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The long-term and frequent use of pyrethroid insecticides has led to the development of pyrethroid resistance in many insect populations around the world. Specific mutations in the voltage-gated sodium channel (VGSC) have been well documented to be responsible for knockdown resistance (kdr) to pyrethroids and dichlorodiphenyltrichloroethane (DDT) in a variety of arthropods. However, reports regarding naturally occurring kdr mutation in field populations of the German cockroach Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae) in China have remained scarce. In this study, a survey was conducted to detect the presence and frequency of kdr mutations in field populations of B. germanica across Neijiang, Sichuan province of China. In addition to the previously reported L993F mutation, a new amino acid substitution L993S was discovered for the first time. Overall, the classical 993F was the dominant allele with frequencies ranging from 61.8 to 89.6%, while the frequencies of the novel L993S mutation were in the range between 2.5 and 15.0%. Notably, high frequencies (50.0–79.2%) of resistant homozygotes were detected in our samples, indicating high levels of pyrethroid resistance in these B. germanica populations. The results suggest that alternative insecticides with a mode of action different from pyrethroids should be considered in the control of German cockroaches in these regions. [ABSTRACT FROM AUTHOR]

The cotton aphid, Aphis gossypii Glover, is one of the most important agricultural insect pests. Pyrethroid and neonicotinoid insecticides have generally shown excellent control of A. gossypii, but many populations of this pest have developed resistance against these classes of insecticides. The success of insecticide resistance management strategies requires detailed knowledge of both phenotype and genotype of the target insect pest. In this study, we attempted to understand the molecular status of insecticide resistance in cotton aphid populations in Xinjiang Uygur Autonomous Region of China, the major cotton planting region of China. In addition to the previously reported M918L mutation, we discovered another substitution (M918V) in the voltage‐gated sodium channel (VGSC). Moreover, we developed a molecular assay that could be used to detect precisely the R81T mutation in the nicotinic acetylcholine receptor (nAChR). This survey revealed that 918L was the predominant VGSC allele with a frequency ranging from 50.0% to 56.7%. Notably, appreciable frequencies (between 10% and 40%) of the resistance 81T allele of the nAChR gene were detected in three investigated populations. The prevalent co‐occurrence of both VGSC 918L/V and nAchR 81T indicates a worrisome situation of multiple resistance to both pyrethroids and neonicotinoids. [ABSTRACT FROM AUTHOR]

Four putative knockdown resistance (kdr) mutations have been documented in the voltage-gated sodium channel (VGSC) gene of Cimex hemipterus from several countries. However, no information regarding kdr mutations in any Chinese tropical bed bug population is available to date. In this study, a double-mutation（M918I + L1014F）kdr allele was identified in six C. hemipterus populations across Guangxi Zhuang Autonomous Region of China. The frequency of this allele was 100% in all the six examined populations. In addition, only two cytochrome c oxidase I (COI) gene haplotypes, with one synonymous nucleotide variation, were identified in a total of 48 individuals from six locations. The fixation and broad geographic distribution of this resistant allele questions the continued use of pyrethroids in the treatment of tropical bed bug infestations. The very low genetic diversity within and among these populations indicates that these bed bugs may have a single origin. [ABSTRACT FROM AUTHOR]

Background: In South Asia, the epidemiology of malaria is complex, and transmission mainly occurs in remote areas near international borders. Vector control has been implemented as a key strategy in malaria prevention for decades. A rising threat to the efficacy of vector control efforts is the development of insecticide resistance, thus it is important to monitor the type and frequency of insecticide resistant alleles in the disease vectors such as An. sinensis along the China-Vietnam border. Such information is needed to synthesize effective malaria vector control strategies. Methods: A total of 208 adults of An. sinensis, collected from seven sites in southwest Guangxi along the China-Vietnam border, were inspected for the resistance-conferring G119S mutation in acetylcholinesterase (AChE) by PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) and kdr mutations in the voltage-gated sodium channel (VGSC) by sequencing. In addition, the evolutionary origin of An. sinensis vgsc gene haplotypes was analyzed using Network 5.0. Results: The frequencies of mutant 119S of AChE were between 0.61–0.85 in the seven An. sinensis populations. No susceptible homozygote (119GG) was detected in three of the seven sites (DXEC, LZSK and FCGDX). Very low frequencies of kdr (0.00–0.01) were detected in the seven populations, with most individuals being susceptible homozygote (1014LL). The 1014F mutation was detected only in the southeast part (FCGDX) at a low frequency of 0.03. The 1014S mutation was distributed in six of the seven populations with frequencies ranging from 0.04 to 0.08, but absent in JXXW. Diverse haplotypes of 1014L and 1014S were found in An. sinensis along the China-Vietnam border, while only one 1014F haplotype was detected in this study. Consistent with a previous report, resistant 1014S haplotypes did not have a single origin. Conclusions: The G119S mutation of AChE was present at high frequencies (0.61–0.85) in the An. sinensis populations along the China-Vietnam border, suggesting that the vector control authorities should be cautious when considering carbamates and organophosphates as chemicals for vector control. The low frequencies (0.00–0.11) of kdr in these populations suggest that pyrethroids remain suitable for use against An. sinensis in these regions. [ABSTRACT FROM AUTHOR]

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas. [ABSTRACT FROM AUTHOR]

Abstract: 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 [ABSTRACT FROM AUTHOR]

Planetary gear is widely used in engineering and usually has symmetrical structure. As the number of teeth in contact changes during rotation, the time-varying mesh stiffness parametrically excites the planetary gear and may cause severe vibrations and instabilities. Taking speed fluctuations into account, the time-varying mesh stiffness is frequency modulated, and therefore sideband instabilities may arise and original instabilities are significantly affected. Considering two different speed fluctuations, original and sideband instabilities are numerically and analytically investigated. A rotational lumped-parameter model of the planetary gear is developed, in which the time-varying mesh stiffness, input speed fluctuations, and damping are considered. Closed-form approximations of instability boundaries for primary and combination instabilities are obtained by perturbation analysis and verified by numerical analysis. The effects of speed fluctuations and damping on parametric instability are systematically examined. Because of the frequency modulation, whether a parametric instability occurs cannot be simply predicted by the planet meshing phase which is applicable to constant speed. Besides adjusting the planet meshing phase, speed fluctuation supplies a new thought to minimize certain instability by adjusting the amplitude or frequency of the speed fluctuation. Both original and sideband instabilities are shrunken by damping, and speed fluctuation further shrinks the original instability. [ABSTRACT FROM AUTHOR]

Insecticide-resistant Drosophila melanogaster strains represent a resource for the discovery of the underlying molecular mechanisms of cytochrome P450 constitutive over-expression, even if some of these P450s are not directly involved in the resistance phenotype. For example, in select 4,4’-dichlorodiphenyltrichloroethane (DDT) resistant strains the glucocorticoid receptor-like (GR-like) potential transcription factor binding motifs (TFBMs) have previously been shown to be associated with constitutively differentially-expressed cytochrome P450s, Cyp12d1, Cyp6g2 and Cyp9c1. However, insects are not known to have glucocorticoids. The only ortholog to the mammalian glucocorticoid receptor (GR) in D. melanogaster is an estrogen-related receptor (ERR) gene, which has two predicted alternative splice isoforms (ERRa and ERRb). Sequencing of ERRa and ERRb in select DDT susceptible and resistant D. melanogaster strains has revealed a glycine (G) codon insertion which was only observed in the ligand binding domain of ERR from the resistant strains tested (ERR-G). Transgenic flies, expressing the ERRa-G allele, constitutively over-expressed Cyp12d1, Cyp6g2 and Cyp9c1. Only Cyp12d1 and Cyp6g2 were over-expressed in the ERRb-G transgenic flies. Phylogenetic studies show that the G-insertion appeared to be located in a less conserved domain in ERR and this insertion is found in multiple species across the Sophophora subgenera. [ABSTRACT FROM AUTHOR]

Background: The German cockroach, Blattella germanica, is an important insect pest that transmits various pathogens mechanically and causes severe allergic diseases. This insect has long served as a model system for studies of insect biology, physiology and ecology. However, the lack of genome or transcriptome information heavily hinder our further understanding about the German cockroach in every aspect at a molecular level and on a genome-wide scale. To explore the transcriptome and identify unique sequences of interest, we subjected the B. germanica transcriptome to massively parallel pyrosequencing and generated the first reference transcriptome for B. germanica. Methodology/Principal Findings: A total of 1,365,609 raw reads with an average length of 529 bp were generated via pyrosequencing the mixed cDNA library from different life stages of German cockroach including maturing oothecae, nymphs, adult females and males. The raw reads were de novo assembled to 48,800 contigs and 3,961 singletons with high-quality unique sequences. These sequences were annotated and classified functionally in terms of BLAST, GO and KEGG, and the genes putatively coding detoxification enzyme systems, insecticide targets, key components in systematic RNA interference, immunity and chemoreception pathways were identified. A total of 3,601 SSRs (Simple Sequence Repeats) loci were also predicted. Conclusions/Significance: The whole transcriptome pyrosequencing data from this study provides a usable genetic resource for future identification of potential functional genes involved in various biological processes. [ABSTRACT FROM AUTHOR]

BACKGROUND Increased insecticide detoxification mediated by cytochrome P450s is a common mechanism of insecticide resistance. Although Cyp6a2 has been observed to be overexpressed in many 4,4′-dichlorodiphenyltrichloroethane ( DDT)-resistant strains of Drosophila melanogaster, how Cyp6a2 is regulated and whether its overproduction confers DDT resistance remain elusive. RESULTS Molecular analysis identified five Cyp6a2 alleles ( Cyp6a2Canton−S-1, Cyp6a2Canton−S-2, Cyp6a291-C, Cyp6a291-R and Cyp6a2Wisconsin− WD) from four D. melanogaster strains, notably differing in the presence or absence of an intact Nrf2/Maf (a transcription factor) binding site in the 5′-promoter core region, a ' G1410' frameshift deletion mutation in the heme-binding region and a long terminal repeat ( LTR) of transposable element 17.6 in the 3′-untranslated region ( UTR). Linkage analysis confirmed that DDT resistance was genetically linked to a Nrf2/Maf-binding-site-containing, LTR-lacking functional allele of Cyp6a2 ( Cyp6a291-R). The qRT-PCR results showed that overexpression of functional Cyp6a2 was consistently associated with DDT resistance. Luciferase reporter gene assays revealed that an intact Nrf2/Maf binding site in the 5′-promoter core region enhanced the constitutive transcription of Cyp6a2. CONCLUSION The results suggest that the Nrf2/Maf binding-site-containing functional Cyp6a2 allele is associated with DDT resistance in the D. melanogaster strains under study. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background Moderate to high DDT resistance in generally associated with overexpression of multiple genes and therefore has been considered to be polygenic. However, very little information is available about the molecular mechanisms that insect populations employ when evolving increased levels of resistance. The presence of common regulatory motifs among resistance-associated genes may help to explain how and why certain suites of genes are preferentially represented in genomic-scale analyses. Results A set of commonly differentially expressed genes associated with DDT resistance in the fruit fly was identified on the basis of genome-wide microarray analysis followed by qRT-PCR verification. More genes were observed to be overtranscribed in the highly resistant strain ( 91-R) than in the moderately resistant strain ( Wisconsin) and susceptible strain ( Canton-S). Furthermore, possible transcription factor binding sites that occurred in coexpressed resistance-associated genes were discovered by computational motif discovery methods. Conclusion A glucocorticoid receptor ( GR)-like putative transcription factor binding motif ( TFBM) was observed to be associated with genes commonly differentially transcribed in both the 91-R and Wisconsin lines of DDT-resistant Drosophila. © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Insecticide resistance is a model phenotype that can be used to investigate evolutionary processes underlying the spread of alleles across a global landscape, while offering valuable insights into solving the problems that resistant pests present to human health and agriculture. Pyrethroids are one of the most widely used classes of insecticides world-wide and they exert their toxic effects through interactions with the voltage-sensitive sodium channel (Vssc). Specific mutations in Vssc (kdr, kdrhis and super-kdr) are known to cause resistance to pyrethroid insecticides in house flies. In order to determine the number of evolutionary origins of kdr, kdr-his and super-kdr, we sequenced a region of Vssc from house flies collected in the USA, Turkey and China. Our phylogenetic analysis of Vssc unequivocally supports the hypothesis of multiple independent origins of kdr, super-kdr and kdr-his on an unprecedented geographic scale. The implications of these evolutionary processes on pest management are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Several resistance-associated mutations have been previously documented in the house fly Musca domestica, an important sanitary pest of humans and animals world-wide. Sensitive detection of resistance is crucial for effective insecticide applications. We have developed simple PCR-RFLP assays for reliably detecting the G342A/V mutations in the acetylcholinesterase (Ace), the L1014F/H in voltage sensitive sodium channel (Vssc) and the W251L/S in the carboxylesterase (mdaE7). These accurate diagnostics are useful to monitor the frequency of the resistance mutations, thus, help to avoid ineffective insecticide application in fly control practice. [Copyright &y& Elsevier]

The article presents a study on the identification of new cytochrome P450 genes and analysis of their expression profile and xenobiotic response to interpret the roles of cytochrome P450s in the Helicoverpa armigera. From H. armigera, a novel P450 cDNA, CYP9A17, was separated which showed the highest amino acid identity with CYP9A12. The results of the study indicate that after the gene duplication event, CYP9A12 and CYP9A17 genes in H. armigera might vary via subfunctionalisation.

Abstract: A soil bacterium strain, capable of using p-nitrophenol (PNP) as its sole source of carbon and energy, was isolated by enrichment on minimal salt medium (MSM). On the basis of a phylogenetic analysis of 16S rRNA gene sequences the bacterium is a species of Arthrobacter, closely related to Arthrobacter ureafaciens DSM 20126. This strain has an unusually high substrate tolerance for PNP degradation in MSM. Greatest degradation of PNP was observed at 30°C and under slightly alkaline pH (pH 7–9) conditions. Effective degradation rates slowed as the concentration of PNP was increased. Addition of glucose from 0.1% to 0.5% generally enhanced the degradation of PNP at high concentration (400mg/l) although acidification as a result of glucose metabolism had a negative effect on PNP depletion. Biodegradation of PNP at high concentration was greatly accelerated by β-cyclodextrin at a concentration of 0.5%, indicating that β-cyclodextrin could be a promising addictive for effective PNP bioremediation. [Copyright &y& Elsevier]

Abstract: A Helicoverpa armigera population was collected from Shandong province, China. After 15 generations of selection in the laboratory, the H. armigera strain developed more than 20-fold resistance to spinosad. At LD50 level, no significant cross-resistance was found between spinosad and chlorpyrifos, methomyl, avermectin and chlorfenapyr except for fenvalerate with a low cross-resistance of 2.4-fold. However, LD99 values of fenvalerate against the parental and resistant strains were not different significantly. After inhibitors were used, spinosad resistance could be partially suppressed by piperonylbutoxide (PBO) and triphenylphosphate (TPP), but not by diethylmaleate (DEM). Activities of p-nitroanisole O-demethylase (ODM) developed to 8.26-fold compared with the parental strain, but no obvious changes were found in activities of carboxyl esterase (CarE) and glutathione-S-transferase (GST). The results indicated that resistance to spinosad in the cotton bollworm might be associated with an increase in cytochrome P450 monooxygenase. [Copyright &y& Elsevier]

The potential capacity of a soil methyl parathion-degrading bacterium strain, Ochrobactrum sp. B2, for degrading various aromatic compounds were investigated. The results showed B2 was capable of degrading diverse aromatic compounds, but amino-substituted benzene compounds, at a concentration up to 100 mg L-1 in 4 days. B2 could use 4-nitrocatechol (4-NC) as a sole carbon and energy source with release of nitrite ion. The pathway for 4-NC degradation via 1,2,4-benzenetriol (BT) and hydroquinone (HQ) formation in B2 was proposed based on the identification and quantification of intermediates by gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatography (HPLC). Degradation studies carried out on a plasmid-cured derivative showed that the genes for 4-NC degradative pathway was plasmid-borne in B2, suggesting that B2 degrades both p-nitrophenol and 4-NC by enzymes encoded by genes on the same plasmid. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China. [Copyright &y& Elsevier]

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100mgl−1) was investigated in this study. The greatest growth of B2 was observed at a temperature of 30°C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation. [Copyright &y& Elsevier]

We report a transposase gene (tnpA) upstream of the opdA phosphotriesterase gene of Agrobacterium radiobacter P230, as well as inverted repeats indicative of insertion sequences, flanking the two genes. Both the tnpA gene and the inverted repeats resemble the Tn610 transposon from Mycobacterium fortuitum. Two additional putative open reading frames separate opdA and tnpA with inferred translation products with similarity to two proteins encoded on the Geobacillus stearothermophilus IS5376 transposon. To test the proposition that these genes were contained on a transposon, an artificial composite transposon was constructed. This artificial transposon was then delivered into Escherichia coli DH10β cells. Transposition was demonstrated by the presence of opdA on the E. coli chromosome and confirmation of insertion by inverse polymerase chain reaction. The data presented suggest a possible role of transposition in the distribution of the opd/opdA genes across a wide range of soil bacteria. [Copyright &y& Elsevier]

Helicoverpa armigera and H. assulta are two of the few insects that can feed on hot pepper fruits. Capsaicin and dihydrocapsaicin (i.e. , capsaicinoids) are the principal pungent compounds in hot peppers. To explore possible molecular mechanisms of adaptation that allow these two species to consume capsaicinoids, the capacity of the three detoxification tissues (fat body, midgut, and Malpighian tubule) of the two pests, to metabolically degrade capsaicin and dihydrocapsaicin, was compared. The results showed that capsaicin and dihydrocapsaicin were metabolized by crude enzyme preparations from all three tissues of the two pests. Five metabolites of capsaicin, and five metabolites of dihydrocapsaicin were identified. Tissue and species differences in the degree of capsaicin and dihydrocapsaicin metabolism were observed. The specialist H. assulta had an overall greater capacity to degrade the capsaicinoids compared to the generalist H. armigera. Further, the midgut was the most significant contributor to capsaicinoid metabolism. The notably high specific activity in Malpighian tubules of H. armigera also further highlights the significance of this organ in xenobiotic detoxification. Alkyl hydroxylation and dehydrogenation were the main pathways for the oxidative biotransformation of both capsaicin and dihydrocapsaicin by cytochrome P450s. This study provides evidence that enhanced metabolic decomposition of capsaicinoids may be an adaptation explaining dietary preferences for Capsicum fruits by these two pests. Unlabelled Image • Helicoverpa assulta and H. armigera eat peppers, but mechanisms that allow these pests to tolerate capsaicinoids are unknown. • Species- and tissue-selective differences in capsaicinoid degradation capacity were observed. • The midgut was the most significant contributor in capsaicinoid metabolism for both pests. • Helicoverpa assulta exhibited greater total metabolic capacity than H. armigera. • Enhanced capsaicinoid metabolism may contribute to reported fitness advantages for the specialist H. assulta. [ABSTRACT FROM AUTHOR]

Capsaicin (8-methyl- N -vanillyl-6-nonenamide) is the most abundant capsaicinoids found in hot peppers (Capsicum annum and Capsicum frutescens). It has been well documented that capsaicin plays an important role in the defense against the attack of herbivores or pathogens on Capsicum plants. A few insect herbivores such as Helicoverpa armigera and Helicoverpa assulta have been recorded to be capable of feeding on hot pepper fruits, suggesting that these insects evolve mechanisms against the toxicity of capsaicin. Although cytochrome P450-meidated detoxification is considered to be an important mechanism by which cotton bollworms cope with capsaicin, experimental evidence is lacking. In this study, we compared the capacity of four H. armigera P450s (CYP6B6, CYP9A12, CYP9A14 and CYP9A17) in capsaicin metabolism, and the capsaicin metabolites were screened and tentatively identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). HPLC analyses showed that depletion rates of capsaicin were 21.9 ± 0.1, 11.9 ± 1.5, 16.3 ± 1.4 and 14.8 ± 0.2 min−1 for CYP6B6, CYP9A12, CYP9A14 and CYP9A17 respectively. The transformation of capsaicin was inhibited by the P450 inhibitor piperonyl butoxide. A total of seven products were detected, and hydroxylation (aromatic and aliphatic) and dehydrogenation were found to be two main pathways in capsaicin metabolism. In addition, capsaicin metabolism was enzyme selective: M1 (ω-hydroxylated N-macrocyclic metabolite) and M3 (ω-hydroxylated metabolite) were uniquely detected in the CYP6B6 catalytic reaction, while M4 (ω-n hydroxylated capsaicin), M5 (diene of capsaicin) and M6 (doubly oxidized metabolite of dehydrogenated capsaicin) were only detectable in CYP9A metabolisms. A capsaicin dimer (5, 5′-dicapsaicin) was found to be the major metabolite of CYP9A reactions, but the minor product produced by CYP6B6. An overall more similar behavior in capsaicin metabolism was observed among CYP9As than between CYP6B6 and CYP9As. Our data demonstrate that CYP6B6 and CYP9As have a potent capability to transform capsaicin, and individual P450 produce unique metabolite profile. These findings help us to understand the molecular basis of capsaicin adaptation in H. armigera. Unlabelled Image • Capsaicin can be efficiently metabolized by HaCYP6B6 and HaCYP9A. • Both HaCYP6B6 and HaCYP9A generate multiple metabolites. • Metabolite profiles are enzyme-dependent in P450- mediated capsaicin metabolism. • Hydroxylation and dehydrogenation are two major pathways in capsaicin biotransformation. • More similar behavior occurs among CYP9As than between CYP6B and CYP9A. [ABSTRACT FROM AUTHOR]

The cotton bollworm, Helicoverpa armigera , is a polyphagous pest that has a strong capacity to evolve resistance against various classes of insecticides. Cytochrome P450 enzymes have been suspected involved in pyrethroid metabolism and resistance in this pest. However, how many and which P450s are involved in pyrethroid metabolism is largely unknown. In this study, CYP6B6 and NADPH-cytochrome P450 reductase (HaCPR) from H. armigera were successfully co-expressed in Escherichia coli . Incubation of esfenvalerate with the recombinant CYP6B6-HaCPR monooxygenase complex revealed that CYP6B6 was able to transform esfenvalerate into 4′-hydroxy fenvalerate. Kcat and Km values for the formation of 4′-hydroxyfenvalerate by the E. coli -produced CYP6B6 were determined to be 1.65 ± 0.11 min − 1 and 4.10 ± 0.84 μM respectively. Our results demonstrate that CYP6B6 has the ability to hydroxylate esfenvalerate, thus plays a role in fenvalerate detoxification. [ABSTRACT FROM AUTHOR]

The house fly (Musca domestica L.) (Diptera: Muscidae) is a global vector that can transmit >250 human and animal diseases. The control of house flies has heavily relied on the application of various chemical insecticides. The carbamate insecticide propoxur has been widely used for the control of house flies, and resistance to propoxur has been documented in many house fly populations worldwide. Previous studies have identified several propoxur resistance-conferring mutations in the target protein acetylcholinesterase; however, the molecular basis for metabolic resistance to propoxur remains unknown. In this study, we investigated the involvement of CYP6G4, a cytochrome P450 overexpressed in many insecticide resistant populations of Musca domestica , in propoxur metabolism and resistance by using combined approaches of recombinant protein-based insecticide metabolism and the Drosophila GAL4/UAS transgenic system. The recombinant CYP6G4 and its redox partners (NADPH-dependent cytochrome P450 reductase and cytochrome b 5) were functionally expressed in Escherichia coli. Metabolism experiments showed that CYP6G4 was able to transform propoxur with a turnover rate of around 0.79 min−1. Six metabolites were putatively identified, suggesting that CYP6G4 could metabolize propoxur via hydroxylation, O-depropylation and N -demethylation. Moreover, bioassay results showed that ectopic overexpression of CYP6G4 in fruit flies significantly increased their tolerance to propoxur. Our in vivo and in vitro data convincingly demonstrate that CYP6G4 contributes to propoxur metabolism and resistance. [Display omitted] • The recombinant CYP6G4 and its redox partners were successfully produced in Escherichia coli • The CYP6G4 enzyme is able to metabolize propoxur and generate multiple metabolites • Hydroxylation and O-depropylation are two major pathways in propoxur biotransformation • Ectopic overexpression of CYP6G4 in fruit flies increases tolerance to propoxur [ABSTRACT FROM AUTHOR]

Abstract: A complete cDNA encoding the NADPH–cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis–Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50 =1.69μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90μM) was also observed. [Copyright &y& Elsevier]