Insect Insecticide Research Articles

Suppressing the expression of glutathione S-transferase gene GSTd10 increases the sensitivity of Zeugodacus cucurbitae against β-cypermethrin.

Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agriculturally and economically important pest worldwide that has developed resistance to β-cypermethrin. Glutathione S-transferases (GSTs) have been reported to be involved in the detoxification of insecticides in insects. We have found that both ZcGSTd6 and ZcGSTd10 were up-regulated by β-cypermethrin induction in our previous study, so we aimed to explore their potential relationship with β-cypermethrin tolerance in this study. The heterologous expression of ZcGSTd6 and ZcGSTd10 in Escherichia coli showed significantly high activities against 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters of ZcGSTd6 and ZcGSTd10 were determined by Lineweaver-Burk. The Vmax and Km of ZcGSTd6 were 0.50 μmol/min·mg and 0.3 mM, respectively. The Vmax and Km of ZcGSTd10 were 1.82 μmol/min·mg and 0.53 mM. The 3D modelling and molecular docking results revealed that β-cypermethrin exhibited a stronger bounding to the active site SER-9 of ZcGSTd10. The sensitivity to β-cypermethrin was significantly increased by 18.73% and 27.21%, respectively, after the knockdown of ZcGSTd6 and ZcGSTd10 by using RNA interference. In addition, the inhibition of CDNB at 50% (IC50) and the inhibition constants (Ki) of β-cypermethrin against ZcGSTd10 were determined as 0.41 and 0.33 mM, respectively. The Ki and IC50 of β-cypermethrin against ZcSGTd6 were not analysed. These results suggested that ZcGSTd10 could be an essential regulator involved in the tolerance of Z. cucurbitae to β-cypermethrin.

CncC-Keap1-P450s pathway is involved in the detoxification of emamectin benzoate in the spongy moth Lymantria dispar

The detoxification of insecticides in insects is dependent on the expression and activity of multiple detoxification enzymes. As an important modulator of detoxification enzymes, the CncC-Keap1 pathway was involved in the detoxification of various pesticides. However, whether the CncC-Keap1 pathway is involved in the detoxification of emamectin benzoate (EMB) is unclear. In this study, we cloned the LdCncC and LdKeap1 from spongy moths (Lymantria dispar). Our results showed that EMB exposure induced oxidative stress, and activated the CncC-Keap1 pathway at mRNA and protein levels. Removing ROS by N-acetylcysteine remarkably decreased H2O2 levels and restored the expression of LdCncC and LdKeap1. The silencing LdCncC, not LdKeap1, by dsRNA significantly decreased the cytochrome P450 activities, and increased the sensitivity of larvae to EMB. Besides, the expression of CYP6B7v1, CYP321A7 and CYP4S4v1 were significantly decreased after silencing LdCncC. Notably, the knockdown of CYP6B7v1, CYP321A7 or CYP4S4v1 significantly increased the mortality induced by EMB exposure. Therefore, we proposed that activation of CncC-Keap1 pathway induced by ROS increased the detoxification of EMB in spongy moths by regulating the expression of CYP6B7v1, CYP321A7 and CYP4S4v1. Our study strengthened the understanding of the detoxification of EMB from the perspective of CncC-Keap1-P450s pathway.

Endosymbionts mediated detoxification of insecticides in insects

Endosymbionts mediated detoxification of insecticides in insects

Fitness costs of resistance to insecticides in insects

The chemical application is considered one of the most crucial methods for controlling insect pests, especially in intensive farming practices. Owing to the chemical application, insect pests are exposed to toxic chemical insecticides along with other stress factors in the environment. Insects require energy and resources for survival and adaptation to cope with these conditions. Also, insects use behavioral, physiological, and genetic mechanisms to combat stressors, like new environments, which may include chemicals insecticides. Sometimes, the continuous selection pressure of insecticides is metabolically costly, which leads to resistance development through constitutive upregulation of detoxification genes and/or target-site mutations. These actions are costly and can potentially affect the biological traits, including development and reproduction parameters and other key variables that ultimately affect the overall fitness of insects. This review synthesizes published in-depth information on fitness costs induced by insecticide resistance in insect pests in the past decade. It thereby highlights the insecticides resistant to insect populations that might help design integrated pest management (IPM) programs for controlling the spread of resistant populations.

Comprehensive analysis of the overexpressed cytochrome P450-based insecticide resistance mechanism in Spodoptera litura

Cytochrome P450s play critical roles in the metabolic resistance of insecticides in insects. Previous findings showed that enhanced P450 activity was an important mechanism mediating indoxacarb resistance, and multiple P450 genes were upregulated in indoxacarb resistant strains of Spodoptera litura. However, the functions of these P450 genes in insecticide resistance remain unknown. Here, the P450 inhibitor PBO effectively decreased the resistance of S. litura to indoxacarb. Ten upregulated P450 genes were characterized, all of which were overexpressed in response to indoxacarb induction. Knockdown of nine P450 genes decreased cell viability against indoxacarb, and further silencing of three genes (CYP339A1, CYP340G2, CYP321A19) in larvae enhanced the sensitivity to indoxacarb. Transgenic overexpression of these three genes increased resistance to indoxacarb in Drosophila melanogaster. Moreover, molecular modeling and docking predicted that these three P450 proteins could bind tightly to indoxacarb and N-decarbomethoxylated metabolite (DCJW). Interestingly, these three P450 genes may also mediate cross-resistance to chlorantraniliprole, λ-cyhalothrin and imidacloprid. Additionally, heterologous expression and metabolic assays confirmed that three recombinant P450s could effectively metabolize indoxacarb and DCJW. This study strongly demonstrates that multiple overexpressed mitochondrial and microsomal P450 genes were involved in insecticide resistance in S. litura.

Identification, expression profiles and involvement in insecticides tolerance and detoxification of carboxylesterase genes in Bactrocera dorsalis

Carboxylesterases (CarEs) are a multifunctional superfamily of enzymes and play an important role in detoxification of various insecticides in insects. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive agricultural pests and has developed different degrees of resistance to organophosphates in field. However, the involvement of BdCarEs in tolerance or resistance to other alternative insecticides are still unclear. In the present study, 33 BdCarEs genes were identified based on the genome database of B. dorsalis. Phylogenetic analysis demonstrated that they were classified into nine clades, with abundance of α-esterases. Meanwhile, the sequence characterization and the chromosome distribution were also analyzed. The spatiotemporal expression analysis of BdCarEs genes suggested that the diversity of potential function in different physiological processes. With the exception of BdCarE21, all BdCarEs genes responded to at least one insecticide exposure, and BdCarE20 was found to be up-regulated after exposure to all five tested insecticides individually. Eight BdCarEs genes were overexpressed in MR strain when compared to that in SS strain. Subsequently, knockdown the expression of representative BdCarEs genes significantly increased the susceptibility of the oriental fruit fly to corresponding insecticides, which indicated that the tested BdCarEs genes contributed to one or multiple insecticide detoxification. These findings provide valuable insights into the potential role in respond to tolerance or resistance to insecticides with different mode of action, and will facilitate development of efficiency management strategy for B. dorsalis.

Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core).

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+ /K+ -ATPase, it is unclear how closely related species such as the non-sequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defenses might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.

Identification and characterization of UDP-glycosyltransferase genes and the potential role in response to insecticides exposure in Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis (Hendel) is a worldwide pest damaging a wide range of hosts. Due to the long-term indiscriminate use of insecticides, B. dorsalis has developed serious resistance to several insecticides. UDP-glycosyltransferases (UGTs) are secondary metabolic enzymes involved in biotransformation and play an important role in the metabolism of plant secondary metabolites and synthetic insecticides in insects. Thus, we suspect that UGTs in B. dorsalis play an important role in insecticide tolerance. In this study, 31 UGT genes were identified in the genome of B. dorsalis, belonging to 13 subfamilies. Real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that 12 UGT genes were highly expressed in the antennae, midgut, Malpighian tubule and fat body. The mRNA expressions of 17 UGT genes were up-regulated upon exposure to λ-cyhalothrin, imidacloprid, abamectin and chlorpyrifos. Knockdown of the selected five UGT genes (BdUGT301D2, BdUGT35F2, BdUGT36K2, BdUGT49D2, BdUGT50B5) by RNA interference increased the mortality of B. dorsalis from 9.29% to 27.22% upon exposure to four insecticides. The abundance of UGTs in B. dorsalis is similar to other insect species, and 12 out of 31 UGTs were specifically expressed in metabolic tissues, suggesting a key role in detoxification. Down-regulation of five selected UGT genes increased the susceptibility of B. dorsalis to various insecticides, indicating that UGTs may play an important role in tolerance of B. dorsalis to multiple insecticides. © 2022 Society of Chemical Industry.

Gut Symbionts: Hidden Players of Pesticide Resistance in Insects

Development of resistance to insecticides in insects is a global concern. Understanding the mechanisms is critical for effective plant protection and human health. Challenge in dealing the matter is that, resistance can occur via direct organism response (metabolic, physiological, and target-site changes) or via gut microbiome. Insects are constantly evolving like any other organisms; they are adopting various measures to overcome the chemicals sprayed to control them. Increasing evidence suggest that the gut microbiome can promote pesticide resistance in pests. Possible mechanisms by which gut bacteria play role in insecticide resistance are, direct acquisition of pesticide degrading microbes from the environment, difference in gut bacterial composition and diversity, difference in xenobiotic degrading enzymes and presence of microbial xenobiotic degradation pathways and cross-acclimatization to related insecticides.

Identification and Functional Characterization of a Sigma Glutathione S-Transferase CpGSTs2 Involved in λ-Cyhalothrin Resistance in the Codling Moth Cydia pomonella.

The codling moth, Cydia pomonella (L.), is a quarantine pest of global significance impacting pome fruits and walnuts. It has evolved resistance to many commonly used insecticides including λ-cyhalothrin. Glutathione S-transferases (GSTs) are multifunctional enzymes playing a crucial role in the detoxification of insecticides in insects. However, the role of specific GST gene in λ-cyhalothrin resistance in C. pomonella is unclear. In this study, we identified three sigma-class genes (CpGSTs1, CpGSTs2, and CpGSTs3). These genes were ubiquitously expressed at all developmental stages, and of these, the expression level of CpGSTs2 in the larval stage was significantly higher than in the egg, pupal, and adult stages. Moreover, CpGSTs2 was predominantly expressed in the fat body while lower levels in the cuticle. In addition to exposure of larvae to LD10 of λ-cyhalothrin elevating the expression level of CpGSTs2, mRNA levels of CpGSTs2 in a field population (ZW_R) from northeast China, which has developed moderate level resistance to λ-cyhalothrin, was significantly higher than that of susceptible strains. In vitro inhibition assays demonstrated that λ-cyhalothrin inhibited the conjugating activities of recombinant CpGSTs2, and metabolic assays indicated that λ-cyhalothrin could be depleted by recombinant CpGSTs2. These results bring evidence for the involvement of CpGSTs2 in C. pomonella in resistance to λ-cyhalothrin.

Insecticides induce the co-expression of glutathione S-transferases through ROS/CncC pathway in Spodoptera exigua

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that are involved in detoxification of electrophilic toxic compounds. Although the co-induced expression of GST genes by insecticides in insects has been documented in recent years, the underlying regulatory mechanisms are not understood. In this study, a total of thirty-one cytosolic S. exigua GSTs (SeGSTs) was cloned and identified. The bioinformatics and gene expression patterns were also analyzed. Out of them, SeGSTe9, SeGSTs6, SeGSTe1, SeGSTe6, SeGSTe8, SeGSTe14, and SeGSTd1 were significantly co-expressed following exposure to three insecticides (lambda-cyhalothrin, chlorpyrifos and chlorantraniliprole). The analysis of upstream sequences revealed that all of these seven SeGSTs harbored CncC/Maf binding site. The luciferase reporter assay showed that the pGL3-SeGST promoter construct exhibited a significant increase in luciferase activities after exposure to insecticides, and mutation of CncC/Maf binding site diminish the induction effect. These data indicate that CncC/Maf pathway regulates the co-expression of GST genes in response to different insecticides in S. exigua. Insecticides significantly enhanced the ROS content and treatment with the ROS inhibitor N-acetylcysteine (NAC) decreased the insecticide-induced luciferase activities of the PGL3-GSTe6 promoter construct, but not the CncC-mutated construct. These results indicate that ROS mediates GST gene expression after exposure to insecticides through CncC/Maf pathway. Overall, these data show that insecticides induce the co-expression of glutathione S-transferases through the ROS/CncC pathway in S. exigua.

Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides.

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain.

Multiple ATP-binding cassette transporters are involved in insecticide resistance in the small brown planthopper, Laodelphax striatellus.

ATP-binding cassette (ABC) transporters are membrane-bound proteins involved in the movement of various substrates, including drugs and insecticides, across the lipid membrane. Demonstration of the role of human ABC transporters in multidrug resistance has led to speculation that they might be an important mechanism controlling the fate of insecticides in insects. However, the role of ABC transporters in insects remains largely unknown. The small brown planthopper, Laodelphax striatellus Fallén, has developed resistance to most of the insecticides used for its control. Our goals were to identify the ABC transporters in La. striatellus and to examine their involvement in resistance mechanisms, using related strains resistant to chlorpyrifos, deltamethrin and imidacloprid, compared with the susceptible strain. Based on the transcriptome of La. striatellus, 40 full-length ABC transporters belonging to the ABCA-ABCH subfamilies were identified. Quantitative PCR revealed that over 20% of genes were significantly up-regulated in different resistant strains, and eight genes from the ABCB/C/D/G subfamilies were up-regulated in all three resistant strains, compared with the susceptible strain. Furthermore, synergism studies showed verapamil significantly enhanced insecticide toxicity in various resistant strains but not in the susceptible strain. These results suggest that ABC transporters might be involved in resistance to multiple insecticides in La. striatellus.

Gene expression analysis and enzyme assay reveal a potential role of the carboxylesterase gene CpCE-1 from Cydia pomonella in detoxification of insecticides

Carboxylesterases (CarEs) are responsible for metabolism of xenobiotics including insecticides in insects. Understanding the expression patterns of a such detoxifying gene and effect of insecticides on its enzyme activity are important to clarify the function of this gene relevant to insecticides-detoxifying process, but little information is available in the codling moth Cydia pomonella (L.). In this study, we investigated the expression profiles of CarE gene CpCE-1 at different developmental stages and in different tissues of C. pomonella, as well as the larvae exposed to chlorpyrifos-ethyl and lambda-cyhalothrin by using absolute real-time quantitative PCR (absolute RT-qPCR). Results indicated that CpCE-1 expression was significantly altered during C. pomonella development stages, and this expression differed between sexes, with a higher transcript in females than males. Meanwhile, CpCE-1 is overexpressed in cuticle, midgut and head than silk gland, fat body and Malpighian tubules. Exposure of third instar larvae to a non-lethal dosage of chlorpyrifos-ethyl and lambda-cyhalothrin resulted in induction of CpCE-1 transcript. The total carboxylesterase enzyme activity was inhibited by chlorpyrifos-ethyl in vivo; in contrast, the activity of Escherichia coli produced recombinant CpCE-1 was significantly inhibited by both lambda-cyhalothrin and chlorpyrifos-ethyl in vitro. These results suggested that CpCE-1 in C. pomonella is potentially involved in the development and in detoxification of chlorpyrifos-ethyl and lambda-cyhalothrin.

Two homologous carboxylesterase genes from Locusta migratoria with different tissue expression patterns and roles in insecticide detoxification

Carboxylesterases (CarEs) play a crucial role in detoxification of xenobiotics and resistance to insecticides in insects. In this study, two cDNAs of CarE genes (LmCesA4 and LmCesA5) were sequenced from the migratory locust, Locusta migratoria. The cDNAs of LmCesA4 and LmCesA5 putatively encoded 538 and 470 amino acid residues, respectively. The deduced amino acid sequences of the two CarE genes showed 45.0% identities, possessed highly conserved catalytic triads (Ser-Glu-His), and clustered in phylogenetic analysis. These results suggest that they are homologous genes. Both CarE genes were expressed throughout the developmental stages. However, LmCesA4 was predominately expressed in the midgut (including the gastric caeca) and fat bodies, whereas LmCesA5 was mainly expressed in the gastric caeca. The in situ hybridization results showed that the transcripts of the two genes were localized in apical and basal regions of the columnar cells in the gastric caeca. Gene silencing followed by insecticide bioassay increased the mortalities of deltamethrin-, malathion-, and carbaryl-treated locusts by 29.5%, 31.0% and 20.4%, respectively, after the locusts were injected with LmCesA4 double-stranded RNA (dsRNA). In contrast, the injection of LmCesA5 dsRNA did not significantly increase the susceptibility of the locusts to any of these insecticides. These results suggest that these genes not only show different tissue expression patterns but also play different roles in insecticide detoxification.

Pesticides and asthma: challenges for epidemiology.

DO PESTICIDES CAUSE ASTHMA? Pesticides are chemical or biological agents used to kill or incapacitate unwanted organisms both indoors and outdoors. In and around the home, these agents are commonly and increasingly used to control rats, ants, cockroaches, flies, moths, mites, weeds, and molds. Pesticides are usually grouped into families not only depending on the organism they target (e.g., fungicides for fungi, insecticides for insects) but also according to their chemical characteristics (e.g., organophosphates, carbamates, pyrethroids). Asthma is a common chronic inflammatory disease of the airways characterized by variable and recurring respiratory symptoms (wheezing, breathlessness, chest tightness, and dry cough), airflow obstruction, and increased bronchial responsiveness (1, 2). However, it is increasingly recognized that this is not a homogeneous disease since multiple subtypes (e.g., early-onset allergic, late-onset eosinophilic, and exercise-induced asthma) have been described (3). Some of the factors that have been linked to asthma include allergies, tobacco smoking, air pollution, viral and bacterial infections, changes in sex hormone levels, obesity, and occupational exposures (2–5). Variants in several genes (e.g., CHI3L1, IL6R, DENND1B, IL1RL1– IL18R1, PDE4D, RAD50–IL13, HLA-DQ, IL33, SMAD3, ORMDL3–GSDMB, IL2RB, RORA, TSLP, and PYHIN1) have also been associated with increased susceptibility to this disease (2). Asthma affects more than 300 million people worldwide (6), and in the last decades, its prevalence has been increasing especially in low and middle income countries (7–9). Over the same period, the use of multiple chemicals such as pesticides for crop protection and home disinfestation has also increased (10–12). Associations between pesticide exposure and asthma in children, adults, and occupational groups have been reported, but these have not yet been shown to be causal and the biological explanation tying the two remains unclear – it could lie in the mechanisms such as irritation (13), inflammation (14), immunosuppression (15, 16), endocrine disruption (17), or a combination of these. So far, it is also unknown how pesticides interact with genes that increase susceptibility to asthma.

Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization

Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni(2+)-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k cat/K m of 100 s(-1) μM(-1) for α-NA, and the value is 29.78 s(-1) μM(-1) for β-naphthyl acetate. The V max and K m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu(2+) and Mg(2+) significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.

Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl‐coenzyme A carboxylase

Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.

Heterologous expression and characterization of a sigma glutathione S-transferase involved in carbaryl detoxification from oriental migratory locust, Locusta migratoria manilensis (Meyen)

Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and resistance to insecticides in insects. In the present study, a sigma-class GST gene (LmGSTs3) was identified from the locust, Locusta migratoria manilensis. Its full-length cDNA sequence is 828bp containing an open reading frame (ORF) of 612bp that encodes 204 amino acid residues. The predicted protein molecular mass and pI are 23.4kDa and 7.62, respectively. Recombinant LmGSTs3 was heterologously expressed in Escherichia coli as a soluble fusion protein. Its optimal activity was observed at pH 8.0. Incubation for 30min at temperatures below 40°C scarcely affected activity. The LmGSTs3 at pH values between 4.0 and 11.0 retained more than 80% of its original activity. Ethacrynic acid and cibacron blue were very effective inhibitors of LmGSTs3 with I50-values 1.7 and 3.7μM, respectively. In response to heavy metal (CuSO4, CdCl2) exposure there was a concentration-dependent and time-dependent decrease in activity. The nymph mortalities after carbaryl treatment increased 38.7% after LmGSTs3 were silenced. These results suggest that LmGSTs3 may be involved in carbaryl detoxification in L. migratoria manilensis.

The molecular biology of knockdown resistance to pyrethroid insecticides

The term “knockdown resistance” is used to describe cases of resistance to diphenylethane (e.g. DDT) and pyrethroid insecticides in insects and other arthropods that result from reduced sensitivity of the nervous system. Knockdown resistance, first identified and characterized in the house fly ( Musca domestica) in the 1950’s, remains a threat to the continued usefulness of pyrethroids in the control of many pest species. Research since 1990 has provided a wealth of new information on the molecular basis of knockdown resistance. This paper reviews these recent developments with emphasis on the results of genetic linkage analyses, the identification of gene mutations associated with knockdown resistance, and the functional characterization of resistance-associated mutations. Results of these studies identify voltage-sensitive sodium channel genes orthologous to the para gene of Drosophila melanogaster as the site of multiple knockdown resistance mutations and define the molecular mechanisms by which these mutations cause pyrethroid resistance. These results also provide new insight into the mechanisms by which pyrethroids modify the function of voltage-sensitive sodium channels.