Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Overexpression of an Integument Esterase Gene LbEST-inte4 Infers the Malathion Detoxification in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.

A Novel Approach to Integrate Human Biomonitoring Data with Model Predicted Dietary Exposures: A Crop Protection Chemical Case Study Using Lambda-Cyhalothrin.

The appropriate use of human biomonitoring data to model population chemical exposures is challenging, especially for rapidly metabolized chemicals, such as agricultural chemicals. The objective of this study is to demonstrate a novel approach integrating model predicted dietary exposures and biomonitoring data to potentially inform regulatory risk assessments. We use lambda-cyhalothrin as a case study, and for the same representative U.S. population in the National Health and Nutrition Examination Survey (NHANES), an integrated exposure and pharmacokinetic model predicted exposures are calibrated to measurements of the urinary metabolite 3-phenoxybenzoic acid (3PBA), using an approximate Bayesian computing (ABC) methodology. We demonstrate that the correlation between modeled urinary 3PBA and the NHANES 3PBA measurements more than doubled as ABC thresholding narrowed the acceptable tolerance range for predicted versus observed urinary measurements. The median predicted urinary concentrations were closer to the median measured value using ABC than using current regulatory Monte Carlo methods.

Biochemistry and transcriptomic analyses of Phthorimaea absoluta (Lepidoptera: Gelechiidae) response to insecticides.

Phthorimaea absoluta is an invasive solanaceous plant pest with highly devastating effects on tomato plant. Heavy reliance on insecticide use to tackle the pest has been linked to insecticide resistance selection in P. absoluta populations. To underline insights on P. absoluta insecticide resistance mechanisms to diamides and avermectins, we evaluated the transcriptomic profile of parental (field-collected) and F8 (lab-reared) populations. Furthermore, to screen for the presence of organophosphate and pyrethroid resistance, we assessed the gene expression levels of acetylcholinesterase (ace1) and para-type voltage-gated sodium channel (VGSG) genes in the F1 to F8 lab-reared progeny of diamide and avermectin exposed P. absoluta field-collected populations. The VGSG gene showed up-regulation in 12.5% and down-regulation in 87.5% of the screened populations, while ace1 gene showed up-regulation in 37.5% and down-regulation in 62.5% of the screened populations. Gene ontology of the differentially expressed genes from both parental and eighth generations of diamide-sprayed P. absoluta populations revealed three genes involved in the metabolic detoxification of diamides in P. absoluta. Therefore, our study showed that the detoxification enzymes found could be responsible for P. absoluta diamide-based resistance, while behavioural resistance, which is stimulus-dependent, could be attributed to P. absoluta avermectin resistance.

Heterologous expression, biochemical characterization and prospects for insecticide biosensing potential of carboxylesterase Ha006a from Helicoverpa armigera.

Enzymes have attracted considerable scientific attention for their crucial role in detoxifying a wide range of harmful compounds. In today's global context, the extensive use of insecticides has emerged as a significant threat to the environment, sparking substantial concern. Insects, including economically important pests like Helicoverpa armigera, have developed resistance to conventional pest control methods through enzymes like carboxyl/cholinesterases. This study specifically focuses on a notable carboxyl/cholinesterase enzyme from Helicoverpa armigera (Ha006a), with the goal of harnessing its potential to combat environmental toxins. A total of six insecticides belonging to two different classes displayed varying inhibitory responses towards Ha006a, thereby rendering it effective in detoxifying a broader spectrum of insecticides. The significance of this research lies in discovering the bioremediation property of Ha006a, as it hydrolyzes synthetic pyrethroids (fenvalerate, λ-cyhalothrin and deltamethrin) and sequesters organophosphate (paraoxon ethyl, profenofos, and chlorpyrifos) insecticides. Additionally, the interaction studies between organophosphate insecticides and Ha006a helped in the fabrication of a novel electroanalytical sensor using a modified carbon paste electrode (MCPE). This sensor boasts impressive sensitivity, with detection limits of 0.019 µM, 0.15 µM, and 0.025 µM for paraoxon ethyl, profenofos, and chlorpyrifos, respectively. This study provides a comprehensive biochemical and biophysical characterization of the purified esterase Ha006a, showcasing its potential to remediate different classes of insecticides.

Glutathione S-transferase directly metabolizes imidacloprid in the whitefly, Bemisia tabaci.

The whitefly Bemisia tabaci poses a significant threat to various crops and ornamental plants and causes severe damage to the agricultural industry. Over the past few decades, B. tabaci has developed resistance to several pesticides, including imidacloprid. Therefore, elucidating the mechanism that leads to insecticide detoxification is very important for controlling B. tabaci and managing whitefly resistance to neonicotinoid insecticides. Among insect detoxification enzymes, glutathione S-transferase (GST) is an important phase II detoxification enzyme that helps detoxify exogenous toxic substances. In this study, we cloned the BtGSTz1 gene and observed that its expression level was greater in imidacloprid-resistant populations than sensitive populations of B. tabaci. By silencing BtGSTz1 via RNA interference, we found a significant increase in the mortality of imidacloprid-resistant B. tabaci. Additionally, prokaryotic expression and in vitro metabolism studies revealed that the recombinant BtGSTz1 protein could metabolize 36.36% of the total imidacloprid, providing direct evidence that BtGSTz1 plays a crucial role in the detoxification of imidacloprid. Overall, our study elucidated the role of GSTs in physiological activities related to insecticide resistance, which helps clarify the resistance mechanisms conferred by GSTs and provides useful insights for sustainable integrated pest management.

Functional characterization of Helicoverpa assulta CYP6B6 in insecticide metabolism.

The oriental tobacco budworm Helicoverpa assulta (Lepidoptera: Noctuidae) is a specialist pest that may cause serious damages to important crops such as chili pepper and tobacco. Various man-made insecticides have been applied to control the infestation of this pest. To understand how this pest copes with insecticides, it is required to identify key players involved in insecticide transformation. In this study, a P450 gene of CYP6B subfamily was identified in the oriental tobacco budworm, and its expression pattern was revealed. Moreover, the activities of HassCYP6B6 against 12 insecticides were explored using recombinant enzymes produced in the facile Escherichia coli. Data from metabolic experiments showed that HassCYP6B6 was able to metabolize conventional insecticides including organophosporates (diazinon, malathion, phoxim), carbamate propoxur, and pyrethroid esfenvalerate, while no significant metabolism was observed towards new-type pesticides such as neonicotinoids (acetamiprid, imidacloprid), diamides (chlorantraniliprole, cyantraniliprole), macrocyclic lactone (emamectin benzoate, ivermectin), and metaflumizone. Structures of metabolites were proposed based on mass spectrometry analyses. The results demonstrate that HassCYP6B6 plays important roles in the transformation of multiple insecticides via substrate-dependent catalytic mechanisms including dehydrogenation, hydroxylation and oxidative desulfurization. The findings have important applied implications for the usage of insecticides.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

The fate and transport of neonicotinoid insecticides and their metabolites through municipal wastewater treatment plants in South China.

Neonicotinoid insecticides (NEOs) have gained widespread usage as the most prevalent class of insecticides globally and are frequently detected in the environment, posing potential risks to biodiversity and human health. Wastewater discharged from wastewater treatment plants (WWTPs) is a substantial source of environmental NEOs. However, research tracking NEO variations in different treatment units at the WWTPs after being treated by the treatment processes remains limited. Therefore, this study aimed to comprehensively investigate the fate of nine parent NEOs (p-NEOs) and five metabolites in two municipal WWTPs using distinct treatment processes. The mean concentrations of ∑NEOs in influent (effluent) for the UNITANK, anaerobic-anoxic-oxic (A2/O), and cyclic activated sludge system (CASS) processes were 189 ng/L (195 ng/L), 173 ng/L (177 ng/L), and 123 ng/L (138 ng/L), respectively. Dinotefuran, imidacloprid, thiamethoxam, acetamiprid, and clothianidin were the most abundant p-NEOs in the WWTPs. Conventional wastewater treatment processes were ineffective in removing NEOs from wastewater (-4.91% to -12.1%), particularly major p-NEOs. Moreover, the behavior of the NEOs in various treatment units was investigated. The results showed that biodegradation and sludge adsorption were the primary mechanisms responsible for eliminating NEO. An anoxic or anaerobic treatment unit can improve the removal efficiency of NEOs during biological treatment. However, the terminal treatment unit (chlorination disinfection tank) did not facilitate the removal of most of the NEOs. The estimated total amount of NEOs released from WWTPs to receiving waters in the Pearl River of South China totaled approximately 6.90-42.6 g/d. These findings provide new insights into the efficiency of different treatment processes for removing NEOs in current wastewater treatment systems.

Effects of biochar and compost on microbial community assembly and metabolic processes in glyphosate, imidacloprid and pyraclostrobin polluted soil under freezethaw cycles.

Biochar and organic compost are widely used in agricultural soil remediation as soil immobilization agents. However, the effects of biochar and compost on microbial community assembly processes in polluted soil under freezingthawing need to be further clarified. Therefore, a freezethaw cycle experiment was conducted with glyphosate (herbicide), imidacloprid (insecticide) and pyraclostrobin (fungicide) polluted to understand the effect of biochar and compost on microbial community assembly and metabolic behavior. We found that biochar and compost could significantly promote the degradation of glyphosate, imidacloprid and pyraclostrobin in freezethaw soil decrease the half-life of the three pesticides. The addition of immobilization agents improved soil bacterial and fungal communities and promoted the transformation from homogeneous dispersal to homogeneous selection. For soil metabolism, the combined addition of biochar and compost alleviated the pollution of glyphosate, imidacloprid and imidacloprid to soil through up-regulation of metabolites (DEMs) in amino acid metabolism pathway and down-regulation of DEMs in fatty acid metabolism pathway. The structural equation modeling (SEM) results showed that soil pH and DOC were the main driving factors affecting microbial community assembly and metabolites. In summary, the combined addition of biochar and compost reduced the adverse effects of pesticides residues.

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Key residues involved in the interaction between chlorpyrifos and a chemosensory protein in Rhopalosiphum padi: Implication for tracking chemical residues via insect olfactory proteins.

The development of advanced biosensors for tracking chemical residues and detecting environmental pollution is of great significance. Insect chemical sensory proteins, including chemosensory proteins (CSPs), are easy to synthesize and purify and have been used to design proteins for specific biosensor applications. Chlorpyrifos is one of the most commonly used chemicals for controlling insect pests in agriculture. This organophosphate is harmful to aquatic species and has long-term negative consequences for the ecosystem. CSPs can bind and carry a variety of environmental chemicals, including insecticides. However, the mechanism by which CSPs bind to insecticides in aphids has not been clarified. In this study, we discovered that RpCSP1 from Rhopalosiphum padi has a higher affinity for chlorpyrifos, with a Ki value of 4.763 ± 0.491 µM. Multispectral analysis revealed the physicochemical binding mechanism between RpCSP1 and chlorpyrifos. Computational simulation analysis demonstrated that the main factor promoting the development of the RpCSP1-chlorpyrifos complex is polar solvation energy. Four residues (Arg33, Glu94, Gln145, Lys153) were essential in facilitating the interaction between RpCSP1 and chlorpyrifos. Our research has improved knowledge of the relationship between CSPs and organophosphorus pesticides. This knowledge contributes to the advancement of biosensor chips for tracking chemical residues and detecting environmental pollution through the use of CSPs.

Asp-tRNAAsn/Glu-tRNAGln amidotransferase A subunit-like amidase mediates the degradation of insecticide flonicamid by Variovorax boronicumulans CGMCC 4969.

The main metabolic product of the pyridinecarboxamide insecticide flonicamid, N-(4-trifluoromethylnicotinyl)glycinamide (TFNG-AM), has been shown to have very high mobility in soil, leading to its accumulation in the environment. Catabolic pathways of flonicamid have been widely reported, but few studies have focused on the metabolism of TFNG-AM. Here, the rapid transformation of TFNG-AM and production of the corresponding acid product N-(4-trifluoromethylnicotinoyl) glycine (TFNG) by the plant growth-promoting bacterium Variovorax boronicumulans CGMCC 4969 were investigated. With TFNG-AM at an initial concentration of 0.86 mmol/L, 90.70 % was transformed by V. boronicumulans CGMCC 4969 resting cells within 20 d, with a degradation half-life of 4.82 d. A novel amidase that potentially mediated this transformation process, called AmiD, was identified by bioinformatic analyses. The gene encoding amiD was cloned and expressed recombinantly in Escherichia coli, and the enzyme AmiD was characterized. Key amino acid residue Val154, which is associated with the catalytic activity and substrate specificity of signature family amidases, was identified for the first time by homology modeling, structural alignment, and site-directed mutagenesis analyses. When compared to wild-type recombinant AmiD, the mutant AmiD V154G demonstrated a 3.08-fold increase in activity toward TFNG-AM. The activity of AmiD V154G was greatly increased toward aromatic L-phenylalanine amides, heterocyclic TFNG-AM and IAM, and aliphatic asparagine, whereas it was dramatically lowered toward benzamide, phenylacetamide, nicotinamide, acetamide, acrylamide, and hexanamid. Quantitative PCR analysis revealed that AmiD may be a substrate-inducible enzyme in V. boronicumulans CGMCC 4969. The mechanism of transcriptional regulation of AmiD by a member of the AraC family of regulators encoded upstream of the amiD gene was preliminarily investigated. This study deepens our understanding of the mechanisms of metabolism of toxic amides in the environment, providing new ideas for microbial bioremediation.

Resistance management and integrated pest management insights from deployment of a Cry3Bb1+ Gpp34Ab1/Tpp35Ab1 pyramid in a resistant western corn rootworm landscape.

In Nebraska USA, many populations of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, now exhibit some level of resistance to all corn rootworm-active Bacillus thuringiensis Berliner (Bt) proteins expressed in commercial hybrids. Therefore, a study was conducted in northeast Nebraska from 2020-2022 to reevaluate current corn rootworm management options in continuous maize (consecutive planting for ≥2 years). Results from on-farm experiments to evaluate a standard soil-applied insecticide (Aztec® 4.67G) in combination with non-rootworm Bt or rootworm-active Bt pyramided maize (Cry3Bb1 + Gpp34Ab1/Tpp35Ab1) are reported within the context of WCR Bt resistance levels present. Corrected survival from Bt pyramid single-plant bioassays (<0.3, 0.3-0.49, >0.5) was used to place populations into 3 resistance categories. Variables evaluated included root injury, adult emergence, proportion lodged maize, and grain yield. Key results: A composite analysis of all populations across resistance levels indicated that addition of soil insecticide to Bt pyramid significantly reduced adult emergence and lodging but did not significantly increase root protection or yield. Within and among resistance category analyses of root injury revealed that the Bt pyramid remained highly efficacious at any non-rootworm Bt root injury level when resistance was absent or low. When corrected survival was >0.3, mean Bt pyramid root injury tracked more closely in a positive linear fashion with mean non-rootworm Bt root injury (rootworm density x level of resistance interaction). Similar trends were obtained for adult emergence but not yield. Mean Bt pyramid root injury rating was <0.75 in most populations with Bt resistance, which contributed to no significant yield differences among categories. Results are discussed within the context of IPM:IRM tradeoffs and the need to reduce WCR densities in this system to decrease the impact of the density x resistance interaction to bridge use of current pyramids with new technologies introduced over the next decade.

Knockout of ecdysis triggering hormone receptor (ETHr) gene adversely affects the nymphal molting and adult reproduction in Bemisia tabaci.

Bemisia tabaci (Gennadius) is one of the most dangerous polyphagous pests in the world causing damage to various crops by sucking sap during the nymphal and adult stages. Chemical management of whiteflies is challenging because of the emergence of pesticide resistance. RNA interference has been well established in whitefly to study the functions of various genes. G-protein coupled receptors (GPCRs) are important targets for development of new generation insecticides. In this study, Ecdysis triggering hormone receptor (ETHr) gene expression was recorded in different stages of whitefly and its function has been studied through RNAi. The expression of ETHr is highest in third-instar nymphs followed by other nymphal instars, pupae and newly emerged adults. Silencing of ETHr resulted in significantly higher adult mortality (68.88%), reduced fecundity (4.46 eggs /female), reduced longevity of male and female (1.05 and 1.40 days, respectively) when adults were fed with dsETHr @ 1.0 µg/µl. Silencing of ETHr in nymphs lead to significantly higher mortality (81.35%) as compared to control. This study confirms that ETHr gene is essential for growth and development of whitefly nymphs and adults. Hence, it can be future target for developing dsRNA based insecticides for management of whitefly.

Three chemosensory proteins enriched in antennae and tarsi of Rhaphuma horsfieldi differentially contribute to the binding of insecticides.

Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 µM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.

Investigation of dissipation kinetics and half-lives of fipronil and thiamethoxam in soil under various conditions using experimental modeling design by Minitab software.

To determine the extent of pesticide buildup and their environmental contamination, the environmental half-lives of pesticides are examined. The influence of the factors affecting the half-lives of fipronil and thiamethoxam including soil type, sterilization, temperature, and time and their interactions was studied using experimental modeling design by Minitab software. Based on the dissipation kinetics data, fipronil concentrations reduced gradually over 60 days while thiamethoxam concentrations decreased strongly. Also, fipronil and thiamethoxam dissipated more rapidly in calcareous soil than in alluvial soil. Thiamethoxam, however, disappeared more rapidly than fipronil in all treatments. Incubation at 50 °C leads to rapid the pesticide degradation. For prediction of the dissipation rate, model 5 was found to be the best fit, Residue of insecticide (%) = 15.466 - 11.793 Pesticide - 1.579 Soil type + 0.566 Sterilization - 3.120 Temperature, R2 = 0.94 and s = 3.80. Also, the predicted DT50 values were calculated by a model, DT50 (day) = 20.20 - 0.30 Pesticide - 7.97 Soil Type + 0.07 Sterilization - 2.04 Temperature. The shortest experimental and predicted DT50 values were obtained from treatment of thiamethoxam at 50 °C in calcareous soil either sterilized (7.36 and 9.96 days) or non-sterilized (5.92 and 9.82 days), respectively. The experimental DT50 values of fipronil and thiamethoxam ranged from 5.92 to 59.95 days while, the modeled values ranged from 9.82 to 30.58 days. According to the contour plot and response surface plot, temperature and sterilization were the main factors affecting the half-lives of fipronil and thiamethoxam. The DT50 values of fipronil and thiamethoxam increased in alluvial soil and soil with low temperature. In general, there is a high agreement between the experimental results and the modeled results.

Development of a Safe and Effective Bacillus thuringiensis-Based Nanobiopesticide for Controlling Tea Pests.

Mg(OH)2 was used as the nanocarrier of the Bacillus thuringiensis (Bt) Cry1Ac protein, and the synthesized Cry1Ac-Mg(OH)2 composites were regular and uniform nanosheets. Nano-Mg(OH)2 could effectively improve the insecticidal effect of the Cry1Ac protein toward Ectropis obliqua. It could enhance the damage degree of the Cry1Ac protein to intestinal epithelial cells and microvilli, induce and enrich the production of reactive oxygen species (ROS) in the midgut, and enhance the degradation of the Cry1Ac protein into active fragments. Furthermore, an anti-rinsing assay showed that the Cry1Ac-Mg(OH)2 composites were bound to the notch structure of the tea leaf surface. The retention of the Cry1Ac protein increased by 11.45%, and sprayed nano-Mg(OH)2 was rapidly absorbed by different tissues of tea plants. Moreover, nano-Mg(OH)2 and composites did not significantly affect non-target organisms. These results show that nano-Mg(OH)2 can serve as a safe and effective biopesticide carrier, which provides a new approach for stable and efficient Bt preparation.

Comparison of the performance of multiple whole-genome sequence-based tools for the identification of Bacillus cereus sensu stricto biovar Thuringiensis.

The Bacillus cereus sensu stricto (s.s.) species comprises strains of biovar Thuringiensis (Bt) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins, also known as Bt toxins, or (ii) based on the presence of cry, cyt, and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt identification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90).IMPORTANCEStrains of Bacillus cereus sensu stricto (s.s.) biovar Thuringiensis (Bt) are used as organic biopesticides. Bt is differentiated from the foodborne pathogen Bacillus cereus s.s. by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of whole-genome sequencing (WGS)-based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, identification of pesticidal sequences performed best overall at WGS-based Bt identification.

Mesoporous silica nanospheres-mediated insecticide and antibiotics co-delivery system for synergizing insecticidal toxicity and reducing environmental risk of insecticide.

Mesoporous silica nanoparticles (MSNs) are efficient carriers of drugs, and are promising in developing novel pesticide formulations. The cotton aphids Aphis gossypii Glover is a world devastating insect pest. It has evolved high level resistance to various insecticides thus resulted in the application of higher doses of insecticides, which raised environmental risk. In this study, the MSNs based pesticide/antibiotic delivery system was constructed for co-delivery of ampicillin (Amp) and imidacloprid (IMI). The IMI@Amp@MSNs complexes have improved toxicity against cotton aphids, and reduced acute toxicity to zebrafish. From the 16S rDNA sequencing results, Amp@MSNs, prepared by loading ampicillin to the mesoporous of MSNs, greatly disturbed the gut community of cotton aphids. Then, the relative expression of at least 25 cytochrome P450 genes of A. gossypii was significantly suppressed, including CYP6CY19 and CYP6CY22, which were found to be associated with imidacloprid resistance by RNAi. The bioassay results indicated that the synergy ratio of ampicillin to imidacloprid was 1.6, while Amp@MSNs improved the toxicity of imidacloprid by 2.4-fold. In addition, IMI@Amp@MSNs significantly improved the penetration of imidacloprid, and contributed to the amount of imidacloprid delivered to A. gossypii increased 1.4-fold. Thus, through inhibiting the relative expression of cytochrome P450 genes and improving penetration of imidacloprid, the toxicity of IMI@Amp@MSNs was 6.0-fold higher than that of imidacloprid. The greenhouse experiments further demonstrated the enhanced insecticidal activity of IMI@Amp@MSNs to A. gossypii. Meanwhile, the LC50 of IMI@Amp@MSNs to zebrafish was 3.9-fold higher than that of IMI, and the EC50 for malformation was 2.8-fold higher than IMI, respectively, which indicated that the IMI@Amp@MSNs complexes significantly reduced the environmental risk of imidacloprid. These findings encouraged the development of pesticide/antibiotic co-delivery nanoparticles, which would benefit pesticide reduction and environmental safety.