Six-year monitoring of pesticide resistance in the Colorado potato beetle (Leptinotarsa decemlineata Say) during a neonicotinoid restriction period.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is an important potato pest with known resistance to pyrethroids and organophosphates in Czechia. Decreased efficacy of neonicotinoids has been observed in last decade. After the restriction of using chlorpyrifos, thiacloprid and thiamethoxam by EU regulation, growers seek for information about the resistance of CPB to used insecticides and recommended antiresistant strategies. The development of CPB resistance to selected insecticides was evaluated in bioassays in 69 local populations from Czechia in 2017-2022 and in 2007-2022 in small plot experiments in Zabcice in South Moravia. The mortality in each subpopulation in the bioassays was evaluated at the field-recommended rates of insecticides to estimate the 50% and 90% lethal concentrations (LC50 and LC90, respectively). High levels of CPB resistance to lambda-cyhalothrin and chlorpyrifos were demonstrated throughout Czechia, without significant changes between years and regions. The average mortality after application of the field-recommended rate of lambda-cyhalothrin was influenced by temperature before larvae were sampled for bioassays and decreased with increasing temperature in June. Downwards trends in the LC90 values of chlorpyrifos and the average mortality after application of the field-recommended rate of acetamiprid in the bioassay were recorded over a 6-year period. The baseline LC50 value (with 95% confidence limit) of 0.04 mg/L of chlorantraniliprole was established for Czech populations of CPBs for the purpose of resistance monitoring in the next years. Widespread resistance to pyrethroids, organophosphates and neonicotinoids was demonstrated, and changes in anti-resistant strategies to control CPBs were discussed.

Two cuticle-enriched chemosensory proteins confer multi-insecticide resistance in Spodoptera frugiperda.

Recent studies revealed that insect chemosensory proteins (CSPs) both play essential roles in insect olfaction and insect resistance. However, functional evidence supporting the crosslink between CSP and insecticide resistance remains unexplored. In the present study, 22 SfruCSP transcripts were identified from the fall armyworm (FAW) and SfruCSP1 and SfruCSP2 are enriched in the larval cuticle and could be induced by multiple insecticides. Both SfruCSP1 and SfruCSP2 are highly expressed in the larval inner endocuticle and outer epicuticle, and these two proteins exhibited high binding affinities with three insecticides (chlorfenapyr, chlorpyrifos and indoxacarb). The knockdown of SfruCSP1 and SfruCSP2 increased the susceptibility of FAW larvae to the above three insecticides, and significantly increased the penetration ratios of these insecticides. Our in vitro and in vivo evidence suggests that SfruCSP1 and SfruCSP2 are insecticide binding proteins and confer FAW larval resistance to chlorfenapyr, chlorpyrifos and indoxacarb by an insecticide sequestration mechanism. The study should aid in the exploration of larval cuticle-enriched CSPs for insect resistance management.

Grape seed extract prevents chlorpyrifos-induced toxicity in rat liver through the modulation of phase I detoxification pathway.

Chlorpyrifos (CPF) poisoning is a public health problem for which there is not currently any effective prophylaxis. In this study, we investigated the protective effect of grape seed extract (GSE) against CPF-induced hepatotoxicity. Rats were daily treated either with CPF (2 mg/kg) or CPF and GSE (20 mg/kg) for 1 week, sacrificed, and their livers dissected for biochemical, molecular, and histopathological analyses. CPF generated liver dysfunction by altering carbohydrate, lipid, amino acid, ammonia and urea metabolism, and provoked mitochondrial impairment through disturbing tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and mitochondrial viability. CPF also induced cholinergic excitotoxicity along with oxidative stress and histopathological alterations. Interestingly, treatment with GSE prevented all the detrimental effects of CPF through the regulation of cytochrome P450 (CYP450) gene expression. Molecular docking analysis indicated that GSE-containing polyphenols acted as epigenetic modulators through inhibiting DNA (cytosine-5)-methyltransferase 1 (DNMT1), thus favoring the CYP2C6 detoxification pathway. Thereby, GSE might be a promising strategy in the protection of the liver against CPF toxicity.

An investigation of the acaricidal activity of benzyl alcohol on Rhipicephalus annulatus and Rhipicephalus sanguineus and its synergistic or antagonistic interaction with commonly used acaricides.

The most economically significant ectoparasites in the tropics and subtropics are ixodid ticks, especially Rhipicephalus annulatus and Rhipicephalus sanguineus. Years of extensive use of the readily available acaricides have resulted in widespread resistance development in these ticks, as well as negative environmental consequences. Benzyl alcohol (BA) has been frequently used to treat pediculosis and scabies, and it may be an effective alternative to commonly used acaricides. The main aim of the present study was to evaluate the acaricide activity of BA and its combination with the regularly used chemical acaricides against R. annulatus and R. sanguineus. Different concentrations of BA alone and in combination with deltamethrin, cypermethrin and chlorpyrifos were tested in vitro against adult and larvae of both tick species. The results showed that BA is toxic to R. annulatus and R. sanguineus larvae, with 100% larval mortality at concentrations of ≥50 mL/L, and LC50 and LC90 attained the concentrations of 19.8 and 33.8 mL/L for R. annulatus and 18.8 and 31.8 mL/L for R. sanguineus, respectively. Furthermore, BA in combination with deltamethrin, cypermethrin and chlorpyrifos exhibited synergistic factors of 2.48, 1.26 and 1.68 against R. annulatus larvae and 1.64, 11.1 and 1.14 against R. sanguineus larvae for deltamethrin + BA, cypermethrin + BA and chlorpyrifos + BA, respectively. BA induced 100% mortality in adult R. annulatus at concentrations of ≥250 mL/L with LC50 and LC90 reached the concentrations of 111 and 154 mL/L, respectively. Additionally, BA had ovicidal activity causing complete inhibition of larval hatching at 100 mL/L. The combination of BA with deltamethrin and cypermethrin increased acetylcholinesterase inhibition, whereas the combination of BA with chlorpyrifos decreased glutathione (GSH) activity and malondialdehyde levels. In the field application, the combination of BA 50 mL/L and deltamethrin (DBA) resulted in a significant reduction in the percentage of ticks by 30.9% 28 days post-treatment when compared with groups treated with deltamethrin alone. In conclusion, BA causes mortality in laboratory and field studies alone and in combination with cypermethrin or deltamethrin. BA can be used for control of ticks of different life stages, that is, eggs and larvae, through application to the ground.

Cross-resistance and genetics of field-evolved resistance to chlorfenapyr in Plutella xylostella.

Chlorfenapyr is a broad-spectrum halogenated pyrrole insecticide with a unique mode of action. Due to the misuse and overuse of this chemical, resistance has been reported in several arthropods, including Plutella xylostella, which is one of the most destructive insect pests afflicting crucifers worldwide. A better understanding of the cross-resistance and genetics of field-evolved chlorfenapyr resistance could effectively guide resistance management practices. Here, the chlorfenapyr resistance of a field-derived population of P. xylostella was introgressed into the susceptible IPP-S strain using a selection-assisted multigenerational backcrossing approach. The constructed near-isogenic strain, TH-BC5F2, shared 98.4% genetic background with the recurrent parent IPP-S strain. The TH-BC5F2 strain showed 275-fold resistance to chlorfenapyr, but no significant cross-resistance to spinosad, abamectin, chlorpyrifos, ß-cypermethrin, indoxacarb, chlorantraniliprole, or broflanilide (no more than 4.2-fold). Genetic analysis revealed that resistance was autosomal, incompletely dominant, and conferred by 1 major gene or a few tightly linked loci. The synergism of metabolic inhibitors (PBO, DEM, and DEF) to chlorfenapyr was very weak (<1.7-fold), and the metabolic enzyme activities in the TH-BC5F2 strain were not significantly elevated compared with the IPP-S strain. The results enhances our understanding of the genetic traits of chlorfenapyr resistance, and provides essential information for improving resistance management strategies.

Tubermycin B coated on Galactosylated Chitosan Nanoparticles synthesized by eco-friendly method ameliorates intracellular free radical production and reduces oxidative stress, reducing phosphorylation of IKKα/ß/pIкBα/NF-кB pathway.

OBJECTIVE: The objective of the current study was to investigate the cytotoxic potentials of Galactosylated Chitosan Nanoparticles. Specifically, the study aimed to develop Tubermycin B coated on Galactosylated Chitosan Nanoparticles using a new green method that replaces sodium borohydride in the reduction process. MATERIALS AND METHODS: The study synthesized Tubermycin B coated on Galactosylated Chitosan Nanoparticles through a new green method. The cytotoxicity of these nanoparticles was evaluated in a mice intestinal tract model that had been induced with chlorpyrifos, which causes oxidative stress-related enterotoxicity. Multiple activities, including the apoptosis of intestinal macrophages and the activation of Ikappa α/ß kinase (IKKα/ß), were examined as indicators of the nanoparticles' efficacy. The stability of the synthesized Chitosan Nanoparticles was also assessed. Additionally, the encapsulation efficiency of Boscia angustifalia and Boscia senegalensis extracts within the nanoparticles was determined. RESULTS: The results of the study showed that Tubermycin B coated on Galactosylated Chitosan Nanoparticles effectively alleviated the oxidative stress-related enterotoxicity in the mice intestinal tract induced by chlorpyrifos. The nanoparticles prevented the apoptosis of intestinal macrophages and inhibited the activation of IKKα/ß. The synthesized chitosan nanoparticles exhibited high stability. The encapsulation efficiency of Boscia angustifalia extract was recorded as 46.58%, whereas for Boscia senegalensis extract, it was 9.77%. The nanoparticles showed no cytotoxicity at all tested concentrations and demonstrated a medium-level anticancer effect. CONCLUSIONS: Based on the findings, it can be concluded that Tubermycin B coated on Galactosylated Chitosan Nanoparticles has the potential to alleviate oxidative stress-related enterotoxicity in the mice intestinal tract. The nanoparticles showed high stability and exhibited a medium-level anticancer effect. Furthermore, the study demonstrated that Boscia angustifalia extract exhibited higher anti-hepatitis C virus antibodies (anti-HCV) activity compared to Boscia senegalensis extract in an in-vitro system. Therefore, Boscia angustifalia could be considered a promising candidate for the development of an anti-HCV drug for future in-vivo studies.

Assessing phytotoxicity and cyto-genotoxicity of two insecticides using a battery of in-vitro biological assays.

Intensive use of chemical pesticides in agriculture poses environmental risks and may have negative impacts on agricultural productivity. The potential phytotoxicity of two chemical pesticides, chlorpyrifos (CPS) and fensulfothion (FSN), were evaluated using Cicer arietinum and Allium cepa as model crops. Different concentrations (0-100 µgmL-1) of both CPS and FSN decreased germination and biological attributes of C. arietinum. High pesticide doses significantly (p ≤ 0.05) caused membrane damage by producing thiobarbituric acid reactive substances (TBARS) and increasing proline (Pro) content. Pesticides elevated ROS levels and substantially increased the superoxide anions and H2O2 concentrations, thus aggravating cell injury. Plants exposed to high pesticide dosages displayed significantly higher antioxidant levels to combat pesticide-induced oxidative stress. Ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) increased by 48%, 93%, 71%, 52% and 94%, respectively, in C. arietinum roots exposed to 100 µgFSNmL-1. Under CLSM, pesticide-exposed C. arietinum and 2',7'-dichlorodihydrofluorescein diacetate (2'7'-DCF) and 3,3'-diaminobenzidine stained roots exhibited increased ROS production in a concentration-dependent manner. Additionally, enhanced Rhodamine 123 (Rhd 123) and Evan's blue fluorescence in roots, as well as changes in mitochondrial membrane potential (ΔΨm) and cellular apoptosis, were both associated with high pesticide dose. Allium cepa chromosomal aberration (CAs) assay showed a clear reduction in mitotic index (MI) and numerous chromosomal anomalies in root meristematic cells. Additionally, a-dose-dependent increase in DNA damage in root meristematic cells of A. cepa and conversion of the super-coiled form of DNA to open circular in pBR322 plasmid revealed the genotoxic potential of pesticides. The application of CPS and FSN suggests phytotoxic and cyto-genotoxic effects that emphasize the importance of careful monitoring of current pesticide level in soil before application and addition at optimal levels to soil-plant system. It is appropriate to prepare both target-specific and slow-release agrochemical formulations for crop protection with concurrent safeguarding of agroecosystems.

Does Leptinotarsa decemlineata larval survival after pesticide treatment depend on microbiome composition?

BACKGROUND: The microbiomes of some arthropods are believed to eliminate pesticides by chemical degradation or stimulation of the host immune system. The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is an important agricultural pest with known resistance to used pesticides. We sought to analyze microbiome composition in CPB larvae from different sites and to identify the effect of pesticides on the microbiome of surviving and dead larvae after chlorpyrifos treatment in laboratory. Changes in the Lactococcus lactis community in larvae treated with chlorpyrifos and fed by potato leaves with L. lactis cover were studied by manipulative experiment. The microbiome was characterized by sequencing the 16S RNA gene. RESULTS: The microbiome of L. decemlineata larvae is composed of a few operational taxonomic units (OTUs) (Enterobacteriaceae, Pseudocitrobacter, Acinetobacter, Pseudomonas, L. lactis, Enterococcus, Burkholderia and Spiroplasma leptinotarsae). The microbiome varied among the samples from eight sites and showed differences in profiles between surviving and dead larvae. The survival of larvae after chlorpyrifos treatment was correlated with a higher proportion of L. lactis sequences in the microbiome. The S. leptinotarsa profile also increased in the surviving larvae, but this OTU was not present in all sampling sites. In manipulative experiments, larvae treated with L. lactis had five-fold lower mortality rates than untreated larvae. CONCLUSION: These results indicate that the microbiome of larvae is formed from a few bacterial taxa depending on the sampling site. A member of the gut microbiome, L. lactis, is believed to help overcome the toxic effects of chlorpyrifos in the larval gut. © 2023 Society of Chemical Industry.

Status of Acaricide Resistance and Detecting the Knockdown Resistance Mutation T2134A in the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae) from Northeastern Mexico.

Rhipicephalus microplus is the most important tick in veterinary medicine, given its repercussions on animal production. The principal strategy to avoid adverse effects associated with R. microplus is the chemical control of tick populations through organosynthetic acaricides. Therefore, monitoring susceptibility to acaricides is paramount in any control program. This study aimed to analyze the resistance status of 2 populations of R. microplus from northeastern Mexico to the organochlorine (OC) lindane, organophosphates (OP) coumaphos, chlorfenvinphos, diazinon, and chlorpyrifos, and the synthetic pyrethroids (SPs) flumethrin, deltamethrin, and cypermethrin. Discriminating doses (DD) of each acaricide were used in the larval packet bioassay (LPT). Additionally, the presence of the knockdown resistance (kdr) mutation T2134A associated with pyrethroid resistance was evaluated using allele-specific polymerase chain reaction (PCR). The populations of R. microplus showed a high frequency of resistance to SP, with mortality rates of less than 5%; they also showed resistance to the OPs (diazinon and chlorpyrifos) with mortality rates ranging from 1.29% to 34.62%; meanwhile, they were susceptible to coumaphos and chlorfenvinphos. Mortality rates higher than 66% were observed for lindane, indicating susceptibility. The mutant allele of the kdr mutation T2134A was detected in 75% and 100% of the pools analyzed. The populations studied presented a highly resistant profile to pyrethroids, with the presence of the kdr mutant allele A2134. The susceptibility to the organophosphates such as coumaphos and chlorfenvinphos of R. microplus from northeastern Mexico should be noted.

Chlorpyrifos and chlorfenapyr resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) relies on UDP-glucuronosyltransferases.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), has become an important agricultural pest worldwide. S. frugiperda is mainly controlled by the chemical insecticides, whereas the frequent application of insecticides would result in the resistance development. Insect uridine diphosphate-glucuronosyltransferases (UGTs), as phase II metabolism enzymes, play vital roles in the breakdown of endobiotic and xenobiotics. In this study, 42 UGT genes were identified by RNA-seq, including 29 UGT genes were elevated compared to the susceptible population, and the transcript levels of 3 UGTs (UGT40F20, UGT40R18, and UGT40D17) were increased by more than 2.0-fold in the field populations. Expression pattern analysis revealed that S. frugiperda UGT40F20, UGT40R18, and UGT40D17 were increased by 6.34-, 4.26-, and 8.28-fold, compared the susceptible populations, respectively. The expression of UGT40D17, UGT40F20, and UGT40R18 was affected after exposure to phenobarbital, chlorpyrifos, chlorfenapyr, sulfinpyrazone, and 5-nitrouracil. The induced expression of UGT genes may have improved UGT enzymatic activity, while the inhibition of UGTs genes expression may decreased UGT enzymatic activity. Sulfinpyrazone, and 5-nitrouracil, significantly increased the toxicity of chlorpyrifos and chlorfenapyr, as well as phenobarbital significantly reduced the toxicity of chlorpyrifos and chlorfenapyr against the susceptible populations and field populations of S. frugiperda. The suppression of UGTs (UGT40D17, UGT40F20, and UGT40R18) significantly increased the insensitivity of the field populations to chlorpyrifos and chlorfenapyr. These findings strongly supported our viewpoint that UGTs may play a critical role in insecticide detoxification. This study provides a scientific basis for the management of S. frugiperda.

Thirty-minute test to detect insecticide resistance under field conditions: the case of the Asian citrus psyllid.

BACKGROUND: Diaphorina citri Kuwayama is one of the most destructive citrus pests worldwide. It is controlled mainly through applications of conventional insecticides. Methodologies used to estimate resistance to insecticides do not correlate with field efficacy, and do not provide timely and reliable information to make decisions at a site where spraying is needed. The use of diagnostic doses with 30-min exposure is proposed for estimating the resistance of D. citri to imidacloprid, spinosad, malathion and chlorpyrifos at the orchard level. RESULTS: Under laboratory conditions, we estimated the lowest doses that caused 100% mortality within 30 min of exposure (diagnostic dose) in a susceptible D. citri colony. The diagnostic doses for imidacloprid, spinosad, malathion and chlorpyrifos were 7.4, 4.2, 1.0 and 5.5 mg a.i. L-1 , respectively. Under field conditions, we applied the diagnostic doses to D. citri feeding on Citrus aurantifolia Swingle at five localities in Michoacan state, Mexico (Nueva Italia, Santo Domingo, El Varal, Gambara and El Ceñidor). Additionally, the field efficacy of these insecticides against these populations was evaluated. A significant correlation between field efficacy and mortality was observed with the diagnostic doses for imidacloprid, malathion and chlorpyrifos (R2 ≥ 0.93). The correlation for spinosad could not be estimated because the mortality caused by the diagnostic dose and its field efficacy at all study sites was consistently >98%. CONCLUSIONS: Field efficacy and resistance were estimated based on the field diagnostic doses with 30-min exposure for all tested insecticides. Consequently, growers and pest management technicians can estimate the performance of the evaluated insecticides at the orchard level and before insecticide application. © 2023 Society of Chemical Industry.

Application of soil amendments mitigates phytotoxic effects on Solanum melongena L. and Lycopersicon esculentum L. seedlings exposed to chlorpyrifos and dimethoate pesticides.

This field study was done to study the effects of pesticides chlorpyrifos and dimethoate singly and in combination with soil amendments like chemical fertilizer (CF), farmyard manure (FM), and 50% CF + 50% FM (CM) on various indices of growth, physio-biochemical parameters of brinjal, and their residual effect in tomato seedlings. As compared to the control, the decrease of 9.5 and 5.5%, 8.9 and 5.0% in fresh weight, dry weight respectively was recorded in the pesticide-only treatment in the brinjal crop. Pesticides when applied in combination with soil amendments depicted the highest growth of 105.4 and 118.2%, 104.1 and 115.1% in pesticides + CF treatment, 72.7 and 85.1%, 68.1 and 78.1% in pesticides + CM treatment, and 64.4 and 74.0%, 62.7 and 65.7% in pesticides + FM treatment compared to control. In tomato seedlings, the pesticides + CF treatment exhibited the lowest growth indices (25.5 and 31.9%, 26.4 and 28.8%) across the combined treatments while pesticide-only treatment depicted minimum growth compared to the control. In the case of photosynthesis rate and antioxidant activity, the combined treatments showed the trend as pesticides + CF > pesticides + CM > pesticides + FM in the brinjal crop; however, the trend became somewhat reversed in the tomato crop. The results indicated that soil-amended practices modulated pesticide-induced damage by upregulating photosynthetic performance, chlorophyll a fluorescence, and antioxidant balancing which might be associated with the mitigation of ROS-induced pesticide toxicity, and the effect was more pronounced with CM. Furthermore, our study was supported by non-metric-multidimensional scaling (NMDS)-constructed ordination plots by showing spatial patterns in different variables. The study might help in taking management decision to design mitigation actions for government and non-government agency at the farmers' level.

From laboratory to field: laboratory-measured pesticide resistance reflects outcomes of field-based control in the redlegged earth mite, Halotydeus destructor.

Resistance to pesticides is typically identified via laboratory bioassays after field control failures are observed, but the results of such assays are rarely validated through experiments under field conditions. Such validation is particularly important when only a low-to-moderate level of resistance is detected in the laboratory. Here we undertake such a validation for organophosphate resistance in the agricultural pest mite Halotydeus destructor, in which low-to-moderate levels of resistance to organophosphorus pesticides have evolved in Australia. Using data from laboratory bioassays, we show that resistance to the organophosphate chlorpyrifos is higher (around 100-fold) than resistance to another organophosphate, omethoate (around 7-fold). In field trials, both these chemicals were found to effectively control pesticide-susceptible populations of H. destructor. However, when applied to a resistant mite population in the field, the effectiveness of chlorpyrifos was substantially decreased. In contrast, omethoate remained effective when tested alone or as a mixture with chlorpyrifos. We also show that two novel (non-pesticide) treatments, molasses and wood vinegar, are ineffective in controlling H. destructor when sprayed to pasture fields at rates of 4 L/ha. These findings suggest a close link between levels of resistance quantified through laboratory bioassays and the field effectiveness of pesticides; however, in the case of H. destructor, this does not necessarily mean all field populations possessing organophosphate resistance will respond similarly given the potentially complex nature of the underlying resistance mechanism(s).

CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae).

BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens. RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2- and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight. CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. © 2023 Society of Chemical Industry.

Susceptibility monitoring and comparative gene expression of susceptible and resistant strains of Spodoptera frugiperda to lambda-cyhalothrin and chlorpyrifos.

BACKGROUND: Spodoptera frugiperda (J. E. Smith) is a widespread agricultural pest with several records of resistance to different insecticides and Bt proteins, including the neurotoxic insecticides chlorpyrifos (organophosphate) and lambda-cyhalothrin (pyrethroid). Here, we (i) characterized and monitored the susceptibility of field populations of S. frugiperda to chlorpyrifos (194 populations) and lambda-cyhalothrin (197 populations) collected from major maize-growing regions of Brazil from 2003 to 2016, and (ii) compared gene expression levels of laboratory-selected, chlorpyrifos- and lambda-cyhalothrin-resistant strains to a susceptible reference strain (Sf-ss) of S. frugiperda. RESULTS: The susceptibility monitoring detected average survival ranging from 29.3% to 36.0% for chlorpyrifos, and 23.1% to 68.0% for lambda-cyhalothrin. The resistance ratio of the chlorpyrifos-resistant strain (Clo-rr) was 25.4-fold and of the lambda-cyhalothrin-resistant strain (Lam-rr) was 21.5-fold. We identified 1098 differentially expressed genes (DEGs) between Clo-rr and Sf-ss, and 303 DEGs between Lam-rr and Sf-ss. Functional analyses of the DEGs revealed the up-regulation of several detoxification enzymes, mainly cytochrome P450 belonging to CYP3 and CYP6 clans. Genes associated with regulatory processes, such as the forkhead box class O (FoxO) transcription factor were also up-regulated. Variant analysis of target-site mutations for both pesticides identified the A201S and F290V mutations in acetylcholinesterase-1, both occurring in heterozigosis in the Clo-rr S. frugiperda strain. CONCLUSION: Our data show that the overexpression of the enzymatic detoxification machinery is the main difference to explain the resistance of Clo-rr and Lam-rr strains of S. frugiperda to chlorpyrifos and lambda-cyhalothrin, although a target-site mutation also contributes to the Clo-rr resistance to chlorpyrifos. © 2023 Society of Chemical Industry.

Contribution of UDP-glycosyltransferases to chlorpyrifos resistance in Nilaparvata lugens.

As a multigene superfamily of Phase II detoxification enzymes, uridine diphosphate (UDP)-glycosyltransferases (UGTs) play important roles in the metabolism of xenobiotics including insecticides. In this study, 5-nitrouracil, an inhibitor of UGT enzyme activity, effectively increased the toxicity of chlorpyrifos to the chlorpyrifos-resistant strain of Nilaparvata lugens, one of the most resistant rice pests. The enzyme content of UGT in the resistant strain was significantly higher than that in the susceptible strain. Among 20 identified UGT genes, UGT386H2, UGT386J2, UGT386N2 and UGT386P1 were found significantly overexpressed in the resistant strain and can be effectively induced by chlorpyrifos. These four UGT genes were most highly expressed in the midgut and/or fat body, two main insect detoxification tissues. Amino acid sequence alignments revealed that these four UGTs contained a variable N-terminal substrate-binding domain and a conserved C-terminal sugar donor-binding domain. Furthermore, homology modeling and molecular docking analyses showed that these UGTs could stably bind to chlorpyrifos and chlorpyrifos oxon, with the binding free energies from -19.4 to -110.62 kcal mol-1. Knockdown of UGT386H2 or UGT386P1 by RNA interference dramatically increased the susceptibility of the resistant strain to chlorpyrifos. These findings suggest that overexpression of these two UGT genes contributes to chlorpyrifos resistance in N. lugens.

Chlorpyrifos induces apoptosis and necroptosis via the activation of CYP450s pathway mediated by nuclear receptors in LMH cells.

Chlorpyrifos (CPF), an organophosphorus pesticide, is detected commonly in environments, where it is thought to be highly toxic to non-target organisms. However, the mechanism of CYP450s pathway mediated by nuclear receptors on CPF-induced apoptosis and necroptosis at the cellular level and the effect of CPF on the cytotoxicity of the chicken hepatocarcinoma cell line (LMH) has also not been reported in detail. Therefore, this experiment aims to explore whether CPF can improve apoptosis and necroptosis in LMH cells by activating the nuclear receptors/CYP450s axis. LMH cells, the subject of this study, were exposed to 5 µg/mL, 10 µg/mL, and 15 µg/mL doses of CPF. With the increase of CPF concentration, the increase of nuclear receptor level led to the up-regulation of CYP450s activity. With the massive production of ROS, the expression of apoptotic pathway genes (Bax, Caspase9, and Caspase3) enhanced, while Bcl-2 expression dropped sharply. The expression of programmed necroptosis genes (RIPK1, RIPK3, and MLKL) heightened, and Caspase8 reduced considerably. In short, our data suggests that excessive activation of nuclear receptors and CYP450s induced by CPF promotes ROS production, which directs apoptosis and programmed necroptosis in LMH cells.

Chlorpyrifos stimulates ABCC-mediated transport in the intestine of the rainbow trout Oncorhynchus mykiss.

The organophosphorus pesticide chlorpyrifos, detected in water and food worldwide, has also been found in the Río Negro and Neuquén Valley, North Patagonia, Argentina, where the rainbow trout, Oncorhynchus mykiss, is one of the most abundant fish species. We analyzed whether chlorpyrifos affects the transport activity of the ATP-binding cassette protein transporters from the subfamily C (ABCC), which are critical components of multixenobiotic resistance. We exposed ex vivo O. mykiss middle intestine strips (non-polarized) and segments (polarized) for one hour to 0 (solvent control), 3, 10, and 20 µg L-1 and to 0, 10, and 20 µg L-1 chlorpyrifos, respectively. We estimated the Abcc-mediated transport rate by measuring the transport rate of the specific Abcc substrate 2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, we measured the enzymatic activity of cholinesterase, carboxylesterase, glutathione-S-transferase, and 7-ethoxyresorufin-O-deethylase (EROD, indicative of the activity of cytochrome P450 monooxygenase 1A, CYP1A). We also measured lipid peroxidation using the thiobarbituric acid reactive substances method and the gene expression of Abcc2 and genes of the AhR pathway, AhR, ARNT, and cyp1a, by qRT-PCR. Chlorpyrifos induced the DNP-SG transport rate in middle intestine strips in a concentration-dependent manner (49-71%). In polarized preparations, the induction of the DNP-SG transport rate was observed only in everted segments exposed to 20 µg L-1 chlorpyrifos (40%), indicating that CPF only stimulated the apical (luminal) transport flux. Exposure to chlorpyrifos increased GST activity by 42% in intestine strips and inhibited EROD activity (47.5%). In addition, chlorpyrifos exposure inhibited cholinesterase (34-55%) and carboxylesterase (33-42.5%) activities at all the concentrations assayed and increased TBARS levels in a concentration-dependent manner (71-123%). Exposure to 20 µgL-1 chlorpyrifos did not affect the mRNA expression of the studied genes. The lack of inhibition of DNP-SG transport suggests that chlorpyrifos is not an Abcc substrate. Instead, CPF induces the activity of Abcc proteins in the apical membrane of enterocytes, likely through a post-translational pathway.

Herbivore-induced tomato plant volatiles lead to the reduction of insecticides susceptibility in Spodoptera litura.

Herbivore-induced plant volatiles (HIPVs) have been associated with plant-plant-herbivorous-natural enemies communication and an enhanced response to the subsequent attack. Spodoptera litura is a serious cosmopolitan pest that has developed a high level of resistance to many insecticides. However, the underlying molecular and biochemical mechanism by which HIPV priming reduces S. litura larval sensitivity to insecticides remains largely unknown. This study was conducted to explore the potential of volatile from undamaged, or artificially damaged, or S. litura-damaged tomato plants on the susceptibility of S. litura to the insecticides beta-cypermethrin indoxacarb and chlorpyrifos. We found that larvae exposed to volatile from S. litura-damaged or artificially damaged tomato plants were significantly less susceptible to the three insecticides than those exposed to volatile from undamaged tomato plants. Elevated activities of detoxifying enzymes [cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and esterases (ESTs)], were expressed in S. litura larvae exposed to volatile from S. litura-damaged tomato plants than those exposed to volatile from undamaged tomato plants. Similarly, seven detoxification-related genes [GSTs (SlGSTe1, SlGSTo1, and SlGSTe3) and P450s (CYP6B48, CYP9A40, CYP321A7, and CYP321B1)] in the midgut and fat body of larvae were up-regulated under exposure to volatile from S. litura-damaged tomato plants. Increased volatile organic compounds emissions were detected in the headspace of tomato plants damaged by S. litura compared to the undamaged plants. Collectively, these findings suggest that HIPVs can considerably reduce caterpillar susceptibility to insecticides, possibly through induction-enhanced detoxification mechanisms, and provide valuable information for implementing an effective integrated pest management strategy.

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.