Chlorantraniliprole Research Articles

Calmodulin Modulation of Insect Ryanodine Receptors

Ryanodine receptor (RyR) is a giant calcium release channel located on the membrane of the endoplasmic reticulum (ER). Here, we report the regulation of RyRs from two major agricultural pests, diamondback moth and fall armyworm, by insect calmodulin (CaM). The recombinantly expressed full-length insect RyR could be pulled down by insect CaM in the presence of Ca2+, but the efficiency is lower compared to rabbit RyR1 and insect RyR with the CaM-binding domain (CaMBD) replaced by rabbit RyR1 sequence. Interestingly, the enhanced binding of CaM in the mutant insect RyR resulted in an increased sensitivity to the diamide insecticide chlorantraniliprole (CHL), suggesting that this CaM-CaMBD interface could be targeted by potential synergists acting as molecular glue. The thermodynamics of the binding between insect CaM and CaMBD was characterized by isothermal titration calorimetry, and the key residues responsible for the insect-specific regulation were identified through mutagenesis studies.

A Binary Mixture of Emamectin Benzoate and Chlorantraniliprole Supplemented with an Adjuvant Effectively Controls Spodoptera frugiperda.

The fall armyworm (FAW) Spodoptera frugiperda is a notorious pest, causing severe crop damage worldwide and prompting effective prevention and control. Over-reliance on and intensive use of insecticides are prone to leading to the rapid evolution of insecticide resistance, urging rational insecticide application. One effective way of rational insecticide application is to apply insecticides of different modes of action in combination or supplemented with adjuvants. In this study, we assessed the efficacies of two individual insecticides, emamectin benzoate (EB) and chlorantraniliprole (CT), and their mixture, supplemented with and without the oil adjuvant Jijian® to control FAW in laboratory bioassays and a field trial. Both EB and CT showed high toxicities to FAW. The EB × CT mixture at a mass ratio of 9:1 yielded a remarkable synergistic effect, with the co-toxicity coefficient (CTC) being 239.38 and the median lethal concentration (LC50) being 0.177 mg/L. In leaf-spray bioassays, the addition of the adjuvant reduced the LC50 values of both the individual insecticides and the EB × CT mixture by more than 59%, significantly improving the efficacies. The field trial confirmed the synergistic effects of the adjuvant, which reduced the amount of EB × CT mixture by 80%. This study provides an effective and promising insecticide-adjuvant mixture to control S. frugiperda.

Stable Isotope Analysis of Residual Pesticides via High Performance Liquid Chromatography and Elemental Analyzer-Isotope Ratio Mass Spectrometry.

To broaden the range of measurable pesticides for stable isotope analysis (SIA), we tested whether SIA of the anthranilic diamides cyantraniliprole (CYN) and chlorantraniliprole (CHL) can be achieved under elemental analyzer/isotope ratio mass spectrometry with compound purification in high-performance liquid chromatography (HPLC). Using this method, carbon isotope compositions were measured in pesticide residues extracted from plants (lettuce) grown indoors in potting soil that were treated with 500 mg/kg CHL and 250 mg/kg CYN and were followed up for 45 days. Our results show that the CYN and CHL standard materials did not have significant isotope differences before and after clean-up processing in HPLC. Further, when applied to the CYN product and CHL product in soil, stable isotope differences between the soil and plant were observed at <1.0‱ throughout the incubation period. There was a slight increase in the variability of pesticide isotope ratio detected with longer-term incubation (CHL, on average 1.5‱). Overall, we measured the carbon isotope ratio of target pesticides from HPLC fraction as the purification and pre-concentration step for environmental and biological samples. Such negligible isotopic differences in pesticide residues in soils and plants 45 days after application confirmed the potential of CSIA to quantify pesticide behavior in environments.

Biodegradation of Chlorantraniliprole and Flubendiamide by Some Bacterial Strains Isolated from Different Polluted Sources

This study aimed to isolate, purify, and identify some bacteria from different sources known to be contaminated with pesticides and evaluate their ability to degrade two important pesticides, chlorantraniliprole (CAP), and flubendiamide (FBD). In our study, six isolates showed maximum growth in the presence of CAP and FBD in the growth media as a sole carbon source. The isolates were purified and then identified by biochemical and morphological tests, MALD-TOF-MS, and 16S rRNA techniques, as Bacillus subtilis subsp. subtilis AZFS3, Bacillus pumilus AZFS5, Bacillus mojavensis AZFS15, Bacillus paramycoides AZFS18, Pseudomonas aeruginosa KZFS4, and Alcaligenes aquatilis KZFS11. The degradation ability of studied bacterial strains against pesticides was estimated under different conditions (temperatures, pH, salt, and incubation time). The results reveal that the optimal conditions for all bacterial strains’ growth were 30–35 °C, pH 7.0, 0.0–0.5% NaCl, and an incubation period of 11 days at 150 rpm in the presence of diamide insecticides at 50 mg/L. The capacity of six bacterial strains of CO2 production and degradation ability against various diamide pesticides and other pesticide groups (Profenofos, Cypermethrin, Carbofuran, and Malathion) were evaluated. The results show that the Pseudomonas aeruginosa KZFS4 (LC599404.1) strain produced the highest CO2 content, about 1.226 mg CO2/16 day, with efficacy in the biodegradation of FBD-CAP (78.6%), while the absorbance of bacterial growth (OD 600) on various pesticides ranged from 1.542 to 1.701. Additionally, Consortium-(No. 3)-mix-6-strains gave 1.553 mg CO2/16 days with efficacy (99.6%) and turbidity of 2.122 to 2.365 (OD 600) on various pesticides. In conclusion, the six bacterial strains could play an important role in the biodegradation process of pollutants in soils.

Sublethal chlorantraniliprole exposure induces autophagy and apoptosis through disrupting calcium homeostasis in the silkworm Bombyx mori.

The intensive application of chlorantraniliprole (CAP) leaves residues in the environment, posing a potential threat to non-target organisms. In the present study, we investigated the adverse effects of sublethal CAP exposure on Bombyx mori. Sublethal CAP (0.02 mg/L) was shown to induce the release of intracellular Ca2+ in BmN cells. Meanwhile, Ca2+ -dependent genes were induced in the midgut at 72 h after CAP (0.01 mg/L) exposure, and damaged mitochondria, autophagosomes, nuclear membrane rupture and condensed chromatin were observed. Moreover, the key genes in the oxidative phosphorylation pathway were significantly down-regulated. The transcript levels of autophagy-related genes ATG6 and ATG8 were significantly up-regulated, and the protein levels of LC3-II and ATG7 were significantly increased by 3.72- and 3.33-fold, respectively. Additionally, the transcript levels of the upstream genes in the apoptosis pathway (calpain and Apaf-1) were significantly up-regulated, the protein levels of the downstream gene caspase 3 and its cleaved form were significantly up-regulated by 1.97- and 4.55-fold, respectively, consistent with the elevated caspase 3 activity at 72 h. Collectively, these findings demonstrate that intracellular Ca2+ release induced by sublethal CAP inhibits oxidative phosphorylation pathway, which causes mitochondrial dysfunction, leading to autophagy and apoptosis in the midgut of B. mori.

Mechanism of autophagy induced by low concentrations of chlorantraniliprole in silk gland, Bombyx mori

Chlorantraniliprole (CAP) is widely used in the control of agricultural pests, and its residues can affect the formation of silkworm (Bombyx. mori) cocoon easily. To accurately evaluate the toxicity of CAP to silkworms and clarify the mechanism of its effect on silk gland function, we proposed a novel toxicity evaluation method based on the body weight changes after CAP exposure. We also analyzed the Ca2+-related ATPase activity, characterized energy metabolism and transcriptional changes about the autophagy key genes on the downstream signaling pathways. The results showed that after a low concentration of CAP exposed for 96 h, there were CAP residues in the silk glands of B. mori, the activities of Ca2+-ATPase and Ca2+-Mg2+-ATPase decreased significantly (P ≤ 0.01), and the activation of AMPK-related genes AMPK-α and AMPK-β were up-regulated by 6.39 ± 0.02-fold and 12.33 ± 1.06-fold, respectively, reaching a significant level (P ≤ 0.01)). In addition, the autophagy-related genes Atg1, Atg6, Atg5, Atg7, and Atg8 downstream AMPK were significantly up-regulated at 96 h (P ≤ 0.05). The results of immunohistochemistry and protein expression assay for autophagy marker Atg8 further confirmed the occurrence of autophagy. Overall, our results indicate that CAP exposure leads to autophagy in the silk gland of B. mori and affects their physiological functions, which provides guidance for the evaluation of toxicity of low concentration environmental CAP residues to insects.

A thioredoxin peroxidase protects Pieris rapae from oxidative stress induced by chlorantraniliprole exposure.

Chlorantraniliprole (CAP) is an insecticide widely used to control the small white butterfly (SWB), Pieris rapae. Exposure to CAP can cause oxidative injury in SWB; however, it is unclear if antioxidant enzymes are involved in the defense process. In this study, a thioredoxin peroxidase (PrTPX1) gene was identified from SWB by using a homology search method. The gene encoded a 195 amino-acid PrTPX1 protein. Sequence characteristics and phylogenetic analysis indicated that PrTPX1 was a typical "2-Cys" TPX, and the PrTPX1 gene consisted of four exons and three introns. Reverse transcription-quantitative polymerase chain reaction analysis indicated that the messenger RNA levels of PrTPX1 were highest in third-, fourth- and fifth-instar larval stages and in the larval midgut. Treatment with sublethal doses (LD20 and LD50 ) of CAP for 6, 12, 18, and 24 h resulted in increased H2 O2 concentration in SWB larvae, indicating insecticide-induced oxidative stress. The transcriptional levels of PrTPX1 were significantly enhanced in larvae exposed to CAP. Recombinant PrTPX1 protein was expressed in Escherichia coli. Enzymatic assay revealed that the protein displayed antioxidant activity and was able to protect against oxidative challenge. These results indicated that PrTPX1 plays an important role in oxidative stress responses and may contribute to the CAP tolerance in SWB.

Halloysite Nanotube-Based Pesticide Formulations with Enhanced Rain Erosion Resistance, Foliar Adhesion, and Insecticidal Effect.

The incorporation of green and sustainable nanomaterials in pesticide formation is an effective method to lower the use of conventional pesticides without adverse effects on productivity. Here pesticide Pickering emulsions stabilized by halloysite nanotubes (HNTs) were developed for low cost, less environmental pollution, low toxic effects, and better emulsion stability. HNTs were added to chlorantraniliprole (CAP) emulsions, and good stability was exhibited due to the adsorption and aggregation of HNTs at the interface of CAP oil droplets, forming a three-dimensional network structure that prevented the emulsion from aggregation. In addition, Spodoptera frugiperda was used as a pest model and corn was used as a plant model to explore the washout resistance, insecticidal effect, and biological safety of HNTs-CAP emulsion. After spraying emulsion on corn leaves and washing for 10 min, the HNTs-CAP emulsion (5 wt % HNTs) pesticide residue rate was 2.7 times that of pristine CAP emulsion. When the HNT dispersion concentration was 2 wt %, the larva mortality was 83%, which was 1.5 times that of the CAP emulsion group. These results demonstrated that HNTs-CAP emulsion showed good foliar adhesion, rainfall resistance, and insecticidal effect. The tubular clay-based nanopesticide formulations show potential applications in the control of crop pests with modern agriculture technology.

Impact of sublethal chlorantraniliprole on epidermis of Bombyx mori during prepupal-pupal transition

The silkworm Bombyx mori, an economically important insect with a long domestication history, exhibits high sensitivity to chemical pesticides. Extensive application of chlorantraniliprole (CAP) in control of pests of agricultural crops and mulberry plants causes residue toxicity to silkworm. We have demonstrated that sublethal concentration of CAP exposure causes defects in the formation of new epidermis and incomplete shedding of old epidermis during prepupal-pupal transition of B. mori. However, the underlying mechanism still remains unclear. Here, we investigated the transcriptional responses of the epidermis of B. mori on day 2 at prepupal stage to sublethal CAP exposure using digital gene expression (DGE) profiling sequencing. We identified 5823 differentially expressed genes (DEGs), with 4830 genes up-regulated and 993 genes down-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that CAP exposure induced disruption of energy homeostasis, oxidative stress, autophagy and apoptosis in the epidermis of B. mori. Meanwhile, trehalose content was increased while most of the genes involved in trehalose metabolism were down-regulated. In addition, chitin contents in CAP-exposed silkworms were decreased. Taken together, these results reveal that sublethal concentration of CAP probably targets trehalose metabolism to impair chitin synthesis, leading to perturbation of pupation metamorphosis in B. mori.

Toxicity effects of chlorantraniliprole in zebrafish (Danio rerio) involving in liver function and metabolic phenotype

Chlorantraniliprole (CAP), a representative bisamide insecticide, is widely used in rice fields around the world, posing potential toxicity risks to aquatic organisms. In this study, we examined the effects of exposure to CAP on growth and metabolic phenotype of zebrafish (Danio rerio) and oxidative stress and apoptosis in the liver of zebrafish (Danio rerio). First, we identified that CAP had a low bioaccumulation in zebrafish. Subsequently, growth phenotype analysis revealed that CAP could significantly increase liver weight and liver index in zebrafish. In addition, we found that CAP exposure could cause significant changes in indicators of oxidative stress, resulting in a significant increase in the content of malondialdehyde (MDA), causing oxidative stress in the liver of zebrafish. Meanwhile, the expression levels of apoptosis-related genes were also significantly changed and apoptosis was promoted in the liver of zebrafish with CAP exposure. Importantly, the results of metabolomics analysis shown that CAP exposure could significantly disrupt the metabolic phenotype of zebrafish, interfering with multiple metabolic pathways, mainly including valine, leucine and isoleucine biosynthesis and degradation, alanine, aspartate and glutamate metabolism and d-glutamine and D-glutamate metabolism. Last but not least, correlation analysis identified strong links between changes in liver function involving oxidative stress and apoptosis and changes in metabolic phenotype of zebrafish following CAP exposure. In brief, these results indicate that potential environmental risks of CAP to aquatic organisms should receive more attention.

Toxicological and transcriptomic effects in Mythimna separata (Lepidoptera: Noctuidae) exposed to chlorantraniliprole and functional characterization of glutathione S-transferases.

Chlorantraniliprole (CAP) is an efficient anthranilic diamide insecticide against economically important pests such as the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae). Resistance to CAP may develop due to enhanced enzymatic detoxification. The glutathione S-transferase (GST) superfamily in M. separata has not been systematically characterized. The aim of this study was therefore to explore the effects of lethal and sublethal doses of CAP on M. separata larvae, screen differentially expressed genes (DEGs) responding to CAP exposure, identify and characterize the GST superfamily, and analyze the metabolism of CAP by recombinant GSTs. The toxicity bioassay showed that CAP was active against M. separata third-instar larvae. LC50 was 17.615, 3.127, and 1.336 mg/L after 24, 48, and 72 h, respectively. Poisoned larvae showed contracted somites and disrupted midgut. Total GST activity in larvae was significantly elevated 24 h after CAP exposure. RNA-sequencing generated 43 055 unigenes with an average length of 1010 bp, and 567 up-regulated and 692 down-regulated DEGs responding to CAP treatment were screened. Thirty-five GST genes were identified from unigenes, including 31 cytosolic, three microsomal, and one unclassified. The expression profile of GST genes was analyzed using samples from different developmental stages, adult tissues, and CAP treatments. Metabolic assays indicated that CAP was depleted by recombinant MseGSTe2 and MseGSTs6. This study provides insight into the toxicological and transcriptomic effects in M. separata larvae exposed to CAP. The identification and functional characterization of the GST superfamily will improve our understanding of CAP detoxification by GSTs. © 2022 Society of Chemical Industry.

Comparative Toxicity and Joint Effects of Chlorantraniliprole and Carbaryl Against the Invasive Spodioptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm, Spodoptera frugiperda, is one of the most devastating invasive pests in China. Chlorantraniliprole (CH) is currently the main agent for controlling S. frugiperda. Carbaryl (CA) has been widely used as a foliar treatment to control S. frugiperda, although the pest has become highly resistant to it. This study investigates the comparative toxicity and joint effects of CH and CA on S. frugiperda. Time-toxicity results showed that CH had high toxicity to 1st and 3rd instar larvae, whereas CA had very low toxicity to 1st and 3rd instar larvae. The mixtures of CH and CA at different mass ratios showed strong synergistic effects on toxicity, and the mass ratio of 2:1 exhibited the highest toxicity to S. frugiperda. Furthermore, the synergistic toxicity of CH and CA at the 2:1 mass ratio (CH+CA) was also verified in field populations of S. frugiperda. The life-history parameters showed that CH+CA dramatically decreased the survival rate and fecundity of the parent population (F0) compared with CH treatment at the same concentration. Besides, CH and CH+CA mixture showed induction effect on cytochrome P450s and glutathione-S-transferases (GSTs) activities in S. frugiperda, with cytochrome P450s enzyme responding the fastest. In conclusion, this research found CH+CA provided synergistic effects on the toxicity and the sublethal effect on larvae. The joint effects on the life-history parameters and the detoxifying enzymes in S. frugiperda, may be useful for implementing IPM programs against this Lepidoptera pest.

Seed Treatment with Diamide and Neonicotinoid Mixtures for Controlling Fall Armyworm on Corn: Toxicity Evaluation, Effects on Plant Growth and Residuality.

The diamides, chlorantraniliprole (CHL) and cyantraniliprole (CYA), have been used as seed treatment agents against the fall armyworm (FAW), Spodoptera frugiperda in China. However, large-scale application of these two insecticides is prohibited because of their high cost. The neonicotinoid insecticides, clothianidin (CLO) and thiamethoxam (THI), are cheaper and widely used. In this study, we tested the efficacy of CHL + CLO and CYA + THI as seed treatment agents against FAW larvae both in laboratory and field conditions. Laboratory experiments showed that the two binary mixtures (both 240 g.a.i.100 kg−1 corn seeds) caused FAW mortality exceeded 84.00% at 14 days after seedling emergence (DAE). The mortality of the binary mixtures were similar to either CHL (300 g.a.i.100 kg−1corn seeds) or CYA (144 g a.i.100 kg−1corn seeds), but higher than CLO (120 g.a.i.100 Kg−1corn seeds) or THI (180 g a.i.100 kg−1corn seeds). Two independent field experiments showed that both binary mixtures resulted in above 68.00% control efficacy at 14 DAE, suggesting that these insecticidal combinations could effectively control FAW over a relative long period. In addition, both binary mixtures showed no negative effects on the growth and development of corn seedlings. The residues of binary mixtures in corn leave were also lower at 28 DAE as compared to residues in CHL or CYA alone. Most importantly, the costs of CHL + CLO were reduced up to 50% and CYA + THI up to 20% when compared to singly used chemical. Totally, our results indicated that CHL + CLO and CYA + THI had the same control efficacy as CHL or CYA alone, but with much lower cost.

The Efficacy of Chlorantraniliprole as a Seed Treatment for Mythimna separata (Walker) (Lepidoptera: Noctuidae)

The oriental armyworm (OAW), Mythimna separata (Walker) (Lepidoptera: Noctuidae), is an important pest in China and causes serious economic losses in corn. The anthranilic diamide, chlorantraniliprole (CHL), has been widely used as a seed treatment to control corn pests; however, no information is available on the efficacy of this insecticide as a seed treatment for OAW. In this study, the efficacy of seed treatment with CHL alone and CHL combined with the neonicotinoid insecticide clothianidin (CHL + CLO) was evaluated for controlling OAW larvae in the laboratory and field conditions. Pot experiments demonstrated that seed treatment with CHL and CHL + CLO (both 240 g a.i. 100 kg−1 seeds) resulted in &gt;79% mortality of OAW larvae and a damage rate &lt;20% in corn at 14 days after seed emergence (DAE). Similar to results obtained in pots, the residual toxicity of CHL and CHL + CLO to OAW larvae in the field declined with DAE and larval development. The control efficacy of field plots treated with CHL and CHL + CLO was &gt;70% within 14 DAE, which was significantly higher than CLO alone. These results suggest that CHL and CHL + CLO as seed treatments could effectively reduce OAW larval infestation in corn. This study validates the effectiveness of corn seed treatment for OAW as an alternative to conventional foliar applications.

Residue and Risk Assessment of Imidacloprid and Chlorantraniliprole in Open Field and Greenhouse Celery

The residue levels and risk assessment of imidacloprid (IMI) and chlorantraniliprole (CAP) in celery grown under open field and greenhouse cultivation were investigated. Both pesticides were used through foliar application and soil drench application at the recommended dose (RD) and 10-fold recommended dose (10RD). The half-lives of IMI and CAP in celery were 1.9 to 5.8 days and 4.3 to 6.5 days after foliar application, respectively, and the dietary risk quotients of IMI and CAP were 14.8 to 18.3% and 1.0 to 1.2%, respectively. For soil drench application, the half-lives of IMI and CAP in soil were 17.5 to 28.5 days and 15.1 to 23.7 days, respectively. Celery plants were able to absorb both insecticides from the soil. The highest concentrations of IMI in celery plants were 0.12 to 0.24 mg kg-1 (RD) and 0.34 to 0.39 mg kg-1 (10RD), and those for CAP were 0.0081 to 0.015 mg kg-1 (RD) and 0.028 to 0.057 mg kg-1 (10RD). Based on the highest residues of IMI and CAP in celery, the dietary risk quotients of IMI and CAP were 15.0% (RD) to 15.6% (10RD) and 1.0% (RD and 10RD) after soil drench application, respectively. The observed bioconcentration factors were 1.38 to 2.11 (IMI) and 0.35 to 0.48 (CAP), indicating that celery accumulated IMI more easily than CAP. The foliar and soil applications of IMI and CAP in celery at the RD and 10RD do not pose a safety risk to consumers.

Diamides conformationally restricted with central amino acid: Design, synthesis, and biological activities

AbstractDiamide insecticides, represented by chlorantraniliprole (CHL), were widely applied in the control of lepidopteran insects. In efforts to develop bioactive diamides with novel scaffolds, we design and synthesized a series of diamides containing central amino acids to conformationally simulate CHL. Bioassay results indicated that most compounds containing 1‐aminocyclopropane‐1‐carboxylic acid exhibited excellent larvacidal potency against Mythimna separate and Plutella xylostella. After a systematic structure–activity relationship study, 1–23 was identified as a potential insecticidal candidate with LC50 values of 34.920 mg·L−1 against M. separate and 61.992 mg·L−1 on P. xylostella. Finally, molecular docking revealed the possible binding mode of 1–23 with the target protein, ryanodine receptors.

Bacillus thuringiensis and Chlorantraniliprole Trigger the Expression of Detoxification-Related Genes in the Larval Midgut of Plutella xylostella.

Background: Diamondback moth (DBM), Plutella xylostella (L.), has developed resistance to many insecticides. The molecular mechanism of DBM resistance to Bt-G033A combined with chlorantraniliprole (CL) remains undefined.Methods: In this study, field-resistant strains of Plutella xylostella to three pesticides, namely, Bacillus thuringiensis (Bt) toxin (Bt-G033A), CL, and a mixture of Bt + CL, were selected to evaluate the resistance level. Additionally, transcriptomic profiles of a susceptible (SS-DBM), field-resistant (FOH-DBM), Bt-resistant (Bt-DBM), CL-resistant (CL-DBM), and Bt + CL-resistant (BtC-DBM) strains were performed by comparative analysis to identify genes responsible for detoxification.Results: The Bt-G033A was the most toxic chemical to all the DBM strains among the three insecticides. The comparative analysis identified 25,518 differentially expressed genes (DEGs) between pairs/combinations of strains. DEGs were enriched in pathways related to metabolic and catalytic activity and ABC transporter in resistant strains. In total, 17 metabolic resistance-related candidate genes were identified in resistance to Bt-G033A, CL, and Bt + CL by co-expression network analysis. Within candidate genes, the majority was upregulated in key genes including cytochrome P450, glutathione S-transferase (GST), carboxylesterase, and acetylcholinesterase in CL- and BtC-resistant strains. Furthermore, aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, trypsin, and ABC transporter genes were eminent as Bt-resistance-related genes. Expression patterns of key genes by the quantitative real-time PCR (qRT-PCR) proved the credibility of transcriptome data and suggest their association in the detoxification process.Conclusion: To date, this study is the most comprehensive research presenting functional transcriptome analysis of DBM using Bt-G033A and CL combined insecticidal activity.

Effects of plants and biochar on the performance of treatment wetlands for removal of the pesticide chlorantraniliprole from agricultural runoff

Chlorantraniliprole (CAP), an emergent insecticide commonly replacing banned neonicotinoids, is used worldwide despite the risk of contaminating water bodies. Treatment wetlands (TWs) have shown great potential for mitigating various pesticides in agricultural runoff, but little is known about CAP removal. The aim of this study was to determine the effect of adding biochar to subsurface flow treatment wetlands (SSF TWs) and the performance of three macrophyte species (Phragmites australis subsp. americanus, Scirpus cyperinus and Sporobolus michauxianus) in CAP removal. Removal efficiency was monitored over a one-month period in water-saturated SSF mesocosms fed with synthetic agricultural runoff containing CAP. To reflect temporal changes in agricultural runoff dynamics, two CAP concentrations were used in influent: a peak concentration (4 μg/L) for the first week and a trace concentration (0.4 μg/L) for the three subsequent weeks. Results showed that mesocosms with biochar were very effective in removing CAP mass (90 to 99%) and remained so throughout the experimental period. On the other hand, the level of CAP removal achieved in planted mesocosms without biochar was low (less than 13%). Evapotranspiration contributed significantly to volume reduction, but no general pattern in CAP mass removal efficiency was detected among the planted treatments without biochar. However, planted treatments acted as buffer zones, accumulating CAP and reducing its peak mass in effluent. Evapotranspiration rates of Scirpus and Phragmites were higher than that of Sporobolus, resulting in a greater buffering effect. This study suggests that addition of biochar to SSF TW substrate is a promising approach for CAP mitigation in agricultural runoff, but long-term efficiency remains to be assessed.

Structural basis for diamide modulation of ryanodine receptor

Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo-voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.

Agricultural surface water, imidacloprid, and chlorantraniliprole result in altered gene expression and receptor activation in Pimephales promelas

The toxicity of single pesticides is likely underestimated when considering complex pesticide mixtures found in agricultural runoff and this is especially true for newer pesticides with little toxicity data on non-target species. The goal of our study was to compare the toxicity of two newer pesticides, imidacloprid (IMI) and chlorantraniliprole (CHL), when an invertebrate and fish were exposed to single compounds, binary mixtures or surface water collected near agricultural fields. A secondary goal was to determine whether changes in select subcellular molecular pathways correspond to the insecticides’ mechanisms of activity in aquatic organisms. We conducted acute (96 h) exposures using a dilution series of field water and environmentally relevant concentrations of single and binary mixtures of IMI and CHL. We then evaluated survival, gene expression and the activity of IMI toward the n-acetylcholine receptor (nAChR) and CHL activity toward the ryanodine receptor (RyR). Both IMI and CHL were detected at all sampling locations for May 2019 and September 2019 sampling dates and exposure to field water led to high invertebrate but not fish mortality. Fish exposed to field collected water had significant changes in the relative expression of genes involved with detoxification and neuromuscular function. Exposure of fish to single compounds or binary mixtures of IMI and CHL led to increased relative gene expression of RyR in fish. Furthermore, we found that IMI targets the nAChR in aquatic invertebrates and that CHL can cause overactivation of the RyR in invertebrates and fish. Overall, our finding suggests that IMI and CHL may impact neuromuscular health in fish. Expanding monitoring efforts to include sublethal and molecular assays would allow the detection of subcellular level effects due to complex mixtures present in surface water near agricultural areas.