthranilic and phthalic diamides, chlorantraniliprole (CAP) and flubendiamide (FLU), respectively, represent a new class of very effective insecticides that activate the ryanodine-sensitive intracellular calcium release channel (ryanodine receptor). This paper reports an analytical method for the simultaneous determination of the two insecticides on fruits and vegetables by liquid chromatography-electrospray tandem mass spectrometry operated in the positive and negative ionization switching mode. The two diamides were extracted with acetonitrile and separated on a Zorbax Column Eclipse XDB C8 (4.6 mm x 150 mm i.d., 3 microm) by isocratic elution with a mobile phase consisting of acetonitrile and water with 0.1% formic acid pumped at a flow rate of 0.4 mL/min. The diamides were selectively detected by multiple reaction monitoring for transitions of proton adduct precursor ions simultaneously: positive m/z 484.3-->285 for CAP, m/z 445.5-->169 for internal standard, and negative m/z 681.4-->253 for FLU. For CAP calibration in the positive mode was linear over a working range of 2 to 1000 microg/L with r > 0.992. The limit of detection (LOD) and limit of quantification (LOQ) for CAP were 0.8 and 1.6 microg/kg, respectively. For FLU in the negative mode the corresponding values were 1-1000 microg/L for linear working range, with r > 0.996 and 0.4 and 0.8 microg/L for LOD and LOQ, respectively. Moreover, the presence of interfering compounds in the fruit and vegetable extracts was found to be minimal. Due to the linear behavior of the MS detector response for the two analytes, it was concluded that the multiple reaction transitions of molecular ions in the ion-switching mode can be used for analytical purposes, that is, for identification and quantification of diamides in fruit and vegetable extracts at trace levels.