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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]

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]

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 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]

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]

Aflatoxin B 1 (AFB 1), a naturally-occurring mycotoxin, can cause severe toxicological and carcinogenic effects in livestock and humans. Given that the chicken is one of the most important food-producing animals, knowledge regarding AFB 1 metabolism and enzymes responsible for AFB 1 transformation in the chicken has important implications for chicken production and food safety. Previously, we have successfully expressed chicken CYP1A5 and CYP3A37 monooxygenases in E. coli , and reconstituted them into a functional CYP system consisting of CYP1A5 or CYP3A37, CPR and cytochrome b 5. In this study, we aimed to investigate the roles of CYP1A5 and CYP3A37 in the bioconversion of AFB 1 to AFM 1. Our results showed that chicken CYP1A5 was able to hydroxylate AFB 1 to AFM 1. The formation of AFM 1 followed the typical Michaelis–Menten kinetics. The kinetics parameters of V max and K m were determined as 0.83 ± 0.039 nmol/min/nmol P450 and 26.9 ± 4.52 μM respectively. Docking simulations further revealed that AFB 1 adopts a "side-on" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom and the production of AFM 1. On the other hand, AFB 1 assumes a "face-on" conformation in chicken CYP3A37, leading to the displacement of the C9a atom from the heme iron and explaining the lack of AFM 1 hydroxylation activity. The results demonstrate that chicken CYP1A5 possesses efficient hydroxylase activity towards AFB 1 to form AFM 1. [Display omitted] • E. coli expressed chicken CYP1A5 is able to hydroxylate aflatoxin B 1 to aflatoxin M 1. • The formation of aflatoxin M 1 followed typical Michaelis–Menten kinetics. • Chicken CYP1A5 is an efficient enzyme catalyzing aflatoxin B 1 hydroxylation. • AFB 1 adopts a "side-on" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom. [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]

Insect cuticular hydrocarbons (CHCs) have dual functions as physical barrier and chemical signals. The last step of CHC biosynthesis is known to be catalyzed by cytochrome P450 CYP4G in a number of insects. Until recently, studies on CYP4Gs in the context of functional evolution are rare. In this study, we analyzed sequence similarity and temporal-spatial expression patterns of the five CYP4G genes in the cotton bollworm Helicoverpa armigera , an important agricultural pest and also typical representative of lepidopteran insects. Moreover, the CRISPR/Cas9-induced knockout was used to clarify the roles of the five CYP4Gs in CHC biosynthesis. Temporal-spatial expression patterns revealed that CYP4G8 was highly expressed at all developmental stages and in most tissues examined. Larvae with CYP4G8 knocked out could not produce methyl-branched CHCs and failed to pupate, while larvae with the other four CYP4G genes knocked out (4G1-type-KO) showed no significant changes in their CHC profiles, weight gain and survival. Comparative transcriptomics revealed that knocking out CYP4G8 affected the global gene expression in larvae, especially down-regulated the expression of genes in the fatty acid biosynthetic pathway, while no significant change in 4G1-type-KO transcriptome was observed. These findings indicate that the five members of the CYP4G subfamily have undergone functional divergence: CYP4G8 maintains the essential function in CHC biosynthesis, while the function of the other four CYP4G genes remains unclear. Intriguingly, CYP4G8 has evolved to be a P450 enzyme responsible for the synthesis of larval methyl-branched hydrocarbons. The observation that CYP4G8 knockout is lethal strongly suggest that CYP4G8 may serve as a candidate target for the development of insecticidal agents for the control of cotton bollworms. [Display omitted] • CRISPR/Cas9 was used to characterize the function of five CYP4G genes from Helicoverpa armigera. • CYP4G paralogs diverge in spatio-temporal expression and function. • CYP4G8, rather than the other four CYP4Gs, is the major player in cuticular hydrocarbon biosynthesis. • CYP4G8 knockout is lethal. [ABSTRACT FROM AUTHOR]

Abstract: The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (Km, Vmax, Vmax/Km) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower Km of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest Vmax/Km ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (<15%) was observed by erythromycin at a concentration of 500μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken. [Copyright &y& Elsevier]

Abstract: Cytochrome P450 monooxygenases-mediated detoxification is a major mechanism of pyrethroid resistance in house flies. Constitutive overexpression of P450 genes is the main cause for this type of insecticide resistance. In this study, three new P450 genes (CYP6A40, CYP6D8 and CYP6G4) over-expressed in a pyrethroid resistance strain (BJD) were identified. Developmental expression analysis showed that CYP6A40 and CYP6D8 were expressed in all life stages, while CYP6G4 was not transcribed in eggs or larvae. Permethrin resistance and expression level of cytochrome P450s in six house fly populations (the BJD strain and five field-derived house fly populations collected across China) were determined. Compared with the susceptible TJS strain, all the six populations exhibited different levels of resistance to permethrin. Piperonyl butoxide (PBO) reduced permethrin resistance level up to 109-fold, indicating that cytochrome P450-mediated detoxification is responsible for pyrethroid resistance in Chinese house flies. Significant overexpression of more than one of the eight P450 genes (CYP6A5v1, CYP6A5v2, CYP6A36, CYP6A40, CYP6D1, CYP6D3, CYP6D8 and CYP6G4) in the six pyrethroid resistant house fly populations was observed. Differential expression patterns of P450s over-represented in the resistance populations suggested evolutionary plasticity of P450-mediated pyrethroid resistance. CYP6G4 and CYP6D1, commonly over-expressed in resistant populations, were hypothesized to be the important P450s contributing to pyrethroid resistance of Chinese house flies. [Copyright &y& Elsevier]

Abstract: Cytochrome P450 monooxygenases have been well known to be responsible for the synthesis of endogenous compounds and the metabolism of exogenous compounds in almost all living organisms, which require NADPH-cytochrome P450 oxidoreductase (POR) as an electron donor to function. In this study, a 2031bp open reading frame of POR gene was cloned from 35-day-old Roman hen liver, encoding an enzyme of 676 amino acids. Sequence analysis showed that chicken POR shares high homology with other vertebrates PORs and possesses the conserved binding domains of FAD, FMN, and NADPH. The genomic sequences of POR genes from chicken and other four vertebrates have highly conserved exon/intron organization structure. By fusion with bacterial signal peptide, chicken POR gene was functionally expressed in E. coli membrane and showed activities in reduction of cytochrome c and oxidation of NADPH. The Km values for cytochrome c and NADPH were 21.9±2.3μM and 2.4±0.3μM respectively. A Ping-Pong mechanism was proposed for chicken POR. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome P450s play a crucial role in insect adaptation to their host plants and in insecticide resistance through metabolic detoxification. The efficacy of P450 mediated detoxification depends on the levels of P450 transcript which may change after exposure to xenobiotics. In this study, expression response of nine cytochrome P450 genes in Helicoverpa armigera, belonging to CYP6 and CYP9 families, were examined by using quantitative real-time PCR method. Results showed that these cytochrome P450 genes were differentially affected by plant chemicals and synthetic compounds, and displayed tissue-, concentration- and inducer-dependent response. Phenobarbital acted as a general inducer for cytochrome P450s, whereas gossypol specifically increased the expression of CYP6AE14. Xanthotoxin showed moderate induction effect on several of the nine genes. Deltamethrin displayed significantly both induction and suppression impact on the expression of cytochrome P450s. These findings provide clues for further elucidating the function and regulation mechanism of these cytochrome P450 genes in H. armigera, a pest responsible for severe losses in many crops. [Copyright &y& Elsevier]

The cotton bollworm, Helicoverpa armigera , is a polyphagous pest threatening many economically important crops worldwide. Until recently, synthetic pyrethroids remain in wide use for controlling pest insects including the cotton bollworm. Understanding the metabolic mechanism of pyrethroids in a given pest can provide significant implication for a smart choice of insecticides, and such information is useful for the development of novel selective and safe insecticides. In this study, we used complexes of recombinant H. armigera cytochrome P450 CYP9A and NADPH-dependent cytochrome P450 reductase to investigate the capacity of three CYP9A paralogs in the transformation of seven structurally different pyrethroids by metabolism assays. The results showed that the three paralogous CYP9As were able to metabolize multiple pyrethroids. Interestingly, all the three CYP9As transformed pyrethrin-resembling pyrethroids (e.g. bioallethrin) more efficiently than the heavily modified ones (e.g. bifenthrin). These findings suggest that herbivorous insects can cope with synthetic insecticides using their physiological systems that initially evolved to survive exposure to the defensive chemicals in their host plants, adding support to the pre-adaptation hypothesis. [Display omitted] • CYP9As have the capacity of metabolizing pyrethroids with varying chemical modifications. • CYP9As possess much higher activity towards pyrethrin-resembling bioallethrin than the heavily modified bifenthrin. • Herbivorous insects can cope with synthetic insecticides using long-term evolved physiological systems. [ABSTRACT FROM AUTHOR]