Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

Pinpointing P450s associated with pyrethroid metabolism in the dengue vector, Aedes aegypti: developing new tools to combat insecticide resistance

Background

Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers.

Methodology/Principal Findings

We have expressed CYP9J32 in Escherichia coli and show that the enzyme can metabolize the pyrethroids permethrin and deltamethrin. In addition, three other Ae. aegypti P450s (CYP9J24, CYP9J26, CYP9J28) were found capable of pyrethroid metabolism, albeit with lower activity. Both Ae. aegypti and Anopheles gambiae P450s (CYP''s 6M2, 6Z2, 6P3) were screened against fluorogenic and luminescent substrates to identify potential diagnostic probes for P450 activity. Luciferin-PPXE was preferentially metabolised by the three major pyrethroid metabolisers (CYP9J32, CYP6M2 and CYP6P3), identifying a potential diagnostic substrate for these P450s.

Conclusions/Significance

P450s have been identified with the potential to confer pyrethroid resistance in Ae.aegypti. It is recommended that over expression of these enzymes should be monitored as indicators of resistance where pyrethroids are used.     

Field-Caught Permethrin-Resistant Anopheles gambiae Overexpress CYP6P3, a P450 That Metabolises Pyrethroids

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non–alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.     

The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis

Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control.Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre.The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools.     

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background

The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.

Methodology/Principal Findings

A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F₁ adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.

Conclusion

This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.     

Insecticide resistance status and mechanisms in Aedes aegypti populations from Senegal

Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal.Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4).All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.     

High Level of Pyrethroid Resistance in an Anopheles funestus Population of the Chokwe District in Mozambique

Background

Although Anopheles funestus is difficult to rear, it is crucial to analyse field populations of this malaria vector in order to successfully characterise mechanisms of insecticide resistance observed in this species in Africa. In this study we carried out a large-scale field collection and rearing of An. funestus from Mozambique in order to analyse its susceptibility status to insecticides and to broadly characterise the main resistance mechanisms involved in natural populations.

Methodology/Principal Findings

3,000 F₁ adults were obtained through larval rearing. WHO susceptibility assays indicated a very high resistance to pyrethroids with no mortality recorded after 1h30min exposure and less than 50% mortality at 3h30min. Resistance to the carbamate, bendiocarb was also noted, with 70% mortality after 1h exposure. In contrast, no DDT resistance was observed, indicating that no kdr-type resistance was involved. The sequencing of the acetylcholinesterase gene indicated the absence of the G119S and F455W mutations associated with carbamate and organophosphate resistance. This could explain the absence of malathion resistance in this population. Both biochemical assays and quantitative PCR implicated up-regulated P450 genes in pyrethroid resistance, with GSTs playing a secondary role. The carbamate resistance observed in this population is probably conferred by the observed altered AChE with esterases also involved.

Conclusion/Significance

The high level of pyrethroid resistance in this population despite the cessation of pyrethroid use for IRS in 1999 is a serious concern for resistance management strategies such as rotational use of insecticides. As DDT has now been re-introduced for IRS, susceptibility to DDT needs to be closely monitored to prevent the appearance and spread of resistance to this insecticide.     

Pyriproxyfen is metabolized by P450s associated with pyrethroid resistance in An. gambiae

Pyrethroid resistance is widespread in the malaria vector Anopheles gambiae leading to concerns about the future efficacy of bednets with pyrethroids as the sole active ingredient. The incorporation of pyriproxyfen (PPF), a juvenile hormone analogue, into pyrethroid treated bednets is being trialed in Africa. Pyrethroid resistance is commonly associated with elevated levels of P450 expression including CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 6Z2 and 9J5. Having expressed these P450s in E. coli we find all are capable of metabolizing PPF. Inhibition of these P450s by permethrin, deltamethrin and PPF was also examined. Deltamethrin and permethrin were moderate inhibitors (IC₅₀ 1–10 μM) of diethoxyfluorescein (DEF) activity for all P450s apart from CYP6Z2 (IC₅₀ > 10 μM), while PPF displayed weaker inhibition of all P450s (IC₅₀ > 10 μM) except CYP's 6Z2 and 6P2 (IC₅₀ 1–10 μM). We found evidence of low levels of cross resistance between PPF and other insecticide classes by comparing the efficacy of PPF in inhibiting metamorphosis and inducing female sterility in an insecticide susceptible strain of An. gambiae and a multiple resistant strain from Cote d’Ivoire.     

Current Perspectives on Plague Vector Control in Madagascar: Susceptibility Status of Xenopsylla cheopis to 12 Insecticides

Plague is a rodent disease transmissible to humans by infected flea bites, and Madagascar is one of the countries with the highest plague incidence in the world. This study reports the susceptibility of the main plague vector Xenopsylla cheopis to 12 different insecticides belonging to 4 insecticide families (carbamates, organophosphates, pyrethroids and organochlorines). Eight populations from different geographical regions of Madagascar previously resistant to deltamethrin were tested with a World Health Organization standard bioassay. Insecticide susceptibility varied amongst populations, but all of them were resistant to six insecticides belonging to pyrethroid and carbamate insecticides (alphacypermethrin, lambdacyhalothrin, etofenprox, deltamethrin, bendiocarb and propoxur). Only one insecticide (dieldrin) was an efficient pulicide for all flea populations. Cross resistances were suspected. This study proposes at least three alternative insecticides (malathion, fenitrothion and cyfluthrin) to replace deltamethrin during plague epidemic responses, but the most efficient insecticide may be different for each population studied. We highlight the importance of continuous insecticide susceptibility surveillance in the areas of high plague risk in Madagascar.     

Re-Visiting Insecticide Resistance Status in Anopheles gambiae from C?te d'Ivoire: A Nation-Wide Informative Survey

Insecticide resistance constitutes a major threat that may undermine current gain in malaria control in most endemic countries. National Malaria Control Programmes (NMCPs) need as much information as possible on the resistance status of malaria vectors and underlying mechanisms in order to implement the most relevant and efficient control strategy. Bioassays, biochemical and molecular analysis were performed on An. gambiae collected in six sentinel sites in Côte d''Ivoire. The sites were selected on the basis of their bioclimatic status and agricultural practices. An. gambiae populations across sites showed high levels of resistance to organochloride, pyrethroid and carbamate insecticides. The kdr and ace-1^R mutations were detected in almost all sentinel sites with mosquitoes on the coastal and cotton growing areas mostly affected by these mutations. At almost all sites, the levels of detoxifying enzymes (mixed-function oxidases (MFOs), non-specific esterases (NSE) and glutathione-S-transferases (GSTs)) in An. gambiae populations were significantly higher than the levels found in the susceptible strain Kisumu. Pre-exposure of mosquitoes to PBO, an inhibitor of MFOs and NSEs, significantly increased mortality rates to pyrethroids and carbamates in mosquitoes but resistance in most cases was not fully synergised by PBO, inferring a residual role of additional mechanisms, including kdr and ace-1 site insensitivity. The large distribution of resistance in Côte d''Ivoire raises an important question of whether to continue to deploy pyrethroid-based long-lasting insecticidal nets (LLINs) and insecticide residual spraying (IRS) towards which resistance continues to rise with no guarantee that the level of resistance would not compromise their efficacy. Innovative strategies that combine insecticide and synergists in LLINs or spatially LLIN and an effective non-pyrethroid insecticide for IRS could be in the short term the best practice for the NMCP to manage insecticide resistance in malaria vectors in Côte d''Ivoire and other endemic countries facing resistance.     

Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa)

An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G116S mutation in An. arabiensis populations collected at 8 sites. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito Anopheles gambiae

Mosquitoes exhibit ∼24 h rhythms in physiology and behavior, regulated by the cooperative action of an endogenous circadian clock and the environmental light:dark cycle. Here, we characterize diel (observed under light:dark conditions) time-of-day changes in metabolic detoxification and resistance to insecticide challenge in Anopheles gambiae mosquitoes. A better understanding of mosquito chronobiology will yield insights into developing novel control strategies for this important disease vector. We have previously identified >2000 rhythmically expressed An. gambiae genes. These include metabolic detoxification enzymes peaking at various times throughout the day. Especially interesting was the identification of rhythmic genes encoding enzymes capable of pyrethroid and/or DDT metabolism (CYP6M2, CYP6P3, CYP6Z1, and GSTE2). We hypothesized that these temporal changes in gene expression would confer time-of-day specific changes in metabolic detoxification and responses to insecticide challenge. An. gambiae mosquitoes (adult female Pimperena and Mali-NIH strains) were tested by gene expression analysis for diel rhythms in key genes associated with insecticidal resistance. Biochemical assays for total GST, esterase, and oxidase enzymatic activities were undertaken on time-specific mosquito head and body protein lysates. To determine for rhythmic susceptibility to insecticides by survivorship, mosquitoes were exposed to DDT or deltamethrin across the diel cycle. We report the occurrence of temporal changes in GST activity in samples extracted from the body and head with a single peak at late-night to dawn, but no rhythms were detected in oxidase or esterase activity. The Pimperena strain was found to be resistant to insecticidal challenge, and subsequent genomic analysis revealed the presence of the resistance-conferring kdr mutation. We observed diel rhythmicity in key insecticide detoxification genes in the Mali-NIH strain, with peak phases as previously reported in the Pimperena strain. The insecticide sensitive Mali-NIH strain mosquitoes exhibited a diel rhythm in survivorship to DDT exposure and a bimodal variation to deltamethrin challenge. Our results demonstrate rhythms in detoxification and pesticide susceptibility in An. gambiae mosquitoes; this knowledge could be incorporated into mosquito control and experimental design strategies, and contributes to our basic understanding of mosquito biology.     

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed

Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is a major threat to malaria control programmes. Cytochome P450-mediated detoxification is an important resistance mechanism. CYP6M2 is over-expressed in wild populations of permethrin resistant A. gambiae but its role in detoxification is not clear. CYP6M2 was expressed in Escherichia coli and a structural model was produced to examine its role in pyrethroid metabolism. Both permethrin and deltamethrin were metabolized. Rates were enhanced by A. gambiae cytochrome b₅ with kinetic parameters of K_M = 11 ± 1 ??M and k_cat = 6.1 ± 0.4 per min for permethrin (1:1 cis-trans) and K_M = 2.0 ± 0.3 ??M and k_cat = 1.2 ± 0.1 per min for deltamethrin. Mass spectrometry and NMR analysis identified 4′-hydroxy deltamethrin and hydroxymethyl deltamethrin as major and minor deltamethrin metabolites respectively. Secondary breakdown products included cyano(3-hydroxyphenyl)methyl deltamethrate and deltamethric acid. CYP6M2 was most highly transcribed in the midgut and Malpighian tubules of adult A. gambiae, consistent with a role in detoxification. Our data indicates that CYP6M2 plays an important role in metabolic resistance to pyrethroids and thus an important target for the design of new tools to combat malaria.