Functional characterization of Helicoverpa assulta CYP6B6 in insecticide metabolism.

The oriental tobacco budworm Helicoverpa assulta (Lepidoptera: Noctuidae) is a specialist pest that may cause serious damages to important crops such as chili pepper and tobacco. Various man-made insecticides have been applied to control the infestation of this pest. To understand how this pest copes with insecticides, it is required to identify key players involved in insecticide transformation. In this study, a P450 gene of CYP6B subfamily was identified in the oriental tobacco budworm, and its expression pattern was revealed. Moreover, the activities of HassCYP6B6 against 12 insecticides were explored using recombinant enzymes produced in the facile Escherichia coli. Data from metabolic experiments showed that HassCYP6B6 was able to metabolize conventional insecticides including organophosporates (diazinon, malathion, phoxim), carbamate propoxur, and pyrethroid esfenvalerate, while no significant metabolism was observed towards new-type pesticides such as neonicotinoids (acetamiprid, imidacloprid), diamides (chlorantraniliprole, cyantraniliprole), macrocyclic lactone (emamectin benzoate, ivermectin), and metaflumizone. Structures of metabolites were proposed based on mass spectrometry analyses. The results demonstrate that HassCYP6B6 plays important roles in the transformation of multiple insecticides via substrate-dependent catalytic mechanisms including dehydrogenation, hydroxylation and oxidative desulfurization. The findings have important applied implications for the usage of insecticides.

Insecticide Susceptibility and KDR Mutations in Aedes Albopictus Collected from Seven Districts of Guangyuan city, Northern Sichuan, China.

The Asian tiger mosquito, Aedes albopictus, is an important vector of chikungunya, dengue, yellow fever, and Zika viruses. Vector control remains an important means for the prevention and control of vector-borne diseases. The development of insecticide resistance has become a serious threat to the efficacy of insecticide-based control programs. To understand the resistance status and the underlying genetic mechanism in mosquitoes in Guangyuan City of Sichuan Province, China, we investigated the susceptibility of Ae. albopictus to four commonly used insecticides. We found that all the examined populations were susceptible to malathion and propoxur. However, Ae. albopictus populations in Guangyuan showed a possible resistance to the two tested pyrethroids (beta-cypermethrin and deltamethrin). Notably, phenotypic resistance to deltamethrin was detected in 2 of the 7 populations. The potential of resistance to pyrethroids was confirmed by the presence of knockdown resistance (kdr) related mutations in the voltage-gated sodium channel. Four kdr mutations (V1016G, I1532T, F1534L, and F1534S) were identified to be present alone or in combination, and their distribution displayed significant spatial heterogeneity. These findings are helpful for making evidence-based mosquito control strategies and highlight the need to regularly monitor the dynamics of pyrethroid resistance in this city.

Chicken CYP1A5 is able to hydroxylate aflatoxin B1 to aflatoxin M1.

Aflatoxin B1 (AFB1), 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 AFB1 metabolism and enzymes responsible for AFB1 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 b5. In this study, we aimed to investigate the roles of CYP1A5 and CYP3A37 in the bioconversion of AFB1 to AFM1. Our results showed that chicken CYP1A5 was able to hydroxylate AFB1 to AFM1. The formation of AFM1 followed the typical Michaelis-Menten kinetics. The kinetics parameters of Vmax and Km were determined as 0.83 ± 0.039 nmol/min/nmol P450 and 26.9 ± 4.52 µM respectively. Docking simulations further revealed that AFB1 adopts a "side-on" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom and the production of AFM1. On the other hand, AFB1 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 AFM1 hydroxylation activity. The results demonstrate that chicken CYP1A5 possesses efficient hydroxylase activity towards AFB1 to form AFM1.

Up-regulation of CYP6G4 mediated by a CncC/maf binding-site-containing insertion confers resistance to multiple classes of insecticides in the house fly Musca domestica.

Populations of many insect species have evolved a variety of resistance mechanisms in response to insecticide selection. Current knowledge about mutations responsible for insecticide resistance is largely achieved from studies on target-site resistance, while much less is known about metabolic resistance. Although it is well known that P450 monooxygenases are one of the major players involved in insecticide metabolism and resistance, understanding mutation(s) responsible for CYP-mediated resistance has been a big challenge. In this study, we used the house fly to pursue a better understanding of P450 mediated insecticide resistance at the molecular level. Metabolism studies illustrated that CYP6G4 had a broad-spectrum metabolic activity in metabolizing insecticides. Population genotyping revealed that the CYP6G4v1 allele harboring a DNA insertion (MdIS1) had been selected in many house fly populations on different continents. Dual luciferase reporter assays identified that the MdIS1 contained a CncC/Maf binding site, and electrophoretic mobility shift assay confirmed that transcription factor CncC was involved in the MdIS1-mediated regulation. This study highlights the common involvement of the CncC pathway in adaptive evolution, and provides an interesting case supportive of parallel evolution in P450-mediated insecticide resistance in insects.

Absence of known knockdown resistance mutations but fixation of CYP337B3 was detected in field populations of Helicoverpa armigera across China.

The cotton bollworm (Helicoverpa armigera) is a worldwide agricultural pest that infests many important crops. Pyrethroids targeting the voltage-gated sodium channel (VGSC) have been long used in the control of the cotton bollworm. Two amino acid substitutions (D1561V and E1565G) in H. armigera VGSC (HaVGSC) and the presence of a chimeric P450 gene (CYP337B3) have been documented to be associated with pyrethroid resistance. To understand the current occurrence of kdr mutations and the CYP337B3 gene in Chinese H. armigera populations, high-throughput amplicon sequencing was adopted to detect potential nucleotide variations in three fragments of the VGSC gene that cover 10 reported knockdown resistance (kdr) sites in insects, and gene-specific PCR was performed to examine the presence of CYP337B3 gene in H. armigera samples collected across China. The nucleotide variation analysis revealed a wealth of nucleotide variations in not only exons but also introns in the VGSC gene in Chinese H. armigera populations. However, neither previously reported kdr-conferring amino acid replacements nor other non-synonymous mutations were observed in a total of 1439 examined individuals. Population genetic analysis suggested that the H. armigera population in Nanchang, Jiangxi Province (JNC) had a moderate genetic differentiation from other populations, while no significant divergence was observed in other populations in northern and northwestern China. The CYP337B3 was present in all the examined individuals, indicating that CYP337B3 is extensively fixed in H. armigera populations across China. These results support that point mutations in VGSC are not a major factor involved in the current pyrethroid resistance in H. armigera. Instead, CYP337B3 plays a prevalent role in the development of resistance to pyrethroids in H. armigera.

Detection of Target Site Mutations in the Acetylcholinesterase and Voltage-Gated Sodium Channel in Field Populations of Culex quinquefasciatus and Cx. tritaeniorhynchus From Southern Sichuan Region of China.

Culex quinquefasciatus and Cx. tritaeniorhynchus are 2 dominant disease vectors in Neijiang City, Sichuan Province, China. Although there is evidence of confirmed resistance against insecticides in mosquito vectors, nothing is known about the existing insecticide resistance-conferring mutations in Cx. quinquefasciatus and Cx. tritaeniorhynchu in this region so far. In this study, the G119S mutation in the acetylcholinesterase (AChE) was detected in Cx. quinquefasciatus at a very low frequency (0.9%) with no resistant homozygotes being observed. Two resistance mutations in the voltage-gated sodium channel (VGSC) (L1014F and L1014S) were found in Cx. quinquefasciatus with frequencies of 88.7% and 8.3%, respectively. By contrast, the AChE F455W mutation was found to be fixed (with a frequency of 100%) in 3 of the 5 studied populations, with an overall frequency being 98.1%. In addition, 1 resistance-conferring VGSC mutation (L1014F) was detected with an overall frequency of 15.2% in Cx. tritaeniorhynchus. These results indicate that the well-recognized insecticide resistance-conferring mutations in both AChE and VGSC are present in the 2 Culex species in Neijiang. The contrasting patterns in the frequency of resistance alleles indicate that species-customized strategies of insecticide resistance management should be considered for the 2 species.

Functional characterization of CYP6G4 from the house fly in propoxur metabolism and resistance.

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 b5) 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.

CYP4G8 is responsible for the synthesis of methyl-branched hydrocarbons in the polyphagous caterpillar of Helicoverpa armigera.

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.

First Report of the L993S Mutation in the Voltage-Gated Sodium Channel in Field Populations of the German Cockroach Blattella germanica.

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.

Functional characterization of a novel, highly expressed ion-driven sugar antiporter in the thoracic muscles of Helicoverpa armigera.

Sugar transporters (STs), which mainly mediate cellular sugar exchanges, play critical physiological roles in living organisms, and they may be responsible for sugar exchanges among various insect tissues. However, the molecular and physiological functions of insect STs are largely unknown. Here, 16 STs of Helicoverpa armigera were identified. A phylogenetic analysis classified the putative HaSTs into 12 sub-families, and those identified in this study were distributed into 6 sub-families. Real-time polymerase chain reaction indicated that the 16 HaSTs had diverse tissue-specific expression levels. One transporter, HaST10, was highly expressed in thoracic muscles. A functional study using a Xenopus oocyte expression system revealed that HaST10 mediated both H+ -driven trehalose and Na+ -driven glucose antiport activities with high transport efficiency and low affinity levels. A HaST10 knockout clearly impaired the performance of H. armigera. Thus, HaST10 may participate in sugar-supply regulation and have essential physiological roles in H. armigera.

Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach.

BACKGROUND: Anopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China. METHODS: To understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China. RESULTS: Nineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F. CONCLUSIONS: A cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis.

Multiple Genetic Mutations Related to Insecticide Resistance are Detected in Field Kazakhstani House Flies (Muscidae: Diptera).

The house fly (Musca domestica Linnaeus) is an important disease vector. Insecticide resistance is an obstacle to effective house fly control. Previous studies have demonstrated that point mutations in acetylcholinesterase (Ace), carboxylesterase (MdαE7) and voltage-sensitive sodium channel (Vssc), and over-expression of CYP6D1v1 confer insecticide resistance in the house fly. However, information about the status and underlying mechanisms of insecticide resistance in Kazakhstani house flies is lacking. In this study, we investigated the occurrence of genetic mutations associated with insecticide resistance in field house flies collected at six different locations in southern Kazakhstan. Four mutations (V260L, G342A/V, and F407Y) in Ace and three mutations (G137D and W251L/S) in MdαE7 were detected with appreciable frequencies. Notably, haplotypes carrying triple-loci mutations in Ace and double mutations in MdαE7 were found in Kazakhstan. The L1014H and L1014F mutations in Vssc, and CYP6D1v1 resistance allele were detected at a low frequency in some of the six investigated house fly populations. Phylogenetic analyses of haplotypes supported multiple origins of resistance mutations in Ace and MdαE7. These observations suggest that house flies in southern Kazakhstan may exhibit significant resistance to organophosphates and carbamates. Regular monitoring of insecticide resistance is recommended to achieve effective house fly control by chemical agents in southern Kazakhstan.

Pyrethrin-resembling pyrethroids are metabolized more readily than heavily modified ones by CYP9As from Helicoverpa armigera.

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.

A survey of insecticide resistance-conferring mutations in multiple targets in Anopheles sinensis populations across Sichuan, China.

BACKGROUND: Sichuan province is located in the southwest of China, and was previously a malaria-endemic region. Although no indigenous malaria case has been reported since 2011, the number of imported cases is on the rise. Insecticide-based vector control has played a central role in the prevention of malaria epidemics. However, the efficacy of this strategy is gravely challenged by the development of insecticide resistance. Regular monitoring of insecticide resistance is essential to inform evidence-based vector control. Unfortunately, almost no information is currently available on the status of insecticide resistance and associated mechanisms in Anopheles sinensis, the dominant malaria vector in Sichuan. In this study, efforts were invested in detecting the presence and frequency of insecticide resistance-associated mutations in three genes that encode target proteins of several classes of commonly used insecticides. METHODS: A total of 446 adults of An. sinensis, collected from 12 locations across Sichuan province of China, were inspected for resistance-conferring mutations in three genes that respectively encode acetylcholinesterase (AChE), voltage-gated sodium channel (VGSC), and GABA receptor (RDL) by DNA Sanger sequencing. RESULTS: The G119S mutation in AChE was detected at high frequencies (0.40-0.73). The predominant ace-1 genotype was GGC/AGC (119GS) heterozygotes. Diverse variations at codon 1014 were found in VGSC, leading to three different amino acid substitutions (L1014F/C/S). The 1014F was the predominant resistance allele and was distributed in all 12 populations at varying frequencies from 0.03 to 0.86. The A296S mutation in RDL was frequently present in Sichuan, with 296SS accounting for more than 80% of individuals in six of the 12 populations. Notably, in samples collected from Chengdu (DJY) and Deyang (DYMZ), almost 30% of individuals were found to be resistant homozygotes for all three targets. CONCLUSIONS: Resistance-related mutations in three target proteins of the four main classes of insecticides were prevalent in most populations. This survey reveals a worrisome situation of multiple resistance genotypes in Sichuan malaria vector. The data strengthen the need for regular monitoring of insecticide resistance and establishing a region-customized vector intervention strategy.

The double-mutation（M918I + L1014F）kdr allele is fixed in Cimex hemipterus populations in Guangxi, China.

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.

RDL mutations in Guangxi Anopheles sinensis populations along the China-Vietnam border: distribution frequency and evolutionary origin of A296S resistance allele.

BACKGROUND: Malaria is a deadly vector-borne disease in tropical and subtropical regions. Although indigenous malaria has been eliminated in Guangxi of China, 473 confirmed cases were reported in the Northern region of neighbouring Vietnam in 2014. Considering that frequent population movement occurs across the China-Vietnam border and insecticide resistance is a major obstacle in disease vector control, there is a need to know the genotype and frequency of insecticide resistance alleles in Anopheles sinensis populations along the China-Vietnam border and to take action to prevent the possible migration of insecticide resistance alleles across the border. METHODS: Two hundred and eight adults of An. sinensis collected from seven locations in Guangxi along the China-Vietnam border were used in the investigation of individual genotypes of the AsRDL gene, which encodes the RDL gamma-aminobutyric acid (GABA) receptor subunit in An. sinensis. PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis was deployed to genotype codon 345, while direct sequencing of PCR products was conducted to clarify the genotypes for codons 296 and 327 of the AsRDL gene. The genealogical relation of AsRDL haplotypes was analyzed using Network 5.0. RESULTS: Three putative insecticide resistance related mutations (A296S, V327I and T345S) were detected in all the seven populations of An. sinensis in Guangxi along the China-Vietnam border. The resistance-conferring A296S mutation was found to be widely distributed and present at notably high frequencies (78.8% to 100%). Relatively lower frequencies of mutations V327I (26.9% to 53.2%) and T345S (0% to 28.8%) were observed. The V327I or T345S always occurred in the presence of A296S. Evolutionary analysis of 21 AsRDL haplotypes indicated multiple origins of the A296S and V327I mutations. CONCLUSION: The resistance A296S allele was present at high frequencies in the An. sinensis populations along the China-Vietnam border, indicating a risk of resistance to insecticides targeting RDL. The double mutations (A296S + V327I) may have evolved from alleles carrying the A296S mutation by scaffolding the additional mutation V327I, and A296S allele may have multiple evolutionary origins. These findings will help inform strategies for vector control and malaria prevention.

Capsaicinoid metabolism by the generalist Helicoverpa armigera and specialist H. assulta: Species and tissue differences.

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.

First detection of the kdr mutation (L1014F) in the plague vector Xenopsylla cheopis (Siphonaptera: Pulicidae).

BACKGROUND: The oriental rat flea, Xenopsylla cheopis, is the most efficient vector of the plague. Pyrethroid insecticides such as cypermethrin, cyhalothrin and deltamethrin have been often used to limit plague transmission via controlling the vector during outbreaks. However, this strategy is threatened by the development of insecticide resistance. Understanding the mechanisms underlying pyrethroid resistance is the prerequisite for successful flea control. METHODS: Partial DNA sequences of X. cheopis voltage gated sodium channel (VGSC) gene were amplified from a total of 111 individuals, collected from a natural plague epidemic foci in Baise City, Guangxi Zhuang Autonomous Region of China. These DNA fragments were sequenced. The frequency and distribution of kdr mutations were assessed in four X. cheopis populations. The origin of kdr mutations was investigated by phylogenetic and network analysis. RESULTS: The classical knockdown resistance (kdr) mutation (L1014F) was detected in four field populations at frequencies ranging between 0.021-0.241. The mutant homozygote was observed only in one of the four populations. Seven haplotypes were identified, with two of them carrying the resistance L1014F mutation. Phylogenetic tree and network analysis indicated that the L1014F allele was not singly originated. Based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) profiling, an easy-to-use and accurate molecular assay for screening individual fleas for the L1014F mutation was developed. CONCLUSIONS: To our knowledge, this work represents the first report of the L1014F mutation in the plague vector X. cheopis. The incidence of the L1014F allele highlights the need of further studies on the phenotypic effect of this mutation in this plague vector. Early detection and monitoring of insecticide resistance is suggested in order to make effective control strategies in case of plague outbreaks in this region.

Capsaicin is efficiently transformed by multiple cytochrome P450s from Capsicum fruit-feeding Helicoverpa armigera.

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 ±â€¯0.1, 11.9 ±â€¯1.5, 16.3 ±â€¯1.4 and 14.8 ±â€¯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.