SlCarE054 in Spodoptera litura (Lepidoptera: Noctuidae) showed direct metabolic activity to ß-cypermethrin with stereoselectivity.

Carboxylesterases (CarEs) is an important detoxification enzyme system in phase Ⅰ participating in insecticides resistance. In our previous study, SlCarE054, a CarEs gene from lepidoptera class, was screened out to be upregulated in a pyrethroids and organophosphates resistant population. Its overexpression was verified in two field-collected populations of Spodoptera litura (Lepidoptera: Noctuidae) resistant to pyrethroids and organophosphates by qRT-PCR. Spatiotemporal expression results showed that SlCarE054 was highly expressed in the pupae stage and the digestive tissue midgut. To further explore its role in pyrethroids and organophosphates resistance, its metabolism activity to insecticides was determined by UPLC. Its recombinant protein showed significant metabolism activity to cyhalothrin and fenvalerate, but not to phoxim or chlorpyrifos. The metabolic activity of SlCarE054 to ß-cypermethrin showed stereoselectivity, with higher metabolic activity to θ-cypermethrin than the enantiomer α-cypermethrin. The metabolite of ß-cypermethrin was identified as 3-phenoxybenzaldehyde. Further modelling and docking analysis indicated that ß-cypermethrin, cyhalothrin and fenvalerate could bind with the catalytic triad of the 3D structure of SlCarE054. The interaction of ß-cypermethrin with SlCarE054 also showed the lowest binding energy. Our work provides evidence that SlCarE054 play roles in ß-cypermethrin resistance in S. litura.

Genome-wide identification of G-protein coupled receptors (GPCRs) and their expression profile in response to ß-cypermethrin stress in Zeugodacus cucurbitae.

G-protein coupled receptors (GPCRs) are the largest and most diverse transmembrane receptor family in the cell. They are involved in regulating a wide range of biological processes, including behavior, reproduction, and development. However, GPCRs have not yet been identified in Zeugodacus cucurbitae. The current study focuses on the GPCRs identification, classification, distribution, and their expression analysis under ß-cypermethrin stress to uncover novel targets for pest management and assist in the development of effective strategies for controlling the melon fly population. We identified 80 GPCRs genes including 50 GPCRs identified in family A, 17 GPCRs identified in family B, 8 identified in family C, and 5 identified in family F. Z. cucurbitae GPCRs showed significant differences in both the number of genes in families or subfamilies, as well as the sequencing of the genes. Interestingly, newly identified GPCRs genes are expressed differently at various developmental stages of Z. cucurbitae. Further, we evaluated these 80 GPCRs using Realtime quantitative PCR to confirm their expression between ß-cypermethrin-resistant (RS) strain and susceptible strain (SS) of Z. cucurbitae. We identified 50 GPCR genes were highly overexpressed in a RS. Among these genes, eight genes were strongly induced by the 30% lethal concentration (LC) while two genes were significantly increased by the 50% LC of ß-cypermethrin. This first genome-wide profiling and characterization of GPCRs could lay foundation for unraveling detoxification mechanism and target site modifications which may improve the insect resistance and could be effective insecticide targets for Z. cucurbitae management.

Antioxidants improve ß-cypermethrin degradation by alleviating oxidative damage and increasing bioavailability by Bacillus cereus GW-01.

Microbial degradation of pesticide residues has the potential to reduce their hazards to human and environmental health. However, in some cases, degradation can activate pesticides, making them more toxic to microbes. Here we report on the ß-cypermethrin (ß-CY) toxicity to Bacillus cereus GW-01, a recently described ß-CY degrader, and effects of antioxidants on ß-CY degradation. GW-01 exposed to ß-CY negatively affected the growth rate. The highest maximum specific growth rate (µm) appeared at 25 mg/L ß-CY. ß-CY induced the oxidative stress in GW-01. The activities of superoxide dismutase (SOD), catalyse (CAT), and glutathione-S-transferase (GST) were significantly higher than that in control (p < 0.01); but they are decreased as growth phase pronged, which is contrary to the ß-CY degradation by GW-01 cells obtaining from various growth phase. Ascorbic acid (Vc), tea polyphenols (TP), and adenosine monophosphate (AMP) improved the degradation through changing the physiological property of GW-01. TP and AMP prompted the expression of gene encoding ß-CY degradation in GW-01, while Vc does the opposite. Biofilm formation was significantly inhibited by ß-CY, while was significantly enhanced by certain concentrations of TP and AMP (p < 0.05); while cell surface hydrophobicity (CSH) was negatively associated with ß-CY concentrations from 25 to 100 mg/L, and these 4 antioxidants all boosted the CSH. Cells grown with ß-CY had lower levels of saturated fatty acids but increased levels of some unsaturated and branched fatty acids, and these antioxidants alleviated the FA composition changes and gene expression related with FA metabolism. We also mined transcriptome analyses at lag, logarithmic, and stationary phases, and found that ß-CY induced oxidative stress. The objective of this study was to elaborate characteristics in relation to the microbial resistance of pesticide poisoning and the efficiency of pesticide degradation, and to provide a promising method for improving pesticide degradation by microbes.

Transcriptome analysis revealed that short-term stress in Blattella germanica to ß-cypermethrin can reshape the phenotype of resistance adaptation.

Previous studies on insect resistance have primarily focused on resistance monitoring and the molecular mechanisms involved, while overlooking the process of phenotype formation induced by insecticide stress. In this study, we compared the expression profiles of a beta-cypermethrin (ß-CYP) resistant strain (R) and a susceptible strain (S) of Blattella germanica after ß-CYP induction using transcriptome sequencing. In the short-term stress experiment, we identified a total of 792 and 622 differentially expressed genes (DEGs) in the S and R strains. Additionally, 893 DEGs were identified in the long-term adaptation experiment. To validate the RNA-Seq data, we performed qRT-PCR on eleven selected DEGs, and the results were consistent with the transcriptome sequencing data. These DEGs exhibited down-regulation in the short-term stress group and up-regulation in the long-term adaptation group. Among the validated DEGs, CUO8 and Cyp4g19 were identified and subjected to knockdown using RNA interference. Subsequent insecticide bioassays revealed that the mortality rate of cockroaches treated with ß-CYP increased by 69.3% and 66.7% after silencing the CUO8 and Cyp4g19 genes (P<0.05). Furthermore, the silencing of CUO8 resulted in a significant thinning of the cuticle by 59.3% and 53.4% (P<0.05), as observed through transmission electron microscopy and eosin staining, in the S and R strains, respectively. Overall, our findings demonstrate that the phenotypic plasticity in response to short-term stress can reshape the adaptive mechanisms of genetic variation during prolonged exposure to insecticides. And the identified resistance-related genes, CUO8 and Cyp4g19, could serve as potential targets for controlling these pest populations.

ß -Cypermethrin promotes the adipogenesis of 3T3-L1 cells via inducing autophagy and shaping an adipogenesis-friendly microenvironment.

The toxicity of synthetic pyrethroids has garnered attention, and studies have revealed that pyrethroids promote fat accumulation and lead to obesity in mice. Nevertheless, the effect of ß-cypermethrin (ß-CYP) on adipogenesis and its underlying mechanism remains largely unknown. In this study, mouse embryo fibroblasts 3T3-L1 cells were exposed to ß-CYP, and the cell viability, intracellular reactive oxygen species (ROS) level, autophagy, and adipogenesis were assessed to investigate the roles of oxidative stress and autophagy in the toxic effects of ß-CYP on adipogenesis. The results demonstrated that treatment with 100 µΜ ß-CYP elevated the ROS level, decreased mitochondrion membrane potential, stimulated autophagy, and enhanced the adipogenesis induced by the mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. However, co-treatment with N-acetyl-L-cysteine partially blocked the abovementioned effects of ß-CYP in 3T3-L1 cells. In addition, co-treatment with rapamycin, an autophagy agonist, enhanced the inductive effect of ß-CYP on adipogenesis, whereas co-treatment with 3-methyladenine blocked the enhancement of adipogenesis caused by ß-CYP. Moreover, ß-CYP also altered the microenvironment of 3T3-L1 cells to an adipogenesis-friendly one by reducing the extracellular expression of miR-34a, suggesting that the culture media of ß-CYP-treated 3T3-L1 cells could shift macrophages to M2 type. Taken together, the data obtained in the present study demonstrated that ß-CYP promoted adipogenesis via oxidative stress-mediated autophagy disturbance, and it caused macrophage M2 polarization via the alteration of miR-34a level in the microenvironment. The study demonstrated the adipogenesis-promoting effect of ß-CYP and unveiled the potential mechanism.

Analysis of Differentially Expressed Transcripts in Apolygus lucorum (Meyer-Dür) Exposed to Different Temperature Coefficient Insecticides.

The existence of a temperature effect of insecticides frustrated the control of the green plant bug Apolygus lucorum (Meyer-Dür). Previous studies mostly focused on the application of insecticides, but the underlying mechanism remains incompletely understood. Here, we report a transcriptome profiling of A. lucorum treated by three kinds of temperature coefficient insecticides (TCIs) (positive TCI: imidacloprid, negative TCI: b-cypermethrin and non-effect TCI: phoxim) at 15 °C, 25 °C and 35 °C by using next- and third-generation RNA-Seq methods. A total of 34,739 transcripts were annotated from 277.74 Gb of clean data. There were more up-regulated transcripts than down-regulated transcripts in all three kinds of TCI treatments. Further Venn diagrams indicate the regulatory transcripts and regulatory modes were different at the three temperatures. The responses to imidacloprid involved more detox and stress response transcripts such as cytochrome P450 (CYP450), carboxylesterase (CarE) and catalase (CAT) at 35 °C, which was the case for beta-cypermethrin at 15 °C. UDP-glucuronyltransferase (UGT) and heat shock protein (HSP) transcripts were heavily involved, and thus deserve particular note in the temperature effect of insecticides. This high-confidence transcriptome atlas provides improved gene information for further study on the insecticide temperature effect related physiological and biochemical processes of A. lucorum.

Copper exposure enhances Spodoptera litura larval tolerance to ß-cypermethrin.

Environmental xenobiotics can influence the tolerance of insects to chemical insecticides. Heavy metals are widespread distributed, can be easily bio-accumulated in plants and subsequently within phytophagous insects via the food chains. However, less attention has been paid to the effect of heavy metal exposure on their insecticide tolerance. In this study, pre-exposure of copper (Cu, 25-100 mg kg-1) significantly enhanced the subsequent tolerance of Spodoptera litura to ß-cypermethrin, a widely used pyrethroid insecticide in crop field. Cytochrome P450 monooxygenases (CYPs) activities were cross-induced in larvae exposed to Cu and ß-cypermethrin, while the activities of glutathione S-transferase (GST) and carboxylesterase (CarE) were not affected. Application of piperonyl butoxide (PBO), a P450 synergist, effectively impaired the tolerance to ß-cypermethrin in Cu-exposed S. litura larvae with a synergistic ratio of 1.72, indicating that P450s contribute to larval tolerance to ß-cypermethrin induced by Cu exposure. Among the four CYP6AB family genes examined, only larval midgut-specific CYP6AB12 was found to be cross-induced by Cu and ß-cypermethrin. RNA interference (RNAi)-mediated silencing of CYP6AB12 effectively decreased the mRNA levels of the target gene, and significantly reduced the larval tolerance to ß-cypermethrin following exposure to Cu. These results showed that pre-exposure of heavy metal Cu enhanced larval tolerance to ß-cypermethrin in S. litura, possibly through the cross-induction of P450s. Our findings provide new insights on the relationship between heavy metals and chemical insecticides that may benefit both the risk evaluation of heavy metal contamination and development of pest management strategies.

Efficient preparative separation of ß-cypermethrin stereoisomers by supercritical fluid chromatography with a two-step combined strategy.

An efficient two-step method has been developed for the separation of ß-cypermethrin stereoisomers by supercritical fluid chromatography with polysaccharide chiral stationary phases. With respect to retention, selectivity, and resolution of ß-cypermethrin, the effects of chiral stationary phases, cosolvents, mobile phases, and column temperature have been studied in detail. Through a two-step separation, ß-cypermethrin was firstly separated by using a cellulose-derived chiral stationary phase to obtain two stereoisomeric pairs, and further resolved on an amylose-based chiral stationary phase to produce four enantiopure stereoisomers. The electronic circular dichroism patterns of the first- and the third-eluted isomers in methanol solution showed the mirror image of each other in the wavelength range 200∼300 nm, indicating that they were a pair of enantiomers. Moreover, the second- and the fourth-eluted isomers were also enantiomers. This proposed two-step strategy showed low solvent consumption, fast separation speed, and high-purity, which may provide an effective approach for preparative separation of compounds with multiple chiral centers and difficult-to-separate multicomponent samples.

ß-Cypermethrin and its metabolite 3-phenoxybenzoic acid exhibit immunotoxicity in murine macrophages.

ß-Cypermethrin (ß-CYP), one of most important pyrethroids, is widely used to control insects, and has been detected in organisms, including human. Pyrethroids have been shown to pose neurotoxicity, hepatotoxicity, endocrine disruption and reproductive risks in mammals. However, research in immunotoxicity of pyrethroids, especially their metabolites, is limited. A common metabolite of pyrethroids is 3-phenoxybenzoic acid (3-PBA) in mammals. Thus, in this study, we evaluated the immunotoxicity of ß-CYP and 3-PBA in mouse macrophages, RAW 264.7 cells. MTT assays showed that both ß-CYP and 3-PBA reduced cell viability in a concentration- and time-dependent manner. Flow cytometry with Annexin-V/PI staining demonstrated that both ß-CYP and 3-PBA induced RAW 264.7 cell apoptosis. Furthermore, our results also showed that N-acetylcysteine partially blocked ß-CYP- and 3-PBA-induced cytotoxicity and apoptosis. Intrinsic apoptotic pathway was stimulated by both ß-CYP and 3-PBA exposure. In addition, we found that ß-CYP and 3-PBA inhibited mRNA levels of pro-inflammatory cytokines with or without LPS stimulation. Phagocytosis assay showed that both ß-CYP and 3-PBA inhibited phagocytic ability of macrophages. Moreover, it was also found that both ß-CYP and 3-PBA increased reactive oxygen species (ROS) levels in RAW 264.7 cells. Accordingly, both ß-CYP and 3-PBA were found to regulate the mRNA levels of oxidative stress-related genes in RAW 264.7 cells. Taken together, the results obtained in this study demonstrated that ß-CYP and 3-PBA may have immunotoxic effect on macrophages and that elevated ROS may underlie the mechanism. The present study will help to understand the health risks caused by ß-CYP and other pyrethroids.

ß-cypermethrin-induced acute neurotoxicity in the cerebral cortex of mice.

A Type II pyrethroid pesticide ß-cypermethrin is widely used in agriculture and domestic applications for pest control. However, the effect of ß-cypermethrin on the glutamate neurotransmitter has not been well-documented. In the current study, mice were treated with 20, 40, or 80 mg/kg ß-cypermethrin by a single oral gavage, with corn oil as a vehicle control. Four hours after treatment, we investigated glutamate levels and glutamate-metabolizing enzyme (phosphate-activated glutaminase, PAG; glutamine synthetase, GS) activities in the cerebral cortex of mice, using a HPLC system with ultraviolet detectors and a colorimetric assay. Glutamate uptake levels in the synaptosomes of cerebral cortex and mRNA expression levels of PAG, GS, and glutamate transporter-1 (GLT-1) in the cerebral cortex were detected by a radioactive labeling method and qRT-PCR, respectively. Toxic symptoms were observed in mice treated with 40 or 80 mg/kg ß-cypermethrin. Compared with the control, significant decreases in glutamate level and GS activity, and an obvious increase in synaptosomal glutamate uptake, were found in the cerebral cortex of mice treated with 80 mg/kg ß-cypermethrin. No significant changes were found among groups in PAG activity or PAG, GS, and GLT-1 mRNA expression levels. These results suggest that ß-cypermethrin treatment may reduce the glutamate level in the mouse cerebral cortex, which is associated with decreased GS activity and increased synaptosomal glutamate uptake. Our findings provide a partial explanation for the neurotoxic effects of synthetic ß-cypermethrin insecticides.

Identification and biochemical characterization of a carboxylesterase gene associated with ß-cypermethrin resistance in Dermanyssus gallinae.

Dermanyssus gallinae is a major hematophagous ectoparasite in layer hens. Although the acaricide ß-cypermethrin has been used to control mites worldwide, D. gallinae has developed resistance to this compound. Carboxylesterases (CarEs) are important detoxification enzymes that confer resistance to ß-cypermethrin in arthropods. However, CarEs associated with ß-cypermethrin resistance in D. gallinae have not yet been functionally characterized. Here, we isolated a CarE gene (Deg-CarE) from D. gallinae and assayed its activity. The results revealed significantly higher expression of Deg-CarE in the ß-cypermethrin-resistant strain (RS) than in the susceptible strain (SS) toward α-naphthyl acetate (α-NA) and ß-naphthyl acetate (ß-NA). These findings suggest that enhanced esterase activities might have contributed to ß-cypermethrin resistance in D. gallinae. Quantitative real-time PCR analysis revealed that Deg-CarE expression levels were significantly higher in adults than in other life stages. Although Deg-CarE was upregulated in the RS, significant differences in gene copy numbers were not observed. Additionally, Deg-CarE expression was significantly induced by ß-cypermethrin in both the SS and RS. Moreover, silencing Deg-CarE via RNA interference decreased the enzyme activity and increased the susceptibility of the RS to ß-cypermethrin, confirming that Deg-CarE is crucial for ß-cypermethrin detoxification. Finally, recombinant Deg-CarE (rDeg-CarE) expressed in Escherichia coli displayed high enzymatic activity toward α/ß-NA. However, metabolic analysis indicated that rDeg-CarE did not directly metabolize ß-cypermethrin. The collective findings indicate that D. gallinae resistance to ß-cypermethrin is associated with elevated CarEs protein activity and increased Deg-CarE expression levels. These findings provide insights into the metabolic resistance of D. gallinae and offer scientific guidance for the management and control of D. gallinae.

Detoxification and neurotransmitter clearance drive the recovery of Arma chinensis from ß-cypermethrin-triggered knockdown.

Natural enemies of arthropods contribute considerably to agriculture by suppressing pests, particularly when combined with chemical control. Studies show that insect recovery after insecticide application is rare. Here, we discovered the recovery of the predatory bug Arma chinensis from knockdown following the application of ß-cypermethrin but not five other insecticides. A. chinensis individuals were more tolerant to ß-cypermethrin than lepidopteran and coleopteran larvae, which did not recover from knockdown. We assessed A. chinensis recovery by monitoring their respiration and tracking locomotion through the entire process. We identified and verified the trans-regulation of detoxifying genes, including those encoding cytochrome P450s and α/ß-hydrolase, which confer recovery from ß-cypermethrin exposure in A. chinensis, by mitogen-activated protein kinase (MAPK) and cAMP response element binding protein (CREB). Furthermore, we discovered a novel mechanism, the neurotransmitter clearance, in vivo during the recovery process, by which the insect initiated the removal of excessive dopamine with a degrading enzyme ebony. Overall, these results provide mechanistic insights into the detoxification and neurotransmitter clearance that jointly drive insect recovery from insecticide exposure.

Insecticidal Activity of Essential Oils and Their Synergistic Effect on Improving the Efficacy of ß-Cypermethrin against Blattella germanica.

We screened the contact activity of 32 commercial essential oils (EOs) and their synergistic effect with ß-cypermethrin against Blattella germanica. Results showed that the most effective EOs against B. germanica were from Illicium verum, Syzygium aromaticum, and Cinnamomum camphora, with LD50 values of less than 500 µg/insect. The most potent synergistic effects of ß-cypermethrin on B. germanica were from Dysphania ambrosioides and Mentha canadensis. Both oils have a co-toxic factor of 133.33. The results of the major compound testing of the EOs showed that trans-anisaldehyde and thymol have the best insecticidal activity against B. germanica, with LD50 values of 141.30 and 138.61 µg/insect, respectively. The compounds with the best synergistic effect on ß-cypermethrin were γ-terpinene and linalool at a concentration of 0.5%. The co-toxic factors for γ-terpinene and linalool were 150 and 133.33, respectively, which were similar to the synergistic effect observed with 2% piperonyl butoxide.

Effect of Brucea javanica Oil on the Toxicity of ß-Cypermethrin Emulsifiable Concentrate Formulation.

Enhancing the performance of traditional pesticide formulations by improving their leaf surface wetting capabilities is a crucial approach for maximizing the pesticide efficiency. This study develops an emulsifiable concentrate (EC) of 4.5% ß-cypermethrin containing Brucea javanica oil (BJO). The incorporation of BJO aims to improve the leaf-wetting properties of the EC formulation and enhance its insecticidal effectiveness. The droplet size and emulsion characteristics of ß-CYP EC emulsion with varying concentrations of the emulsifier were evaluated, and changes after incorporating BJO were assessed to develop the optimal formulation. A comprehensive comparison was conducted among commercial 4.5% ß-cypermethrin EC (ß-CYP EC-1), 4.5% ß-cypermethrin EC with BJO (ß-CYP EC-2), and 4.5% ß-cypermethrin EC without BJO (ß-CYP EC-3). This comparison encompassed various factors including storage stability, insecticidal activity, cytotoxicity, and wetting performance on cabbage leaves. The results indicated that the ideal emulsifier concentration was 15% emulsifier 0201B. ß-CYP EC-2 demonstrated superior wetting properties on cabbage leaves (the wetting performance of ß-CYP EC-2 emulsion on cabbage leaves is 2.60 times that of the ß-CYP EC-1 emulsion), heightened insecticidal activity against the third larvae of Plutella xylostella [diamondback moth (DBM)] [the insecticidal activity of the ß-CYP EC-2 emulsion against the third larvae of DBM is 1.93 times that of the ß-CYP EC-1 emulsion (12 h)], and more obvious inhibitory effects on the proliferation of DBM embryo cells than the other tested formulations. These findings have significant implications for advancing pest control strategies and promoting sustainable and effective agricultural practices.

Oral administration of Bacillus cereus GW-01 alleviates the accumulation and detrimental effects of ß-cypermethrin in mice.

Pyrethroid insecticides negatively affect feed conversion, reproductive fitness, and food safety in exposed animals. Although probiotics have previously been widely studied for their effect on gut health, comparatively little is known regarding the efficacy of probiotic administration in specifically reducing pesticide toxicity in mice. We demonstrated that oral administration of a ß-cypermethrin (ß-CY)-degrading bacterial strain (Bacillus cereus GW-01) to ß-CY-exposed mice reduced ß-CY levels in the liver, kidney, brain, blood, lipid, and feces (18%-53%). Additionally, co-administration of strain GW-01 to ß-CY-exposed mice reduced weight loss (22%-31%) and improved liver function (15%-19%) in mice. Additionally, mice receiving GW-01 had near-control levels of numerous ß-CY-affected gut microbial taxa, including Muribaculaceae, Alloprevotella, Bacteroides, Dubosiella, and Alistipes. The survival and ß-CY biosorption of GW-01 in simulated gastrointestinal fluid conditions were significantly higher than E. coli. These results suggested that GW-01 can reduce ß-CY accumulation and alleviate the damage in mice. This study is the first to demonstrate that a probiotic strain can reduce the toxicity of ß-CY in mice.

Genome-wide screening and expression of glutathione S-transferase genes reveal that GSTe4 contributes to sensitivity against ß-cypermethrin in Zeugodacus cucurbitae.

Glutathione S-transferases (GSTs) are an essential multifunctional protein family with common detoxifying enzymes. In this study, 34 GST genes were identified from the melon fly, Zeugodacus cucurbitae, one of the most destructive pests worldwide. These GSTs include 32 cytosolic genes and two microsomal genes. Furthermore, these cytosolic GSTs were classified into six classes: 11 delta, 13 epsilon, three theta, one sigma, two zeta, and two omega. Most of these showed dynamic expression during the developmental stage, some of which showed stage-specific expression. The expression in various adult tissues showed that most of them were expressed in anti-stress-related tissues. The transcriptional response of the delta and epsilon families was determined when Z. cucurbitae was exposed to three insecticides, abamectin, dinotefuran, and ß-cypermethrin. Seven genes were significantly up-regulated by abamectin exposure. Moreover, five and four genes were significantly up-regulated with dinotefuran and ß-cypermethrin exposure, respectively, demonstrating their involvement in the detoxification of these such toxic substances in Z. cucurbitae. One example of these genes, ZcGSTe4, was randomly selected to explore its function in response to ß-cypermethrin exposure. Over-expressed ZcGSTe4 in E. coli showed significant tolerance to ß-cypermethrin, and RNAi-mediated suppression of ZcGSTe4 also increased the sensitivity of melon fly to this agent. This study provides a foundation for further studies on the mechanism of detoxification metabolism in the melon fly.

Development and characterization of nanoemulsions loaded with essential oil and ß-cypermethrin and their bioefficacy on insect pest of economic and medical importance.

BACKGROUND: The development of novel and ecofriendly tools plays an important role in insect pest management. Nanoemulsions (NEs) based on essential oils (EOs) offer a safer alternative for human health and the environment. This study aimed to elaborate and evaluate the toxicological effects of NEs containing peppermint or palmarosa EOs combined with ß-cypermethrin (ß-CP) using ultrasound technique. RESULTS: The optimized ratio of active ingredients to surfactant was 1:2. The NEs containing peppermint EO combined with ß-CP (NEs peppermint/ß-CP) were polydisperse with two peaks at 12.77 nm (33.4% intensity) and 299.1 nm (66.6% intensity). However, the NEs containing palmarosa EO combined with ß-CP (NEs palmarosa/ß-CP) were monodisperse with a size of 104.5 nm. Both NEs were transparent and stable for 2 months. The insecticidal effect of NEs was evaluated against Tribolium castaneum and Sitophilus oryzae adults, as well as Culex pipiens pipiens larvae. On all these insects, NEs peppermint/ß-CP enhanced pyrethroid bioactivity from 4.22- to 16-folds while NEs palmarosa/ß-CP, from 3.90- to 10.6-folds. Moreover, both NEs maintained high insecticidal activities against all insects for 2 months, although a slight increase of the particle size was detected. CONCLUSION: The NEs elaborated in this work can be considered as highly promising formulations for the development of new insecticides. © 2023 Society of Chemical Industry.

CRISPR/Cas9-Mediated Knockout Reveals the Involvement of CYP304F1 in ß-Cypermethrin and Chlorpyrifos Resistance in Spodoptera litura.

Functions of insect CYP2 clan P450s in insecticide resistance are relatively less reported. In Spodoptera litura, a gene from the CYP2 clan (CYP304F1) was validated to be up-regulated significantly in a pyrethroid- and organophosphate-resistant population (QJ) than a susceptible population by RNA-Seq and qRT-PCR. Spatial-temporal expression indicated the high expression of CYP304F1 in the fourth, fifth, and sixth instar larvae and the metabolism-related tissue fat body and malpighian tubules. CYP304F1 was knocked out by CRISPR/Cas9, and a homozygous population (QJ-CYP304F1) with a G-base deletion at exon 2 was obtained after selection. Bioassay results showed that the LD50 values to ß-cypermethrin and chlorpyrifos in the QJ-CYP304F1 population decreased significantly, and the resistance ratio was both 1.81-fold in the QJ population compared with that in the QJ-CYP304F1 population. The toxicity of fenvalerate, cyhalothrin, or phoxim showed no significant change. These results suggested that CYP304F1 is involved in ß-cypermethrin and chlorpyrifos resistance in S. litura.

ß-Cypermethrin Alleviated the Inhibitory Effect of Medium from RAW 264.7 Cells on 3T3-L1 Cell Maturation into Adipocytes.

Studies have elucidated that pyrethroids induce adipogenesis. It is also known that macrophages can affect the homeostasis of adipose tissue. However, whether and how the ß-cypermethrin (ß-CYP)-mediated inhibition of the macrophages affects adipogenesis remain unknown. To explore the effects of ß-CYP on adipogenesis through modulating the function of macrophages, 3T3-L1 cells, a preadipocyte cell line, were exposed to culture medium from either RAW 264.7 cells, a macrophage cell line (RM), or ß-CYP-treated RAW 264.7 cells (CRM). CRM decreased the inhibitory effects of RM treatment on cell proliferation and adipogenesis, as lipid accumulation, the CEBPA content, and Fasn and Acaca expression in 3T3-L1 cells were higher following CRM treatment than following RM treatment through the higher levels of the demethylated CEBPA promoter in 3T3-L1 cells. However, the medium from ß-CYP- and N-acetyl-L-cysteine-cotreated RAW 264.7 cells (CNRM) partially restored the inhibitory effects of RAW 264.7 cells on 3T3-L1 cells that had been reduced by CRM, indicating that ß-CYP might reduce the cytotoxicity and inhibitory effects of RAW 264.7 cells on the adipogenesis of 3T3-L1 cells through elevating ROS levels in RAW 264.7 cells. Moreover, exposure to ß-CYP downregulated the TNF-α secretion in RAW 264.7 cells. In conclusion, these data demonstrated that ß-CYP affected the function of RAW 264.7 cells, alleviating their inhibitory effects on adipogenesis and CEBPA demethylation in 3T3-L1 cells. ß-CYP might achieve these effects through downregulating the secretion of TNF-α via elevating ROS levels in RAW 264.7 cells. Our experiments provide a new perspective on the obesogenic effect of pyrethroids.

Autophagy protects murine macrophages from ß-cypermethrin-induced mitochondrial dysfunction and cytotoxicity via the reduction of oxidation stress.

The immunotoxicity of synthetic pyrethroid (SPs) has garnered much attention, and our previous research demonstrated that ß-CYP causes immunotoxicity and oxidative stress in macrophages. Nevertheless, the underlying mechanism remains largely unknown. In this study, the murine macrophage RAW 264.7 cells and murine peritoneal macrophages (PMs) were exposed to ß-CYP. The results showed that ß-CYP elevated intracellular ROS levels in both RAW 264.7 cells and PMs. Exposure to ß-CYP also caused mitochondrial dysfunction with reduced mitochondrial membrane potential (MMP), intracellular ATP level and mitochondrial DNA (mtDNA) content in the two cell types. In addition, exposure of RAW 264.7 cells to ß-CYP for 12 h and 24 h enhanced autophagy, with elevated Beclin1, Rab7, Lamp1 and LC3-II expression levels, while 48 h of exposure attenuated autophagy. In contrast, exposure of PMs to ß-CYP for 12 h promoted autophagy, whereas exposure for 24 h and 48 h impaired autophagy. Cotreatment with an antioxidant, N-acetyl-L-cysteine (NAC), partially blocked the reduced MMP, intracellular ATP level and autophagy disturbance. Moreover, cotreatment with an autophagy agonist, rapamycin (RAPA), partially blocked mitochondrial dysfunction and oxidative stress in the two cell types, whereas cotreatment with an autophagy inhibitor, 3-methyladenine (3-MA), augmented the abovementioned toxic effects. Furthermore, mitochondrial ROS levels in both RAW 264.7 cells and PMs were elevated by exposure to ß-CYP, and molecular docking showed that ß-CYP docked with mouse respiratory chain complex I by binding to the ND2, ND4, and ND5 subunits of the protein complex. Taken together, the data obtained in the present study demonstrate that oxidative stress partially mediates mitochondrial dysfunction and autophagy disturbance upon exposure to ß-CYP in macrophages, and autophagy plays a protective role against the toxic effects.