Knockdown of CYP6CR2 and CYP6DE5 reduces tolerance to host plant allelochemicals in the Chinese white pine beetle Dendroctonus armandi.

Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of pesticides and plant allelochemicals. CYP6 family is unique to Insecta, and its biochemical function is basically related to the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of two CYP6 genes from Chinese white pine beetle, Dendroctonus armandi. Spatiotemporal expression profiling revealed that the expression of CYP6CR2 and CYP6DE5 was higher in larval and adult stages of D. armandi than that in other developmental stages, and that two genes predominantly expressed in brain, midgut, fat body, Malpighian tubules or hemolymph. The expression of CYP6CR2 and CYP6DE5 was significantly induced after feeding on the phloem of Pinus armandii and exposure to six stimuli [(±)- α -pinene, (-)-α-pinene, (-)-ß-pinene, (+)-3-carene, (±)-limonene and turpentine]. Importantly, silencing CYP6CR2 and CYP6DE5 separately could increase the sensitivity, led to a significant reduction of the activity of P450, resulting a significant increase in adult mortality after treatment with terpenoids. The comprehensive results of this study showed that in the process of host selection and colonization, the functions of CYPs were mainly to hydrolyze the chemical defense of the host and degrade odor molecules. These findings may help to develop new treatments to control this important pest.

CYP6P9-Driven Signatures of Selective Sweep of Metabolic Resistance to Pyrethroids in the Malaria Vector Anopheles funestus Reveal Contemporary Barriers to Gene Flow.

Pyrethroid resistance in major malaria vectors such as Anopheles funestus threatens malaria control efforts in Africa. Cytochrome P450-mediated metabolic resistance is best understood for CYP6P9 genes in southern Africa in An. funestus. However, we do not know if this resistance mechanism is spreading across Africa and how it relates to broader patterns of gene flow across the continent. Nucleotide diversity of the CYP6P9a gene and the diversity pattern of five gene fragments spanning a region of 120 kb around the CYP6P9a gene were surveyed in mosquitoes from southern, eastern and central Africa. These analyses revealed that a Cyp6P9a resistance-associated allele has swept through southern and eastern Africa and is now fixed in these regions. A similar diversity profile was observed when analysing genomic regions located 34 kb upstream to 86 kb downstream of the CYP6P9a locus, concordant with a selective sweep throughout the rp1 locus. We identify reduced gene flow between southern/eastern Africa and central Africa, which we hypothesise is due to the Great Rift Valley. These potential barriers to gene flow are likely to prevent or slow the spread of CYP6P9-based resistance mechanism to other parts of Africa and would to be considered in future vector control interventions such as gene drive.

Adipokinetic hormone regulates cytochrome P450-mediated imidacloprid resistance in the brown planthopper, Nilaparvata lugens.

Insect resistance to chemical insecticide is a global problem that presents an ongoing threat to sustainable agriculture. Although the increased production of detoxification enzymes has been frequently implicated in resistance development, the mechanisms employed by insecticide-resistant insects for overexpression of these genes remain elusive. Here we report that neuropeptide adipokinetic hormone (AKH) negatively regulates the expression of CYP6ER1 and CYP6AY1, two important cytochrome P450 monooxygenases (P450s) that confer resistance to neonicotinoid imidacloprid in the brown planthopper (BPH). Imidacloprid exposure suppresses AKH synthesis in the susceptible BPH, and AKH is inhibited in the imidacloprid-resistant strain. RNA interference (RNAi) and AKH peptide injection revealed that imidacloprid exposure inhibits the AKH signaling cascade and then provokes reactive oxygen species (ROS) burst. These in turn activate the transcription factors cap 'n' collar isoform-C (CncC) and muscle aponeurosis fibromatosis (MafK). RNAi and ROS scavenger assays showed that ROS induces CYP6ER1 expression by activating CncC and MafK, while ROS mediates induction of CYP6AY1 through another unidentified pathway in the resistant BPH. Collectively, these results provide new insights into the regulation of insecticide resistance and implicate both the neuropeptide AKH-mediated ROS burst and transcription factors are involved in the overexpression of P450 detoxification genes in insecticide-resistant insects.

Alcohol dehydrogenase 5 of Helicoverpa armigera interacts with the CYP6B6 promoter in response to 2-tridecanone.

Alcohol dehydrogenase 5 (ADH5) is a member of medium-chain dehydrogenase/reductase family and takes part in cellular formaldehyde and S-nitrosoglutathione metabolic network. 2-tridecanone (2-TD) is a toxic compound in many Solanaceae crops to defend against a variety of herbivory insects. In the broader context of insect development and pest control strategies, this study investigates how a new ADH5 from Helicoverpa armigera (HaADH5) regulates the expression of CYP6B6, a gene involved in molting and metamorphosis, in response to 2-TD treatment. Cloning of the HaADH5 complementary DNA sequence revealed that its 1002 bp open reading frame encodes 334 amino acids with a predicted molecular weight of 36.5 kD. HaADH5 protein was purified in the Escherichia coli Transetta (pET32a-HaADH5) strain using a prokaryotic expression system. The ability of HaADH5 protein to interact with the 2-TD responsive region within the promoter of CYP6B6 was confirmed by an in vitro electrophoretic mobility shift assay and transcription activity validation in yeast. Finally, the expression levels of both HaADH5 and CYP6B6 were found to be significantly decreased in the midgut of 6th instar larvae after 48 h of treatment with 10 mg/g 2-TD artificial diet. These results indicate that upon 2-TD treatment of cotton bollworm, HaADH5 regulates the expression of CYP6B6 by interacting with its promoter. As HaADH5 regulation of CYP6B6 expression may contribute to the larval xenobiotic detoxification, molting and metamorphosis, HaADH5 is a candidate target for controlling the growth and development of cotton bollworm.

Activation of CncC pathway by ROS burst regulates cytochrome P450 CYP6AB12 responsible for λ-cyhalothrin tolerance in Spodoptera litura.

Frequent insecticide use poses an environmental hazard and also selects for insecticide tolerance. Increased metabolic detoxification by cytochrome P450 monooxygenases (P450s) is the most common mechanism of insecticide tolerance. However, the underlying regulatory mechanisms remain unknown. We studied the midgut-specific P450 gene, CYP6AB12, associated with λ-cyhalothrin tolerance. Its regulatory pathway was investigated in the tobacco cutworm, Spodoptera litura (Fabricius). P450 activities and CYP6AB12 transcript levels increased after λ-cyhalothrin exposure. Inhibiting P450 activities with piperonyl butoxide and silencing CYP6AB12 by double-stranded RNA (dsRNA) injection decreased larval tolerance to λ-cyhalothrin. λ-Cyhalothrin exposure induced the expression of the cap 'n' collar isoform C (CncC) and muscle aponeurosis fibromatosis (Maf), increased hydrogen peroxide (H2O2) contents and elevated antioxidant enzyme activities. CncC knockdown by dsRNA feeding suppressed CYP6AB12 expression and decreased larval tolerance to λ-cyhalothrin. In contrast, application of the CncC agonist curcumin induced CYP6AB12 expression and enhanced insecticide tolerance. Ingestion of the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced H2O2 accumulation, suppressed the expression of CncC, Maf and CYP6AB12 and led to increased larval susceptibility to λ-cyhalothrin. The results demonstrate that in S. litura, λ-cyhalothrin induces cytochrome P450 CYP6AB12 via elicitation of the ROS burst and activation of the CncC pathway.

A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella, is a damaging pest of cruciferous crops, and has evolved resistance to many of the insecticides used for control, including members of the diamide class. Previous work on the molecular basis of resistance to diamides has documented mutations in the target-site, the ryanodine receptor, in resistant populations of P. xylostella worldwide. In contrast the role of metabolic resistance to this insecticide class is significantly less clear. Here we show that overexpression of a flavin-dependent monooxgenase (FMO) confers resistance to the diamide chlorantraniliprole in P. xylostella. Transcriptome profiling of diamide resistant strains, with and without target-site resistance, revealed constitutive over-expression of several transcripts encoding detoxification enzymes compared to susceptible strains. Two of these, CYP6BG1, and PxFMO2 were particularly highly overexpressed (33,000 and 14,700-fold, respectively) in a resistant strain (HAW) lacking target-site resistance. After 17 generations without diamide selection the resistance of the HAW strain fell by 52-fold and the expression of PxFMO2 by > 1300-fold, however, the expression of CYP6BG1 declined by only 3-fold. Generation of transgenic Drosophila melanogaster expressing these genes demonstrated that PxFMO2, but not CYP6BG1, confers resistance in vivo. Overexpression of PxFMO2 in the HAW strain is associated with mutations, including a putative transposable element insertion, in the promoter of this gene. These enhance the expression of a reporter gene when expressed in a lepidopteran cell line suggesting they are, at least in part, responsible for the overexpression of PxFMO2 in the resistant strain. Our results provide new evidence that insect FMOs can be recruited to provide resistance to synthetic insecticides.

Transcription Factors AhR/ARNT Regulate the Expression of CYP6CY3 and CYP6CY4 Switch Conferring Nicotine Adaptation.

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco.

Metabolism of selected model substrates and insecticides by recombinant CYP6FD encoded by its gene predominately expressed in the brain of Locusta migratoria.

The migratory locust, Locusta migartoria, is a major agricultural insect pest and its resistance to insecticides is becoming more prevalent. Cytochrome P450 monooxygenases (CYPs) are important enzymes for biotransformations of various endogenous and xenobiotic substances. These enzymes play a major role in developing insecticide resistance in many insect species. In this study, we heterologously co-expressed a CYP enzyme (CYP6FD1) and cytochrome P450 reductase (CPR) from L. migartoria in Sf9 insect cells. The recombinant enzymes were assayed for metabolic activity towards six selected model substrates (luciferin-H, luciferin-Me, luciferin-Be, luciferin-PFBE, luciferin-CEE and 7-ethoxycoumarin), and four selected insecticides (deltamethrin, chlorpyrifos, carbaryl and methoprene). Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and luciferin-Me, but no detectable activity towards the other luciferin derivatives. Furthermore, the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner. However, the enzyme did not show any detectable activity towards the other three insecticides. Our results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin. This work is a step towards a more complete characterization of the catalytic capabilities of CYP6FD1 and other xenobiotic metabolizing CYP enzymes in L. migratoria.

Functional characterization of CYP6A51, a cytochrome P450 associated with pyrethroid resistance in the Mediterranean fruit fly Ceratitis capitata.

Overexpression of the cytochrome P450 monooxygenase CYP6A51 has been previously associated with pyrethroid resistance in the Mediterranean fruit fly (medfly) Ceratitis capitata, an important pest species worldwide; however, this association has not been functionally validated. We expressed CYP6A51 gene in Escherichia coli and produced a functional enzyme with preference for the chemiluminescent substrate Luciferin-ME EGE. In vitro metabolism assays revealed that CYP6A51 is capable of metabolizing two insecticides that share the same mode of action, λ-cyhalothrin and deltamethrin, whereas no metabolism or substrate depletion was observed in the presence of spinosad or malathion. We further expressed CYP6A51 in vivo via a GAL4/UAS system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. Toxicity bioassays indicated that CYP6A51 confers knock-down resistance to both λ-cyhalothrin and deltamethrin. Detection of CYP6A51 - associated pyrethroid resistance in field populations may be important for efficient Insecticide Resistance Management (IRM) strategies.

Functions of mountain pine beetle cytochromes P450 CYP6DJ1, CYP6BW1 and CYP6BW3 in the oxidation of pine monoterpenes and diterpene resin acids.

The mountain pine beetle (MPB; Dendroctonus ponderosae) is a forest insect pest that attacks several different pine (Pinus) species in its native range of distribution in western North America. MPB are exposed for most of their life cycle to the chemical defenses of their hosts. These defenses are dominated by oleoresin secretions containing mostly various monoterpenes and diterpene resin acids (DRAs). Cytochrome P450 enzymes (P450s) of the MPB are thought to be involved in the metabolism of at least some of these defense compounds. Here we describe the cloning and characterization of three MPB P450s, CYP6DJ1, CYP6BW1 and CYP6BW3, and their functions in the oxidation of various monoterpenes and diterpene resin acids. CYP6DJ1 oxidizes the monoterpenes (+)-(4R)-limonene, (-)-(4S)-limonene and terpinolene and produces (4R,8R)-limonene-8,9-epoxide, (4R,8S)-limonene-8,9-epoxide, (4S,8S)-limonene-8,9-epoxide, (4S,8R)-limonene-8,9-epoxide, perilla alcohol and several unidentified oxidized compounds. These products of CYP6DJ1 were also identified in extracts of MPB treated with the same monoterpenes. CYP6BW1 and CYP6BW3 both oxidize the DRAs abietic acid, dehydroabietic acid, neoabietic acid, levopimaric acid, palustric acid, and isopimaric acid, producing hydroxylated and epoxidized DRAs. CYP6DJ1, CYP6BW1 and CYP6BW3 appear to contribute to the metabolism of oleoresin terpenes as part of the MPB's ability to cope with host defenses.

Transcription factor FTZ-F1 and cis-acting elements mediate expression of CYP6BG1 conferring resistance to chlorantraniliprole in Plutella xylostella.

BACKGROUND: Cytochrome P450-mediated detoxification plays an important role in the development of insecticide resistance. Previous studies have demonstrated that overexpression of CYP6BG1 was responsible for permethrin resistance in Plutella xylostella, and our experiments also showed that upregulation of this gene is associated with chlorantraniliprole resistance in P. xylostella. However, the transcriptional regulation involved in the expression of CYP6BG1 remains unknown. To further investigate the regulation of CYP6BG1 expression, the promoters of this gene were cloned and analyzed from one susceptible and four different resistant populations of P. xylostella. RESULTS: First, the promoter region of P. xylostella CYP6BG1 was compared in five populations, and three types of 5'-flanking region were found. Second, the region between -562 and +49 of CYP6BG1 in a field population (TH) of P. xylostella showed the highest promoter activity and could be induced by chlorantraniliprole. Third, the transcriptional factor FTZ-F1, which is an orphan nuclear receptor and binds to the fushi tarazu (ftz) gene, was predicted by the online software Alggen and Jaspar. It was proved to regulate the expression of CYP6BG1 by RNAi. The expression levels of FTZ-F1 and CYP6BG1 could be induced by chlorantraniliprole and were significantly higher in the resistant populations. CONCLUSIONS: These data give a better understanding of the transcriptional regulation of an important insecticide detoxification enzyme gene, and therefore will help in understanding the molecular mechanisms of insecticide resistance in P. xylostella. © 2018 Society of Chemical Industry.

The use of gene expression to unravel the single and mixture toxicity of abamectin and difenoconazole on survival and reproduction of the springtail Folsomia candida.

Pesticides risk assessments have traditionally focused on the effects on standard parameters, such as mortality, reproduction and development. However, one of the first signs of adverse effects that occur in organisms exposed to stress conditions is an alteration in their genomic expression, which is specific to the type of stress, sensitive to very low contaminant concentrations and responsive in a few hours. The aim of the present study was to evaluate the single and binary mixture toxicity of commercial products of abamectin (Kraft® 36 EC) and difenoconazole (Score® 250 EC) to Folsomia candida. Laboratory toxicity tests were conducted to access the effects of these pesticides on springtail survival, reproduction and gene expression. The reproduction assays gave EC50 and EC10 values, respectively, of 6.3 and 1.4 mg a.s./kg dry soil for abamectin; 1.0 and 0.12 mg a.s./kg dry soil for Kraft® 36 EC; and 54 and 23 mg a.s./kg dry soil for Score® 250 EC. Technical difenoconazole did not have any effect at the concentrations tested. No significant differences in gene expression were found between the abamectin concentrations tested (EC10 and EC50) and the solvent control. Exposure to Kraft® 36 EC, however, significantly induced Cyp6 expression at the EC50 level, while VgR was significantly downregulated at both the EC10 and EC50. Exposure to the simple pesticide mixture of Kraft® 36 EC + Score® 250 EC caused significant up regulation of ABC transporter, and significant down regulation of VgR relative to the controls. GABA receptor also showed significant down-regulation between the EC10 and EC50 mixture treatments. Results of the present study demonstrate that pesticide-induced gene expression effects precede and occur at lower concentrations than organism-level responses. Integrating "omic" endpoints in traditional bioassays may thus be a promising way forward in pesticide toxicity evaluations.

The regulation of three new members of the cytochrome P450 CYP6 family and their promoters in the cotton aphid Aphis gossypii by plant allelochemicals.

BACKGROUND: The expression of P450 genes in insects can be induced by plant allelochemicals. To understand the induction mechanisms, we measured the expression profiles of three P450 genes and their promoter activities under the induction of plant allelochemicals. RESULTS: The inducible expression of CYP6CY19 was the highest among three genes, followed by those of CYP6CY22 and CYP6DA1. The regions from -687 to +586 bp of CYP6DA1, from -666 to +140 bp of CYP6CY19 and from -530 to +218 bp of CYP6CY22 were essential for basal transcriptional activity. The cis-elements for plant allelochemicals induction were identified between -193 and +56 bp of CYP6DA1, between -157 and +140 bp of CYP6CY19 and between -108 and +218 bp of CYP6CY22. These promoter regions were found to contain a potential aryl hydrocarbon receptor element binding site with a conservative sequence motif 5'-C/TAC/ANCA/CA-3'. All these four plant allelochemicals were able to induce the expression of these P450 genes. Tannic acid had a better inductive effect than other three plant allelochemicals. CONCLUSIONS: Our study identified the plant allelochemical responsive cis-elements. This provides further research targets aimed at understanding the regulatory mechanisms of P450 genes expression and their interactions with plant allelochemicals in insect pests. © 2018 Society of Chemical Industry.

Cytochrome P450 CYP6EV11 in Chironomus kiiensis Larvae Involved in Phenol Stress.

Phenol is one of the organic pollutants which can cause water environment pollution. It is not only enriched in aquatic organisms but is also a serious threat to human health. Chironomus kiiensis is very sensitive to the contaminants in water and its cytochrome P450s are usually chosen as biomarkers for water pollution. To examine whether CYP6EV11 plays a role in the oxidative metabolism of phenol, we measured the silencing efficiency of CYP6EV11 and evaluated larval susceptibility to sublethal phenol levels by RNA interference (RNAi) technology. The results showed that the transcription of CYP6EV11 was found significantly up-regulated when the 4th instar C.kiiensis larvae were exposed to three doses of phenol. However, the transcriptional levels of CYP6EV11 were significantly suppressed by 92.7% in the 4th instar C. kiiensis larvae soaked in dsCYP6EV11 compared with those soaked in dsGFP for 6 h. The CYP6EV11 expression and mortality of the 4th instar C. kiiensis larvae with CYP6EV11 silencing were mostly decreased under phenol stress. Therefore, the CYP6EV11 gene may be used as a molecular biomarker for earlier warning and monitoring for water pollution.

The evolution of insecticide resistance in the brown planthopper (Nilaparvata lugens Stål) of China in the period 2012-2016.

The brown planthopper, Nilaparvata lugens, is an economically important pest on rice in Asia. Chemical control is still the most efficient primary way for rice planthopper control. However, due to the intensive use of insecticides to control this pest over many years, resistance to most of the classes of chemical insecticides has been reported. In this article, we report on the status of eight insecticides resistance in Nilaparvata lugens (Stål) collected from China over the period 2012-2016. All of the field populations collected in 2016 had developed extremely high resistance to imidacloprid, thiamethoxam, and buprofezin. Synergism tests showed that piperonyl butoxide (PBO) produced a high synergism of imidacloprid, thiamethoxam, and buprofezin effects in the three field populations, YA2016, HX2016, and YC2016. Functional studies using both double-strand RNA (dsRNA)-mediated knockdown in the expression of CYP6ER1 and transgenic expression of CYP6ER1 in Drosophila melanogaster showed that CYP6ER1 confers imidacloprid, thiamethoxam and buprofezin resistance. These results will be beneficial for effective insecticide resistance management strategies to prevent or delay the development of insecticide resistance in brown planthopper populations.

Deltamethrin is metabolized by CYP6FU1, a cytochrome P450 associated with pyrethroid resistance, in Laodelphax striatellus.

BACKGROUND: Cytochrome P450s (CYPs) are known to play a major role in metabolizing a wide range compounds. CYP6FU1 has been found to be over-expressed in a deltamethrin-resistant strain of Laodelphax striatellus. This study was conducted to express CYP6FU1 in Sf9 cells as a recombinant protein, to confirm its ability to degrade deltamethrin, chlorpyrifos, imidacloprid and traditional P450 probing substrates. RESULTS: Carbon monoxide difference spectrum analysis indicated that the intact CYP6FU1 protein was expressed in insect Sf9 cells. Catalytic activity tests with four traditional P450 probing substrates revealed that the expressed CYP6FU1 preferentially metabolized p-nitroanisole and ethoxyresorufin, but not ethoxycoumarin and luciferin-HEGE. The enzyme kinetic parameters were tested using p-nitroanisole. The michaelis constant (Km ) and catalytic constant (Kcat ) values were 17.51 ± 4.29 µm and 0.218 ± 0.001 pmol min-1 mg-1 protein, respectively. Furthermore, CYP6FU1 activity for degradation of insecticides was tested by measuring substrate depletion and metabolite formation. The chromatogram analysis showed obvious nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of deltamethrin, and formation of the unknown metabolite. Mass spectra and the molecular docking model showed that the metabolite was 4-hydroxy-deltamethrin. However, the recombinant CYP6FU1 could not metabolize imidacloprid and chlorpyrifos. CONCLUSION: These results confirmed that the over-expressed CYP6FU1 contributes to deltamethrin resistance in L. striatellus, and p-nitroanisole might be a potential diagnostic probe for deltamethrin metabolic resistance detection and monitoring. © 2017 Society of Chemical Industry.

Novel cytochrome P450 (CYP6D1) and voltage sensitive sodium channel (Vssc) alleles of the house fly (Musca domestica) and their roles in pyrethroid resistance.

BACKGROUND: The house fly Musca domestica is an important disease vector. Point mutation-mediated target-site insensitivity of the voltage sensitive sodium channel (VSSC) and increased detoxification mediated by cytochrome P450 (CYP6D1) overexpression have been characterized as two major mechanisms of pyrethroid resistance. In this study, genetic mutations in the Vssc and CYP6D1 genes and their contribution to pyrethroid resistance were investigated. RESULTS: Twelve lines of house flies homozygous for four genotypes were established. House flies carrying the VSSC 1014F mutation and overexpressing CYP6D1 had higher resistance to pyrethroids than those carrying 1014F alone. The presence of the 15-bp insert in the promoter region of the CYP6D1 gene did not necessarily result in a significant increase in CYP6D1 mRNA and pyrethroid resistance levels. A novel Vssc allele carrying two mutations (G1924D and G2004S) in combination with the classic 1014F and a novel CYP6D1 allele that is very similar to CYP6D1v1 were identified in Chinese house flies. CONCLUSION: This work demonstrates the effect of genetic mutations in CYP6D1 and Vssc on the susceptibility of house flies to pyrethroids, and verifies that 15-bp insert-containing CYP6D1 alleles have a single origin. These findings offer insights into the evolution of insecticide resistance and have implications for house fly control. © 2017 Society of Chemical Industry.

Phylogenetic and functional characterization of ten P450 genes from the CYP6AE subfamily of Helicoverpa armigera involved in xenobiotic metabolism.

The cotton bollworm, Helicoverpa armigera, is a generalist herbivore widely distributed over the world and is a major lepidopteran pest on cotton. Studies, especially from Asia, show that it relies on cytochrome P450 monooxygenases with broad substrate specificities to protect itself from pesticides. The number of P450s may have expanded in the processes of coping with the wide diversity of phytochemicals that the insect encounters among its numerous host plants. In order to examine the metabolic capabilities of these P450s, we focused here on all ten P450s of the Helicoverpa armigera CYP6AE subfamily, which can be easily induced by plant toxins and pyrethroids. These P450s, along with cytochrome P450 reductase, were heterologously expressed in insect cells and compared functionally. In vitro metabolism showed that all CYP6AE subfamily members can convert esfenvalerate to 4'-hydroxyesfenvalerate efficiently except CYP6AE20. In contrast, none of the recombinant CYP6AE enzymes could metabolise gossypol under our experimental conditions. Epoxidation capabilities were observed in the CYP6AE subfamily, aldrin can be converted to dieldrin at rates up to 0.45 ±â€¯0.04 pmol/min/pmol P450. Seven P450s in this subfamily can metabolise imidacloprid, but with lower efficiency than Bemisia tabaci CYP6CM1vQ. CYP6AE20 had virtually no metabolic competence to these four compounds but could metabolise several model fluorogenic substrates. These results showed the broad substrate spectrum of H. armigera CYP6AE P450s and suggest a limited role of gossypol upon the evolution of H. armigera CYP6AE genes.

Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

BACKGROUND: Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. RESULTS: Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. CONCLUSION: The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

Cytochrome P450 genes from the aquatic midge Chironomus tentans: Atrazine-induced up-regulation of CtCYP6EX3 enhanced the toxicity of chlorpyrifos.

The open reading frames of 19 cytochrome P450 monooxygenase (CYP) genes were sequenced from Chironomus tentans, a commonly used freshwater invertebrate model. Phylogenetic analysis of the 19 CYPs along with a previously reported CYP (CtCYP4G33) revealed that they belong to three different clans, including 3 in CYP4, 15 in CYP3, and 2 in mitochondria clan. When third-instar larvae were exposed to atrazine at 5000 µg/L, the transcription of CtCYP6EX3, CtCYP6EV3, CtCYP9AT1 and CtCYPEX1 was significantly up-regulated. To examine whether CtCYP6EX3 played a role in oxidative activation of chlorpyrifos to chlorpyrifos-oxon, we evaluated larval susceptibility to chlorpyrifos after CtCYP6EX3 transcript was suppressed by RNAi. The larvae fed chitosan/dsCtCYP6EX3 nanoparticles showed a significantly decreased CtCYP6EX3 transcript (53.1%) as compared with the control larvae fed chitosan/dsGFP nanoparticles. When the CtCYP6EX3-silenced larvae were exposed to chlorpyrifos at 6 µg/L or its binary mixture with atrazine (chlorpyrifos at 3 µg/L and atrazine at 1000 µg/L), the larvae became less susceptible to the pesticides as their mortalities decreased by 24.1% and 20.5%, respectively. These results along with our previous findings suggested that the increased toxicity of chlorpyrifos was likely due to an enhanced oxidative process from chlorpyrifos to chlorpyrifos-oxon by CtCYP6EX3 as RNAi of CtCYP6EX3 led to decreased susceptibility of C. tentans larvae to chlorpyrifos alone and the binary mixture of atrazine and chlorpyrifos. However, further study would be necessary to validate our results by functional assays using heterologously expressed CtCYP6EX3 enzyme.