P450 Monooxygenase Activity Research Articles

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

Dissection of the Enzymatic Process for Forming a Central Imidazopiperidine Heterocycle in the Biosynthesis of a Series c Thiopeptide Antibiotic.

Thiopeptide antibiotics are a family of ribosomally synthesized and posttranslationally modified peptide natural products of significant interest in anti-infective agent development. These antibiotics are classified into five subfamilies according to differences in the central 6-membered heterocycle of the thiopeptide framework. The mechanism through which imidazopiperidine, the most heavily functionalized central domain characteristic of a series c thiopeptide, is formed remains unclear. Based on mining and characterization of the genes specifically involved in the biosynthesis of Sch40832, we here report an enzymatic process for transforming a series b thiopeptide into a series c product through a series a intermediate. This process starts with F420-dependent hydrogenation of the central dehydropiperidine unit to a saturated piperidine unit. With the activity of a cytochrome P450 monooxygenase, the piperidine-thiazole motif of the intermediate undergoes an unusual oxygenation-mediated rearrangement to provide an imidazopiperidine heterocycle subjected to further S-methylation and aldehyde reduction. This study represents the first biochemical reconstitution of the pathway forming a stable series c thiopeptide.

Functional analysis of CYP6AE68, a cytochrome P450 gene associated with indoxacarb resistance in Spodoptera litura (Lepidoptera: Noctuidae)

Spodoptera litura (Fabricius) is a widely distributed, highly polyphagous pest that can cause severe damage to a variety of economically important crops. Various populations have developed resistance to different classes of insecticides. In this study, we report on two indoxacarb-resistant S. litura populations, namely Ind-R (resistance ratio = 18.37-fold) derived from an indoxacarb-susceptible (Ind-S) population and a population caught from a field (resistance ratio = 46.72-fold). A synergist experiment showed that piperonyl butoxide (PBO) combined with indoxacarb produced higher synergistic effects (synergist ratio = 5.29) in the Ind-R population as compared to Ind-S (synergist ratio = 3.08). Elevated enzyme activity of cytochrome P450 monooxygenases (P450s) was observed for Ind-R (2.15-fold) and the Field-caught population (4.03-fold) as compared to Ind-S, while only minor differences were noticed in the activities of esterases and glutathione S-transferases. Furthermore, expression levels of P450 genes of S. litura were determined by quantitative reverse transcription PCR to explore differences among the three populations. The results showed that the mRNA levels of CYP6AE68, a novel P450 gene belonging to the CYP6 family, were constitutively overexpressed in Ind-R (32.79-fold) and in the Field-caught population (68.11-fold). CYP6AE68 expression in S. litura was further analyzed for different developmental stages and in different tissues. Finally, we report that RNA interference-mediated silencing of CYP6AE68 increased the mortality of fourth-instar larvae exposed to indoxacarb at the LC50 dose level (increase by 33.89%, 29.44% and 22.78% for Ind-S, Ind-R and the Field-caught population, respectively). In conclusion, the findings of this study indicate that expression levels of CYP6AE68 in S. litura larvae are associated with indoxacarb resistance and that CYP6AE68 may play a significant role in detoxification of indoxacarb.

Mechanism of metabolic resistance to pymetrozine in Nilaparvata lugens: over-expression of cytochrome P450 CYP6CS1 confers pymetrozine resistance.

Pymetrozine is commonly used for the control of Nilaparvata lugens, and resistance to pymetrozine has been frequently reported in the field populations in recent years. However, the mechanism of brown planthopper resistance to pymetrozine is still unknown. In this study, a pymetrozine-resistant strain (PMR) was established, and the potential biochemical resistance mechanism of N. lugens to pymetrozine was investigated. Pymetrozine was synergized by the inhibitor piperonyl butoxide (PBO) in the PMR with 2.83-fold relative synergistic ratios compared with the susceptible strain (Sus). Compared with the Sus, the cytochrome P450 monooxygenase activity of PMR was increased by 1.7 times, and two P450 genes (NlCYP6CS1 and NlCYP301B1) were found to be significantly overexpressed more than 6.0-fold in the PMR. Pymetrozine exposure induced upregulation of NlCYP6CS1 expression in the Sus, but the expression of NlCYP301B1 did not change significantly. In addition, RNA interference (RNAi)-mediated suppression of NlCYP6CS1 gene expression dramatically increased the toxicity of pymetrozine against N. lugens. Moreover, transgenic lines of Drosophila melanogaster expressing NlCYP6CS1 were less susceptible to pymetrozine, and had a stronger ability to metabolize pymetrozine. Taken together, our findings indicate that the overexpression of NlCYP6CS1 is one of the key factors contributing to pymetrozine resistance in N. lugens. And this result is helpful in proposing a management strategy for pymetrozine resistance.

The role of detoxification enzymes in the susceptibility of Brevipalpus californicus exposed to acaricide and insecticide mixtures

The intense spraying of pesticides to control different arthropod pests has resulted in negative side effects for the management of pests. It was previously discovered that exposure to non-acaricidal insecticides alone or in a mixture, results in lower efficiency of the acaricide spirodiclofen used for Brevipalpus spp. control. We investigate here whether the induced expression of detoxification enzymes by non-lethal insecticides may antagonize spirodiclofen toxicity. Brevipalpus californicus mites exposed to the insecticide phosmet alone or in combination with spirodiclofen showed increased activity of P450 monooxygenases (P450s). No antagonistic effects in mite mortality were observed by the combination of phosmet and spirodiclofen. On the other hand, mites exposed to the insecticide imidacloprid alone or in combination with spirodiclofen showed an increase in the activity of P450s, carboxylcholinesterases (CCE), and glutathione-S-transferases (GST). An antagonistic effect on mite mortality was observed when mites were exposed to the LC25 of spirodiclofen combined with the field rate treatment of imidacloprid. The addition of PBO (a P450 monooxygenase inhibitor) to the mixture of spirodiclofen and imidacloprid resulted in a synergistic effect over mite mortality but the addition of DEM (a GST inhibitor) resulted in an antagonist effect. Taken together, this study showed that the combination of imidacloprid with spirodiclofen is antagonistic for the control of B. californicus, and this results from the induction of detoxification enzymes, such as P450s, CCE, and GST. The use of inhibitors highlights the role of these enzymes in the antagonism of the mixture.

Activity of Cytochrome P450 Monooxygenase (CYPs) Metabolic Enzymes as Markers of Insecticide Resistance in Anopheles vagus Muara Enim Mosquitoes, Indonesia

A B S T R A C TThe use of synthetic pyrethroids in insecticide-treated bed nets (LLINs) and sprayform (IRS) has been carried out since 2012 in Muara Emil Village and since 2016 inall villages in Tanjung Agung District, Muara Enim Regency. Biochemical resistancecan occur to the enzymes that play a role in detoxifying the insecticide. This studyaims to identify whether there is an increase in the levels of the enzyme CytochromeP450 monooxygenase (CYPs) through the enzyme biochemical test on the malariavector mosquito Anopheles vagus originating from Pagar Dewa Village and MuaraEmil Village, Muara Enim Regency. The sample of female Anopheles mosquitoes wasnot full. Blood was taken at night using the resting collection method. Mosquitoesthat have been identified as Anopheles vagus species are used to check CYPs enzymelevels using the ELISA method. The results of the biochemical test showed thatenzyme levels increased from the Muara emil and Pagar Dewa village mosquitoeswith a percentage of 71% and 61.53% (with cut off point OD> 0.165). The value ofCYPs enzyme levels for mosquitoes from Muara Emil village was higher than that ofthe mosquitoes from Pagar Dewa Village (mean 0.005027 ± 0.007). The increasedactivity of the CYPs enzyme plays a role in the detoxification of synthetic pyrethroidinsecticides that can cause resistance. The high levels of enzymes from the villagesof Muara Emil and Desa Farah Dewa are in line with intensive insecticide exposurein eradicating malaria vectors in the highest malaria transmission areas in MuaraEnim Regency.

Fenpyroximate resistance in Iranian populations of the European red mite Panonychus ulmi (Acari: Tetranychidae).

The European red mite, Panonychus ulmi (Koch), is one of the most important apple orchard pests worldwide. Fenpyroximate, a mitochondrial electron transport inhibitor of complex I (METI-I), is a commonly used acaricide to control this pest. In this study, we determined fenpyroximate resistance levels for 11 P. ulmi populations from Iran and a spirodiclofen-resistant strain from Germany (PSR-TK). The LC50 values ranged between 121.8 and 5713.9mg a.i. L-1 and the highest resistance ratio (RR) was 47-fold for the Padena population. PBO, TPP and DEM synergist ratios (SRs) were the highest for the PSR-TK (SR = 6.7), Shahin Dej (SR = 6.1) and Semirom3 (SR = 3.6) populations, respectively. In vitro enzyme activity measurements also showed that there was a higher glutathione S-transferases (GSTs) activity in the PSR-TK and Shahin Dej population compared to the most susceptible populations, whereas the esterase and P450 monooxygenase activity were not significantly higher in the resistant populations. Last, we screened all populations for the presence of two mutations previously associated with METI-I resistance in spider mites but none of these mutations could be detected. To conclude, moderate to high levels of fenpyroximate resistance were observed in P. ulmi populations from Iran, with increased detoxification most likely underlying fenpyroximate resistance.

Biochemical basis of insecticides resistance in cotton leafhopper, Amrasca biguttula biguttula (Ishida) (Hemiptera: Cicadellidae)

Cotton is one of world’s most essential fiber crops having global significance. Several biotic and abiotic stresses are constraints in cotton production including the bollworm complex and sucking pests. Among sucking pests, the cotton leafhopper, Amrasca biguttula biguttula (Ishida) is a devastating pest causing both quantitative and qualitative losses. After the introduction of Bt cotton, there was a check to the bollworm complex. But, the sucking pest complex particularly leafhoppers increased gradually reaching economic injury levels. The rampant application of insecticides to control sucking pests has led to the development of pest resistance to insecticides with different mode of action world over. Among the various mechanisms of resistance, the metabolic resistance plays a vital role. In order to determine the enzyme activity viz., cytochrome P450 monooxygenase, carboxyl esterase and glutathione S-transferase, for which the biochemical analysis was done by using field collected and laboratory reared population of this pest. The results revealed that, the activity of detoxification enzymes was significantly higher in the field collected population as compared to the laboratory population. The specific activity of cytochrome P450 monooxygenase (14.88 nmol/min/mg protein), carboxyl esterase (1321.97 nmol/min/mg protein) and glutathione S-transferase (1021.83 nmol/min/mg protein) in field collected population as compared to cytochrome P450 monooxygenase (12.02 nmol/min/mg protein), carboxyl esterase (1022.82 nmol/min/mg protein) and glutathione S-transferase (310.26 nmol/min/mg protein) in susceptible laboratory reared population of A. biguttula biguttula. This clearly shows that, these three xenobiotic enzymes are responsible for the detoxification of insecticides in the field population of this notorious pest.

Mechanisms of Increased Indoxacarb Toxicity in Methoxyfenozide-Resistant Cotton Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae).

Indoxacarb is an important insecticide for the selective control of Helicoverpa armigera. It can be bioactivated to the more effective N-decarbomethoxylated indoxacarb (DCJW) by esterases in pests. It was observed that both field and laboratory selected populations of H. armigera showed negative cross-resistance between indoxacarb and methoxyfenozide. The Handan population exhibited moderate resistance to indoxacarb, but was susceptible to methoxyfenozide; the Baoding and Yishui populations exhibited moderate resistance to methoxyfenozide, but they were susceptible to indoxacarb. Moreover, the toxicity of indoxacarb was enhanced 1.83-fold in the laboratory methoxyfenozide-resistant H. armigera, and susceptibility to methoxyfenozide was increased 2.81-fold in the laboratory indoxacarb-resistant H. armigera. In vivo, DCJW concentrations in the susceptible and methoxyfenozide-selected (laboratory methoxyfenozide-resistant) populations were 4.59- and 4.31-fold greater than in the indoxacarb-resistant Handan population 1 h after dosing. After 2 h, the highest concentrations of DCJW and indoxacarb appeared in the methoxyfenozide-selected population. Meanwhile, increased carboxyl esterase (CarE) and decreased glutathione S-transferase (GST) activities were observed in the methoxyfenozide-selected population. However, the indoxacarb-selected (laboratory indoxacarb-resistant) and Handan populations showed a higher disappearance of indoxacarb and DCJW, and the activity of cytochrome P450 mono-oxygenase in these populations were significantly increased. This study showed that the improved toxicity of indoxacarb, as observed in the methoxyfenozide-selected H. armigera, was correlated with increased CarE activity, decreased GST activity, and the in vivo accumulation of indoxacarb and DCJW. The significantly increased cytochrome P450 activity and higher disappearance of indoxacarb and DCJW in indoxacarb-resistant H. armigera resulted in the decreased toxicity of indoxacarb.

Resistance monitoring to four insecticides and mechanisms of resistance in Lygus lineolaris Palisot de Beauvois (Hemiptera: Miridae) populations of southeastern USA cotton.

Economically damaging infestations of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), have become more frequent in Virginia and North Carolina cotton since 2013 and 2010, respectively. Foliar insecticide use has increased dramatically in response. Efficacy data (LC50 ) are needed to evaluate L. lineolaris susceptibility and resistance levels (RR50 ) to commonly used and recommended insecticides for managing this pest in the southeastern USA. Elevated resistance levels to acephate and bifenthrin were measured in L. lineolaris populations collected from wild and cultivated hosts in Virginia, North Carolina and northern Alabama when compared to a susceptible laboratory population. High levels of bifenthrin resistance were observed in 2018 and 2019. Mixed-function oxidase and esterase (EST) inhibitors, piperonyl butoxide and S,S,S-Tributyl phosphotrithioate, respectively, had a synergistic effect on bifenthrin with resistant populations of L. lineolaris. Bifenthrin-resistant L. lineolaris populations expressed elevated levels of cytochrome P450 (CYP450 ) monooxygenase and general EST activity. Results suggest that insecticide resistance is present in some locations and that CYP450 and EST activity in L. lineolaris contribute to pyrethroid resistance in the southeastern USA. Results can serve as a baseline for continued monitoring of L. lineolaris insecticide resistance and inform insecticide resistance management strategies that help southeastern USA cotton producers to minimize inputs and slow resistance development. © 2020 Society of Chemical Industry.

Sublethal Effects of Triflumezopyrim on Biological Traits and Detoxification Enzyme Activities in the Small Brown Planthopper Laodelphax striatellus (Hemiptera: Delphacidae).

The small brown planthopper [Laodelphax striatellus (Fallén) (Hemiptera, Delphacidae)] is one of the most destructive insect pests of rice and has developed strong resistance to several kinds of chemical insecticides. Triflumezopyrim, a novel mesoionic insecticide developed by Corteva Agriscience (formerly DuPont Crop Protection), has efficient biological activity in controlling sucking insects, such as the planthopper. However, the effects of triflumezopyrim on the growth and reproduction of L. striatellus have not been reported. In this study, an F5 generation was obtained by conducting five rounds of insecticide screening on a sensitive L. striatellus strain (F0 generation). An age-stage life table procedure was used to evaluate the effects of a sublethal concentration (LC50) of triflumezopyrim on the biological parameters of L. striatellus. Compared with those of the F0 generation, the intrinsic rate of increase (r), the finite rate (λ), and the net reproductive rate (R0) of the F5 generation were significantly decreased; nevertheless, the average duration of life (T) was not significantly affected. The results of detoxification enzyme activity assays indicated that the glutathione S-transferase and cytochrome P450 monooxygenase (P450) activities in the F5 generation were significantly higher than those in the F0 generation. The contents of vitellogenin (Vg) and vitellogenin receptor (VgR) were also detected, and the results indicated that the contents of Vg and VgR in the F5 generation were significantly decreased compared to those in the F0 generation. Furthermore, we detected the relative expression of ecdysone receptor (EcR), Vg, and VgR in the F0 and F5 generations and found that the relative expression levels of Vg and VgR in the F5 generation female adults were obviously lower than those in the F0 generation (P < 0.05), whereas the relative expression of EcR was slightly increased, although the difference was not significant (P > 0.05). Based on these results, a sublethal concentration (median lethal concentration, LC50) of triflumezopyrim may inhibit the generational growth and reproduction of L. striatellus. Moreover, our results may provide a reference for further studies of the suitability and resistance mechanisms of L. striatellus subjected to a sublethal dose of triflumezopyrim.

Baseline susceptibility and biochemical mechanism of resistance to flupyradifurone in Bemisia tabaci

Bemisia tabaci (Gennadius) is one of the world's most devastating crop pests, and many populations show high resistance to various insecticides. To determine B. tabaci susceptibility to flupyradifurone and understand mechanisms of resistance, we conducted field monitoring and cross-resistance, synergism, and metabolic enzyme activity assays with two resistant strains: Wuhan (WH) and Xiangyang (XY). Results showed that most field populations were highly susceptible to flupyradifurone. However, WH and XY populations, with resistance ratios of 21.95- and 29.03-fold, respectively, showed a low level of flupyradifurone resistance but no cross-resistance to imidacloprid or cyantraniliprole. Synergism tests demonstrated that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) significantly inhibited flupyradifurone resistance in the XY strain, while PBO significantly inhibited resistance in the WH strain. Increased cytochrome P450 monooxygenase and esterase activity correlated with flupyradifurone resistance in the two resistant strains, indicating putative involvement of these enzymes in detoxification. By rotating flupyradifurone with other chemical agents that lack cross-resistance, development of resistance could be delayed, making flupyradifurone an effective agent for whitefly management in China.

Functional characterization of NADPH-cytochrome P450 reductase and cinnamic acid 4-hydroxylase encoding genes from Scoparia dulcis L.

BackgroundMost plant cytochrome P450 (P450) proteins need to be supplied with electrons from a redox partner, e.g. an NADPH-cytochrome P450 reductase (CPR), for the activation of oxygen molecules via heme. CPR is a flavoprotein with an N-terminal transmembrane domain, which transfers electrons from NADPH to the P450 via coenzymes flavin adenine dinucleotide and flavin mononucleotide.ResultsIn this study, a novel CPR (SdCPR) was isolated from a tropical medicinal plant Scoparia dulcis L. The deduced amino acid of SdCPR showed high homology of > 76% with CPR from higher plants and belonged to the class II CPRs of dicots. Recombinant SdCPR protein reduced cytochrome c, ferricyanide (K3Fe(CN)6), and dichlorophenolindophenol in an NADPH-dependent manner. To elucidate the P450 monooxygenase activity of SdCPR, we isolated a cinnamic acid 4-hydroxylase (SdC4H, CYP73A111) gene from S. dulcis. Biochemical characterization of SdCPR/SdC4H demonstrated that SdCPR supports the oxidation step of SdC4H. Real-time qPCR results showed that expression levels of SdCPR and SdC4H were inducible by mechanical wounding treatment and phytohormone elicitation (methyl jasmonate, salicylic acid), which were consistent with the results of promotor analyses.ConclusionsOur results showed that the SdCPR and SdC4H are related to defense reactions, including the biosynthesis of secondary metabolites.

Rhodopseudomonas capsulata enhances cleaning of chlorfenapyr from environment

Chlorfenapyr is a pesticide, but is not popular. With the massive spraying of chlorfenapyr, its wastewater is produced in large quantities. Microbial degradation method have certain advantages for this problem. The mixed wastewater (SPW and chlorfenapyr) treatment and valuable biochemical production by R. capsulata were explored. In the study, the concentration of chlorfenapyr, the activity of cytochrome P450 monooxygenase (P450), cpm gene were determined via high performance liquid chromatography and RT-PCR, respectively. Chlorfenapyr was cleared and the cleaner production augmented as SPW was added. Molecular analysis displayed that chlorfenapyr could elicit cpm gene expression to prepare P450 through acting on MAPKKKs gene. The elicitation of P450 and cpm occurred 1 d later. There were inadequate organic substances in initial wastewater to maintain R. capsulate growth for more than 1d. The supplement of SPW at three dosages (3, 7.5, 15 mg/L) provided adequate carbon sources for R. capsulata. The novel technology could be employed to deal with mixed wastewater, improve biochemicals and reuse wastewater and R. capsulata as sludge. The high-order non-linear mathematical model for illustrating relevance among Rchl, Xt and Xt/r was established.

Detection and biochemical characterization of acaricide resistance in field populations of tea red spider mite, Oligonychus coffeae (Acari: Tetranychidae), in Assam tea plantation of India

ABSTRACTThe red spider mite, Oligonychus coffeae, (RSM) is one of the most damaging pests of tea in most tea-producing countries. The use of synthetic acaricide, the main strategy for controlling RSM, has increased year by year. In this study, eight field populations of RSM were evaluated for resistance against five different acaricides (ethion, dicofol, propargite, fenazaquin, and fenpropathrin) using the leaf-dip method. The results showed that almost all the tested field populations had developed a high to very high level of resistance to ethion and dicofol. The highest level of resistance for RSM was found to ethion and dicofol with a resistance ratio of 134.27 and 65.38, respectively in the Cachar population as compared to the laboratory susceptible population. Propargite and fenazaquin were very effective against RSM in all tested populations, whereas a minor level of tolerance was evident against fenpropathrin at the recommended dose. Biochemical assays using detoxifying enzymes indicated that general esterase, glutathione-S- transferase, and cytochrome P450 monooxygenase activities were higher in all conventional field populations than in the susceptible population and also had a significantly positive correlation with the LC50 values for ethion and dicofol. Synergism assays were therefore performed using ethion and dicofol in combination with common synergists like piperonylbutoxide (PBO), S.S.S.-Tributylphosphorotrithioate (DEF), and ethacrynic acid (EA). Results showed that mixing of PBO with ethion ensured about 36.73 times higher toxicity against RSM. DEF was moderately effective in revamping the effect of ethion whereas only EA was able to reduce the tolerance of RSM population to dicofol up to 11.71 times. The present studies infer that general esterase and cytochrome P450 monooxygenase may play a major role in imparting resistance to ethion whereas only glutathione-S-transferase plays an important role in dicofol resistance in RSM. The present study indicates the need for adopting integrated resistance management programmes to effectively control RSM in the tea plantations of Assam.

Cytochrome P450 monooxygenases-mediated sex-differential spinosad resistance in house flies Musca domestica (Diptera: Muscidae)

Females developed notably higher resistance than males in a spinosad-resistant house fly strain, however, resistance factors responsible for this phenomenon are poorly understood. In this study, the potential role of cytochrome P450 monooxygenases involved in the sex-differential spinosad resistance in house flies was investigated, using a susceptible strain (SSS) and a spinosad resistant near-isogenic line (N-SRS). Combination of the synergism of spinosad by PBO and increased cytochrome P450 monooxygenase activity in the N-SRS strain implied that cytochrome P450 monooxygenases contributed to spinosad resistance in house flies. Transcriptional levels of eight P450 genes related to insecticide resistance in two genders of the SSS and N-SRS strain were separately evaluated by quantitative real-time PCR. Notably, compared with the corresponding gender of susceptible SSS house flies, CYP4G2 and CYP6A5v2 were overexpressed in resistant N-SRS females, while the expression of these two P450 genes was significantly decreased in resistant N-SRS males. Furthermore, by measuring the expression of CYP4G2 and CYP6A5v2 in female and male house fly populations with different spinosad resistance levels, which were generated from a series of genetic crosses, the genetic linkage between spinosad resistance and P450 gene expression was analyzed. It was found that with increased spinosad resistance, CYP4G2 and CYP6A5v2 were up-regulated in females, while both of them were down-regulated in males, and this suggested their involvement in the female-linked spinosad resistance of house flies. Taken together, our results provide valuable insight into the involvement of cytochrome P450 monooxygenases in the sex-differential spinosad resistance in house flies.

Minimal modulation of macrocyclic lactone susceptibility in Caenorhabditis elegans following inhibition of cytochrome P450 monooxygenase activity

Anthelmintic and in particular macrocyclic lactone (ML) resistance is a widespread problem in trichostrongyloid parasitic nematodes, yet mechanisms of ML resistance are still poorly understood. In the absence of target-site changes in resistant parasite field populations, increased drug extrusion and xenobiotic metabolism have been implicated in modification of susceptibility to MLs. In addition to P-glycoproteins, cytochrome P450 monooxygenases (CYPs) were considered to be involved in ML resistance. CYPs are highly divergent in nematodes with about 80 genes in the model organism Caenorhabditis elegans. Using larval development assays in the C. elegans model, piperonyl butoxide (PBO) and a temperature-sensitive variant of the emb-8 cytochrome reductase were used for chemical and genetic ablation of CYP activity. Additionally, a loss-of-function variant of cyp-14A5 was characterized to determine whether increased expression of this CYP in an ivermectin (IVM)-tolerant C. elegans line might be related to the phenotype. In a preliminary experiment with PBO, susceptibility to 5 nM IVM was synergistically increased by PBO. However, effects of genetic ablation of CYP activity on the EC50 values were small (1.5-fold decrease) for IVM and not significant for moxidectin (MOX). However, due to the steep concentration-response-curves, there were again strong differences between the wild-type and the CYP deficient genotype at individual IVM but not MOX concentrations. Although these results suggest small but significant effects on the susceptibility level of C. elegans to IVM, the cyp14A5 gene proposed by a previous study as candidate was ruled out since it was neither IVM/MOX inducible nor did a strain with a loss-of-function allele show increased susceptibility to either drug. In conclusion, the effect of the CYP system on IVM susceptibility in C. elegans is at best low while effects on MOX susceptibility were not detected. The previously suggested candidate cyp14A5 could be excluded to be involved in ML metabolism.

Cytochrome P450-Mediated λ-Cyhalothrin-Resistance in a Field Strain of Helicoverpa armigera from Northeast China.

Resistance to pyrethroid and organophosphate insecticides has been a growing problem in the management of cotton bollworm Helicoverpa armigera (Hübner) populations in the Yangtze River and Yellow River valleys of China, but resistance status and mechanisms of H. armigera populations from northeast China are less documented. In this study, a field strain collected from Shenyang in northeast China (SYR) is up to 16-fold more resistant than a susceptible strain (SS) to λ-cyhalothrin, while the resistance level to phoxim remained low (2.6-fold). Synergist tests and enzymatic assays show that increased cytochrome P450 monooxygenase (P450) activity is the main mechanism for λ-cyhalothrin resistance. Eight out of 10 genes from CYP6 and CYP9 subfamilies were significantly overexpressed in the SYR strain, and CYP6AE11 was the mostly overexpressed P450 (59-fold). These results suggest that overexpression of multiple P450 enzymes contributes to λ-cyhalothrin resistance in the SYR strain of H. armigera from northeast China.

Characterization of nitenpyram resistance in Nilaparvata lugens (Stål)

Nitenpyram is very effective in controlling Nilaparvata lugens (brown planthopper, BPH), and its resistance has been reported in field populations; however, the resistance mechanism remains unclear. In the present study, cross-resistance and resistance mechanisms in nitenpyram-resistant BPH were investigated. A resistant strain (NR) with a high resistance level (164.18-fold) to nitenpyram was evolved through successive selection for 42 generations from a laboratory susceptible strain (NS). The bioassay results showed that the NR exhibited cross-resistance to imidacloprid (37.46-fold), thiamethoxam (71.66-fold), clothianidin (149.17-fold), dinotefuran (98.13-fold), sulfoxaflor (47.24-fold), cycloxaprid (9.33-fold), etofenprox (10.51-fold) and isoprocarb (9.97-fold) but not to triflumezopyrim, chlorpyrifos and buprofezin. The NR showed a 3.21-fold increase in cytochrome P450 monooxygenase (P450) activity compared to that in the NS, while resistance was also synergized (4.03-fold) with the inhibitor piperonyl butoxide (PBO), suggesting a role of P450. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes by quantitative real-time PCR results indicated that twelve P450 genes were significantly overexpressed in the NR strain, especially CYP6ER1 (203.22-fold). RNA interference (RNAi) suppression of CYP6ER1 through injection of dsCYP6ER1 led to significant susceptibility in the NR strain. The current study expands our understanding of the nitenpyram resistance mechanism in N. lugens, provides an important reference for integrated pest management (IPM), and enriches the theoretical system of insect toxicology.

Biochemical Resistance Mechanisms to Fenvalerate in Plutella xylostella (Lepidoptera: Plutellidae).

The diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae), is one of the most important pests of cruciferous crops in Iran and is controlled mostly by fenvalerate. The susceptibility to fenvalerate was investigated for four populations of P. xylostella. Bioassay results indicated significant differences among the populations tested. The highest level of resistance to fenvalerate was obtained for the Khuzestan (Khz) population (resistance ratio = 9.5). Survival was suppressed by diethyl maleate, piperonylbutoxide, and triphenyl phosphate, which confirmed that resistance to fenvalerate is caused by glutathione S-transferases (GSTs), mixed function oxidases, and esterases, respectively. Up to 8.6-, 2-, 2.7-, and 1.75-fold increases in GSTs, esterase (α-naphthylacerate and β-naphthyl acetate as substrate), and cytochrome P450 monooxygenases activities in resistant strains when compared with the susceptible one, were observed, respectively. The expression of six GST genes of P. xylostella including GSTs1, GSTe2, GSTe4, GSTo4, GSTd4, and GSTd5 were analyzed. The quantitative PCR analysis showed that three of the PxGSTs had the highest expression levels in the Khz population. Two of the GSTs (GSTd4 and PxGSTe2) exhibited highest expression level in both Khz and Alborz (Alb) populations. Therefore, PxGST genes were involved in fenvalerate resistance in P. xylostella. Overall, the mechanisms of insecticide resistance in diamondback moth populations in four regions of Iran were related to GST, esterase, and cytochrome P450 monooxygenase activities.