A brain-specific cytochrome P450 responsible for the majority of deltamethrin resistance in the QTC279 strain of Tribolium castaneum

Cytochrome P450-mediated detoxification is one of the most important mechanisms involved in insecticide resistance. However, the molecular basis of this mechanism and the physiological functions of P450s associated with insecticide resistance remain largely unknown. Here, we exploited the functional genomics and reverse genetic approaches to identify and characterize a P450 gene responsible for the majority of deltamethrin resistance observed in the QTC279 strain of Tribolium castaneum. We used recently completed whole-genome sequence of T. castaneum to prepare custom microarrays and identified a P450 gene, CYP6BQ9, which showed more than a 200-fold higher expression in the deltamethrin-resistant QTC279 strain when compared with its expression in the deltamethrin-susceptible Lab-S strain. Functional studies using both double-strand RNA (dsRNA)-mediated knockdown in the expression of CYP6BQ9 and transgenic expression of CYP6BQ9 in Drosophila melanogaster showed that CYP6BQ9 confers deltamethrin resistance. Furthermore, CYP6BQ9 enzyme expressed in baculovirus metabolizes deltamethrin to 4-hydroxy deltamethrin. Strikingly, we also found that unlike many P450 genes involved in insecticide resistance that were reported previously, CYP6BQ9 is predominantly expressed in the brain, a part of the central nervous system (CNS) containing voltage-gated sodium channels targeted by deltamethrin. Taken together, the current studies on the brain-specific insect P450 involved in deltamethrin resistance shed new light on the understanding of the molecular basis and evolution of insecticide resistance.     

Indication of pyrethroid resistance in the main malaria vector, Anopheles stephensi from Iran

ObjectiveTo investiagte insecticide resistance in target species for better insecticide resistance managemnet in malaria control programs.MethodsThe status of insecticide resistance to different imagicides in Anopheles stephensi (An. stephensi) including DDT 4%, lambdacyhalothrin 0.50%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.50% was performed according to WHO standard method.ResultsThe mortality rate to lambdacyhalothrin, permethrin, cyfluthrin, deltamethrin, etofenprox and DDT was (88.0±3.2), (92.0±2.7), (52.0±5.0), (96.0±2.2), (90.0±3.0) and (41.0±5.7) percent, respectively at diagnostic dose for one hour exposure time followed by 24 h recovery period.ConclusionsThese results showed first indication of pyrethroid resistance in An. stephensi in a malarious area, from southern Iran. There is widespread, multiple resistances in the country in An. stephensi to organochlorine and some report of tolerance to organophosphate insecticides and recently to pyrethroids. However, results of this paper will provide a clue for monitoring and mapping of insecticide resistance in the main malaria vector for implementation of any vector control.     

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.     

Irritability of malaria vector, Anopheles sacharovi to different insecticides in a malaria-prone area

ObjectiveTo determine the susceptibility and irritability level of malaria vector Anopheles sacharovi (An. sachrovi) to different insecticides in a malaria-prone area.MethodsSusceptibility and irritability levels of field collected strain of An. sacharovi to WHO standard papers of DDT 4%, dieldrin 0.4%, malathion 5%, fenitrothion 1%, permethrin 0.75%, and deltamethrin 0.05% were determined in East Azerbaijan of Iran during reemerging of malaria as described by WHO.ResultsResults showed that at the diagnostic dose of insecticides this species exhibited resistance to DDT, tolerant to dieldrin and but somehow susceptible to fenitrothion, malathion, permethrin and deltamethrin. The results of irritability of this species to DDT, lambdacyhalothrin, permethrin cyfluthrin and deltamethrin revealed that DDT had had the most and deltamethrin the least irritancy effect. The average number of take offs/fly/minutes for DDT was 0.8±0.2. The order of irritability for permethrin, lambdacyhalothrin, cyfluthrin and deltamethrin were 0.7±0.2, 0.5±0.2, 0.5±0.3, and 0.2±0.1, respectively.ConclusionsResults of this study reveals the responsiveness of the main malaria vector to different insecticides. This phenomenon is depending on several factors such as type and background of insecticide used previously, insecticide properties, and physiology of the species. Careful monitoring of insecticide resistance and irritability level of species could provide a clue for appropriate selection of insecticide for malaria control.     

Multiple insecticide resistance/susceptibility status of Culex quinquefasciatus, principal vector of bancroftian filariasis from filaria endemic areas of northern India

ObjectiveTo understand the insecticide resistance status of Culex quinquefasciatus Say (Diptera: Culicidae) (Cx. Quinquefasciatus) to deltamethrin, cyfluthrin, permethrin, lambdacyhalothrin, DDT and malathion in filarial endemic areas of Uttar Pradesh, India.MethodsInsecticide susceptibility assays were performed on wild-caught adult female Cx. quinquefasciatus mosquitoes to deltamethrin (0.05%), cyfluthrin (0.15%), permethrin (0.75%), lambdacyhalothrin (0.05%), malathion (5.0%) and DDT (4.0%), the discriminating doses recommended by the World Health Organisation (WHO).ResultsThe data showed that Cx. quinquefasciatus is highly resistant to DDT and malathion; the mortality was 28.33% and 27.5%, respectively and incipient resistance to synthetic pyrethroids (deltamethrin, cyfluthrin, permethrin, and lambdacyhalothrin), where mortality ranged from 95.83% in permethrin to 98.33% in cyfluthrin and lambdacyhalothrin. Knockdown times (KDT₅₀) in response to synthetic pyrethroids varied significantly between different insecticides (P<0.01) from 31.480 min for permethrin to 21.650 for cyfluthrin.ConclusionsThe results presents here provide the status report of the insecticide resistance/susceptibility of Cx. quinquefasciatus in major filaria endemic areas of northern India.     

Country-level operational implementation of the Global Plan for Insecticide Resistance Management

Malaria control is reliant on the use of long-lasting pyrethroid-impregnated nets and/or indoor residual spraying (IRS) of insecticide. The rapid selection and spread of operationally significant pyrethroid resistance in African malaria vectors threatens our ability to sustain malaria control. Establishing whether resistance is operationally significant is technically challenging. Routine monitoring by bioassay is inadequate, and there are limited data linking resistance selection with changes in disease transmission. The default is to switch insecticides when resistance is detected, but limited insecticide options and resistance to multiple insecticides in numerous locations make this approach unsustainable. Detailed analysis of the resistance situation in Anopheles gambiae on Bioko Island after pyrethroid resistance was detected in this species in 2004, and the IRS program switched to carbamate bendiocarb, has now been undertaken. The pyrethroid resistance selected is a target-site knock-down resistance kdr-form, on a background of generally elevated metabolic activity, compared with insecticide-susceptible A. gambiae, but the major cytochrome P450-based metabolic pyrethroid resistance mechanisms are not present. The available evidence from bioassays and infection data suggests that the pyrethroid resistance mechanisms in Bioko malaria vectors are not operationally significant, and on this basis, a different, long-lasting pyrethroid formulation is now being reintroduced for IRS in a rotational insecticide resistance management program. This will allow control efforts to be sustained in a cost-effective manner while reducing the selection pressure for resistance to nonpyrethroid insecticides. The methods used provide a template for evidence-based insecticide resistance management by malaria control programs.Malaria control activities in Africa have been scaled up during the last decade. Disease control is predominantly dependent on the distribution and use of pyrethroid-impregnated long lasting insecticide-treated nets (LLINs) and/or indoor residual spraying (IRS) of insecticides. The choice of insecticides for IRS is currently limited to four classes with only two modes of action. One of these insecticide classes, pyrethroids, is also the only class recommended for use by the World Health Organization (WHO) on LLINs. The recent rapid selection and spread of pyrethroid resistance in malaria vectors has stimulated the WHO to develop a Global Plan for Insecticide Resistance Management (1), encouraging countries to plan and implement insecticide resistance management strategies and to underpin these strategies with proper, timely entomological resistance monitoring and effective data management. This must be implemented in the short term at the same time medium- to long-term efforts are made to expand the available insecticide choice (2). To be effective, resistance management plans need to be closely aligned with local evidence supported by an effective monitoring system.Here we detail how this process has been undertaken in Equatorial Guinea, resulting in a detailed Operational Plan for Insecticide Resistance Management, which is owned by the National Malaria Control Program, Ministry of Health and Social Welfare, Equatorial Guinea. The plan was formally adopted in 2012.Bioko, the main island of Equatorial Guinea, has a population of ∼200,000 people. It is situated 30 miles off the coast of Cameroon, experiences high annual rainfall (∼2,000 mm/y), and has in recent years undergone major economic and infrastructural development as a result of offshore oil and gas production. Malaria is endemic, with Entomological Inoculation Rates of 281 and 787 infective bites per year recorded for Anopheles gambiae and Anopheles funestus, respectively, in 2002 (3), which was before the scaling-up of malaria control activities. Comprehensive malaria control interventions were introduced jointly by the Bioko Island Malaria Control Project (BIMCP) and the Ministry of Health and Social Welfare in 2004, with the aim of drastically reducing disease burden and, ultimately, eliminating malaria from the island. Serological markers suggest heterogeneity of effectiveness of malaria control activities across the island to date (4). The BIMCP is funded by a private sector consortium led by Marathon Oil Company. Malaria vector control, managed by Medical Care Development International, consists primarily of IRS of all houses on the island. The first round of IRS using a pyrethroid (deltamethrin) was carried out between March and July 2004, followed by two rounds per year of bendiocarb spraying from 2005 onward. In 2007, a mass distribution of LLINs [PermaNet (Vestergaard Frandsen) 2.0, containing 55 mg/m² deltamethrin] was undertaken, providing one net per sleeping area. Although net coverage was initially high, numbers on the island declined rapidly as nets were redistributed by the recipient population.During the first round of deltamethrin IRS in 2004, a large proportion of A. gambiae s.s. mosquitoes sampled from window exit traps were shown to possess the West African form of the kdr mutation (leu¹⁰¹⁴–phe), which confers dichlorodiphenyltrichloroethane resistance and a low level of cross-resistance to all pyrethroids through insecticide target-site insensitivity (5). Pyrethroid resistance assessed by WHO susceptibility tests and the kdr mutation were present in both the M and S forms of A. gambiae s.s. on Bioko. The presence or absence of metabolically conferred pyrethroid resistance was not assessed at this time, although data from neighboring Cameroon suggests that this form of pyrethroid resistance is now widespread in the A. gambiae complex (6, 7). The detection of pyrethroid resistance, and an apparent lack of response to the IRS treatment by A. gambiae compared with A. funestus, prompted a switch from deltamethrin to a carbamate insecticide from the second spray round onward. However, malaria indicator surveys carried out both prespraying in February to March 2004 and postspraying in the same months in 2005 showed a large reduction in the prevalence of malaria infection in children aged from 2 to <15 y, going from 46% [95% confidence interval (CI), 40–51%] to 31% [95% CI, 24–40%] (8), indicating that the deltamethrin spray round had a substantial epidemiological effect. Routine entomological surveys from 2004 to 2012 suggested that A. funestus had been eliminated or virtually eliminated from Bioko, leaving the vectors A. gambiae s.s. and Anopheles melas. Subsequent routine bioassays and PCR analysis for the kdr mutation showed that despite replacement of deltamethrin with bendiocarb for IRS during a 7-y period, the frequency of pyrethroid resistance and the kdr mutation remained high in A. gambiae s.s.To determine retrospectively whether the presence of the kdr gene in A. gambiae compromised the operational effectiveness of deltamethrin IRS in Bioko in 2004, an analysis of ∼4,000 A. gambiae specimens caught in window traps after the spray round in 2004 was carried out. The aim was to determine whether sporozoite rates in kdr-positive mosquitoes were higher than in kdr-negative mosquitoes.In addition, the poor alignment of kdr positivity with survival in WHO pyrethroid susceptibility tests suggested that multiple pyrethroid resistance mechanisms were circulating in the Bioko population of A. gambiae. Metabolically based resistance mechanisms were assessed using microarrays and quantitative (q)PCR in A. gambiae specimens collected from Bioko in 2011.The retrospective sporozoite–kdr analysis of the A. gambiae specimens collected after the pyrethroid spray round in 2004, and the results of the metabolic resistance testing of A. gambiae samples collected in 2011, coupled with a reassessment of the mosquito density data from 2004 onward, has allowed us to develop an evidence-based operational resistance management program. This will underpin cost-effective maintenance of operational IRS activity for Equatorial Guinea and serve as a template that other malaria-endemic countries faced with similar insecticide resistance issues could follow.     

Susceptibility status of malaria vectors to insecticides commonly used for malaria control in Tanzania

Objective The aim of the study was to monitor the insecticide susceptibility status of malaria vectors in 12 sentinel districts of Tanzania. Methods WHO standard methods were used to detect knock‐down and mortality in the wild female Anopheles mosquitoes collected in sentinel districts. The WHO diagnostic doses of 0.05% deltamethrin, 0.05% lambdacyhalothrin, 0.75% permethrin and 4% DDT were used. Results The major malaria vectors in Tanzania, Anopheles gambiae s.l., were susceptible (mortality rate of 98–100%) to permethrin, deltamethrin, lambdacyhalothrin and DDT in most of the surveyed sites. However, some sites recorded marginal susceptibility (mortality rate of 80–97%); Ilala showed resistance to DDT (mortality rate of 65% [95% CI, 54–74]), and Moshi showed resistance to lambdacyhalothrin (mortality rate of 73% [95% CI, 69–76]) and permethrin (mortality rate of 77% [95% CI, 73–80]). Conclusions The sustained susceptibility of malaria vectors to pyrethroid in Tanzania is encouraging for successful malaria control with Insecticide‐treated nets and IRS. However, the emergency of focal points with insecticide resistance is alarming. Continued monitoring is essential to ensure early containment of resistance, particularly in areas that recorded resistance or marginal susceptibility and those with heavy agricultural and public health use of insecticides.     

Resistance status of main malaria vector,Liston (Diptera:Culicidae) to insecticides in a malaria Endemic Area,Southern Iran

Objective: To evaluate the susceptibility of Anopheles stephensi(An. stephensi) Liston, the main malaria vector in southern Iran, to WHO recommended insecticides. Methods: Larvae of An. stephensi were collected from three different larval habitats in both urban and rural area of Bandar Abbas city and one rural area in Rudan county southern Iran. WHO standard method was used for evaluation of adult and larval mosquito susceptibility. Bendiocarb, permethrin, lambda-cyhalothrin, deltamethrin as insecticide and temephos and chlorpyriphos as larvicide were used at the diagnostic dosages recommended by WHO. Results: Findings of this study showed all larval populations of An. stephensi were completely susceptible to temephos and candidate for resistance to chlorpyriphos. Adult mosquitoes in rural areas of Bandar Abbas city were resistant to pyrethroid and carbamate insecticides. Conclusion: Comparison of the results of this survey with previous studies indicates that the resistance to pyrethroids and carbamates in this malaria endemic region is increasing. Wide use of pesticides in agriculture is certainly effective in increasing resistance. The inter-sectoral coordination and collaboration in health and agriculture seem to be necessary to manage insecticide resistance in malaria vectors.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Impact of pyrethroid resistance on operational malaria control in Malawi

The impact of insecticide resistance on insect-borne disease programs is difficult to quantify. The possibility of eliminating malaria in high-transmission settings is heavily dependent on effective vector control reducing disease transmission rates. Pyrethroids are the dominant insecticides used for malaria control, with few options for their replacement. Their failure will adversely affect our ability to control malaria. Pyrethroid resistance has been selected in Malawi over the last 3 y in the two major malaria vectors Anopheles gambiae and Anopheles funestus, with a higher frequency of resistance in the latter. The resistance in An. funestus is metabolically based and involves the up-regulation of two duplicated P450s. The same genes confer resistance in Mozambican An. funestus, although the levels of up-regulation differ. The selection of resistance over 3 y has not increased malaria transmission, as judged by annual point prevalence surveys in 1- to 4-y-old children. This is true in areas with long-lasting insecticide-treated nets (LLINs) alone or LLINs plus pyrethroid-based insecticide residual spraying (IRS). However, in districts where IRS was scaled up, it did not produce the expected decrease in malaria prevalence. As resistance increases in frequency from this low initial level, there is the potential for vector population numbers to increase with a concomitant negative impact on control efficacy. This should be monitored carefully as part of the operational activities in country.The push for malaria elimination and eventual eradication will be heavily dependent on our ability to reduce disease transmission. A recent editorial suggests that we have the tools to take on this challenge in African malaria heartlands (1). This is predicated on ensuring that vector control prevention and drug treatment tools are fully deployed, reaching every person at risk. There will need to be improved delivery of these tools and better clinical management of malaria cases. In highly endemic areas our ability to reduce malaria transmission will be dependent on vector control, before the focus can shift to killing the parasite in infected people. Two forms of vector control, indoor residual spraying (IRS) and the distribution of long-lasting insecticide-treated nets (LLINs) have been demonstrated to reduce transmission when properly deployed against insecticide susceptible mosquito populations. The use of both interventions has dramatically increased since 2000 in many malaria endemic countries, with increased donor funding to attain the Roll Back Malaria targets and support the malaria elimination agenda (2).IRS and LLINs function by reducing the female mosquito daily survival rate and human biting frequency. Pyrethroids are the only insecticides recommended for use on LLINs, and only four chemical classes of insecticides that attack two target sites are available for IRS, and again pyrethroids dominate the IRS market. Resistance to pyrethroids has been selected in Anopheles gambiae and Anopheles funestus, the major African malaria vectors, although the frequency and level (fold) resistance conferred can vary dramatically. The impact of this resistance on the ability of either control intervention to reduce disease transmission is poorly understood, and current monitoring and evaluation practices are not sufficiently robust to assess this unless catastrophic failures occur. The perceived threat of pyrethroid resistance is now sufficiently high for the World Health Organization (WHO) to convene an international multidonor effort to counteract this.Operationally significant pyrethroid resistance has the potential to limit effective malaria control, owing to the small number of alternative public health insecticides. Pyrethroid resistance in malaria vectors has increased dramatically over the last decade (3, 4), particularly in Africa, where the bulk of malaria-related mortality occurs. Typically resistance is monitored by bioassays, for which the WHO has defined a diagnostic dosage for each insecticide that kills susceptible anopheline mosquitoes (5). Mosquitoes surviving the diagnostic dosage are an indication that resistance has been selected and that an operational problem may be developing, but bioassays alone do not signify control failure.Little operational monitoring of the underlying mechanisms of resistance occurs. Two mechanisms are predominantly responsible for insecticide resistance: changes in the insecticide target site, reducing binding of the insecticide, and increases in the rate at which the insecticide is metabolized (6). Information on the resistance mechanisms is more predictive than bioassays, providing information on the level of resistance and potential cross-resistance between insecticides. For example, two common mutations in the sodium channel convey low-level resistance to pyrethroids and higher-level resistance to dichlorodiphenyltrichloroethane (DDT) in An. gambiae (7, 8), whereas a cytochrome P450-based metabolic regulatory mechanism conveys very high-level pyrethroid and low-level carbamate resistance in An. funestus (9).Vector control interventions are being rapidly scaled up in Malawi, where malaria is highly endemic. Malaria accounts for 34% of all outpatient hospital visits and is the main cause of hospital admissions in children aged <5 y (10). Before 2007 sporadic WHO bioassays were undertaken, which indicated that the two major malaria vectors, An. gambiae and An. funestus, remained fully susceptible to pyrethroids. In 2007 pyrethroid-impregnated LLINs were distributed through antenatal and under-5 clinics at district and central hospitals countrywide. The numbers distributed were sufficient to achieve the Roll Back Malaria targets of 80% of pregnant women and children aged <5 y sleeping under a treated net. In 2008, a pilot study of IRS with the pyrethroid lambda cyhalothrin (ICON, Syngenta) was initiated in Nkhota Khota District, supported by the President’s Malaria Initiative (PMI). The initial program targeted 26,950 houses, and was expanded to 74,772 houses in 2009. Approximately 4 million LLINs were procured and ∼2 million distributed during this time. In 2010 the PMI-supported IRS was expanded to cover the whole of Nkohta Khota district, and the Malawian Ministry of Health supported IRS in a further six districts.A series of sentinel sites were established during this period to track the effect of this rapid increase in insecticide selection pressure on the local vectors and assess any impact on malaria transmission. This was particularly pertinent owing to the high levels of pyrethroid resistance reported in the southern part of neighboring Mozambique in An. funestus, which had prompted a switch from pyrethroids to carbamates or DDT for IRS in the Lubombo Spatial Development Initiative area of Mozambique (11, 12).     

Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b₅ knockout mouse livers to validate P450 activation and establish the role for b₅ in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control.Pyrethroids are synthetic analogs of pyrethrins, botanical chemicals derived from chrysanthemum flowers (1). They are highly potent insecticides with low mammalian toxicity that are used worldwide in ∼3,500 registered products in agricultural, medicinal, veterinary, and public health sectors. Importantly, they are the only class of insecticide recommended for insecticide-treated nets for malaria control. More than 254 million insecticide-treated nets were distributed across Africa between 2008–2010 (2). Similar to antibiotics, pyrethroids are critical for controlling a diverse spectrum of diseases. Unfortunately, similar to antibiotics, such intense exposure affects health and drives the rapid evolution of insecticide resistance (3).Pyrethroids are structurally highly diverse (4) but share a common architecture comprising a cyclopropane acid group coupled to an alcohol moiety, as exemplified by deltamethrin (Fig. 1A). Traditionally, they are divided into two classes (type 1 and type 2), depending on the absence (type 1) or presence (type 2) of an α-cyano group (Fig. 1B). Pyrethroids work by blocking the voltage-gated sodium channels, causing paralysis in arthropods, and resulting in death (3). Resistance is commonly associated with target site modification or metabolic resistance, in which increased rates of biotransformation, generally by P450s, esterases, and GSTs, reduce toxic potency (3).Open in a separate windowFig. 1.Conversion of deltamethrin into PyABPs. (A) Structure of deltamethrin with constituent acid and alcohol moieties. Primary sites of P450 hydroxylation are indicated by bold arrows at the 2′ and 4′ positions, and minor routes of hydroxylation are indicated with open arrows (1). (B) Conversion of deltamethrin to a PyABPP involves the addition of an alkyne warhead and a clickable handle. The structures of the general probe and the PyABPs synthesized are illustrated. Alkyne warhead groups were located in the 2′ or 4′ positions, whereas alkyne click handles replaced the cyano group (type 1) or terminal bromides (type 2). The general P450 probe 2-EN is boxed parallel to its type 1 pyrethroid analog, P2.Although the toxicity and metabolism of pyrethroids in mammals and insects have been extensively characterized (1), the role of specific enzymes and pathways involved in pyrethroid clearance is unclear. In insects, P450s are key enzymes involved with metabolic degradation, with constitutive overexpression of specific P450s leading to pyrethroid resistance (5, 6). Similarly, in mammals, the toxic potency of pyrethroids is inversely related to their rates of metabolic elimination (7), with both P450 oxidation and carboxyl esterase-mediated hydrolysis playing major roles. Humans have 57 P450 genes, rodents ∼80 P450 genes, and insects up to ∼200 P450 genes (8). Where genome information exists, genetic and microarray-based studies of pyrethroid-resistant versus susceptible populations have been used to identify P450s potentially capable of pyrethroid metabolism (3, 5). However, relatively few P450s have been functionally validated through recombinant P450 expression. Thus, probes able to identify pyrethroid-metabolizing enzymes directly would aid our understanding of the fundamental processes of insecticide–organism interactions, expanding our understanding of the risks of pyrethroid exposure to mammals and the enzymatic mechanisms of metabolism and resistance used by insects.Activity-based protein profiling (ABPP) has been described for the functional profiling of P450s (9, 10). The activity-based probes (ABPs) work in a mechanism-dependent manner to covalently label P450s, whereby the labeling events are detectable by adding a fluorescent reporter group via copper-catalyzed azide-alkyne cycloaddition (“click chemistry”) onto the probe−P450 adducts (9, 10). Furthermore, affinity tags can also be incorporated to pull down and identify probe–P450 adducts. The major advantage of ABPPs is their ability to directly assess enzyme activity. In this article, we have designed and synthesized a group of seven pyrethroid mimetic ABPs (PyABPs) on the basis of the deltamethrin scaffold (Fig. 1B) for the targeted identification of pyrethroid-metabolizing P450s in highly divergent organisms. We have investigated their reactivity profiles against pyrethroid metabolizing and nonmetabolizing recombinant mosquito P450s and mouse and rat liver microsomes. We show that PyABPs can be used to reveal pyrethroid structure–activity relationships, and they also have been used to identify pyrethroid-reactive P450s and related detoxification enzymes in rat liver microsomes, demonstrating their potential for directly assessing pyrethroid-metabolizing enzyme activity.     

Chlorfenapyr: a pyrrole insecticide for the control of pyrethroid or DDT resistant Anopheles gambiae (Diptera: Culicidae) mosquitoes

Owing to the development and spread of pyrethroid resistance in Anopheles gambiae in Africa there is an urgent need to develop alternative insecticides to supplement the pyrethroids. Chlorfenapyr is a pyrrole insecticide first commercialized for the control of agricultural pests and termites. Performance against An. gambiae bearing kdr (pyrethroid and DDT resistance) or Ace-1(R) insensitive acetylcholinesterase (organophosphate and carbamate resistance) mechanisms was studied using a variety of adult bioassay tests including a simulated-experimental hut system (tunnel tests) that allows uninhibited mosquito behaviour/insecticide interactions. Strains resistant to pyrethroids and organophosphates showed no cross resistance to chlorfenapyr. In cone bioassays on treated netting the mortality of adult mosquitoes showed an unexpected curvilinear response, with highest mortality occurring at intermediate dosages. Adults expressed irritability to chlorfenapyr at higher dosages, which might explain the dosage-mortality trend. Toxic activity of chlorfenapyr was slow compared to conventional neurotoxic insecticides and additional mortality occurred between 24h and 72 h. In tunnel tests, the dosage-mortality trend showed a more typical sigmoid response and most mortality occurred during the first 24h. Mosquito penetration through the holed, treated netting showed only limited inhibition and blood-feeding was not inhibited. Mortality rates in the kdr strain exposed to chlorfenapyr treated netting in tunnel tests were much higher than with permethrin treated netting over the same 100-500 mg/m(2) dosage range. Chlorfenapyr has potential for malaria control in treated-net or residual spraying applications in areas where mosquitoes are pyrethroid resistant. For treated-net applications chlorfenapyr might be combined with pyrethroid as a mixture to provide personal protection as well as to give control of resistant mosquitoes.     

Cytochrome P450 Monooxygenases CYP6AY3 and CYP6CW1 Regulate Rice Black-Streaked Dwarf Virus Replication in Laodelphax striatellus (Fallén)

The small brown planthopper, Laodelphax striatellus (Fallén), is an important agricultural pest that causes significant losses by sucking and transmitting multiple plant viruses, such as rice black-streaked dwarf virus (RBSDV). Insecticides are commonly used to control planthoppers and cause the induction or overexpression of cytochrome P450 monooxygenases (P450s) from the CYP3 and CYP4 clades after insecticide application. However, little is known about the roles of insecticides and P450s in the regulation of viral replication in insects. In this study, RBSDV-infected L. striatellus were injected with imidacloprid, deltamethrin, pymetrozine, and buprofezin, respectively. The insecticide treatments caused a significant decrease in RBSDV abundance in L. striatellus. Treatment of piperonyl butoxide (PBO), an effective inhibitor of P450s, significantly increased the RBSDV abundance in L. striatellus. Fourteen P450 candidate genes in the CYP3 clade and 21 in the CYP4 clade were systematically identified in L. striatellus, and their expression patterns were analyzed under RBSDV infection, in different tissues, and at different developmental stages. Among the thirty-five P450 genes, the expression level of CYP6CW1 was the highest, while CYP6AY3 was the lowest after RBSDV infection. Knockdown of CYP6CW1 and CYP6AY3 significantly increased the virus abundance and promoted virus replication in L. striatellus. Overall, our data reveal that CYP6CW1 and CYP6AY3 play a critical role in the regulation of virus replication in L. striatellus.     

Insecticide resistance of Triatoma infestans (Hemiptera,Reduviidae) vector of Chagas disease in Bolivia

Objective To define the insecticide resistance status of Triatoma infestans to deltamethrin (pyrethroid), malathion (organophosphate) and bendiocarb (carbamate) in Bolivia. Methods Fifty populations of T. infestans were sampled in Bolivian human dwellings. Quantal response data were obtained by topical applications of 0.2 μl of insecticide–acetone solutions on nymphs N1 of the F1 generations. For most populations, dose–mortality relationships and resistance ratios (RR) were analysed. Discriminating concentrations were established for each insecticide with a susceptible reference strain and used on the other field populations. A tarsal‐contact diagnostic test using insecticide impregnated papers was designed to rapidly identify deltamethrin‐resistant populations in the field. Results Discriminating concentrations for topical applications were 5, 70 and 120 ng active ingredient per insect for deltamethrin, bendiocarb and malathion, respectively. The diagnostic concentration for deltamethrin was 0.30% for the 1‐h exposure by tarsal contact. All populations sampled in human dwellings exhibited significant levels of resistance to deltamethrin, from 6 to 491 and varied among regions. Resistant populations did not recover complete susceptibility to deltamethrin when the synergist piperonyl butoxide (PBO) was used. None of the sampled populations exhibited significant resistance to bendiocarb (all RR₅₀ < 1.8) or malathion (all RR₅₀ < 2.2). Conclusion In Bolivia, most ‘domestic’T. infestans populations are resistant to deltamethrin. Because insecticide vector control is the only selection pressure, resistance likely originates from it. Switching from pyrethroids to organophosphates or carbamates could be a short‐term solution to control this vector, but other alternative integrated control strategies should also be considered in the long term.     

Fungal infection counters insecticide resistance in African malaria mosquitoes

The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies.     

Resistance of Australian Helicoverpa armigera to fenvalerate is due to the chimeric P450 enzyme CYP337B3

Worldwide, increasing numbers of insects have evolved resistance to a wide range of pesticides, which hampers their control in the field and, therefore, threatens agriculture. Members of the carboxylesterase and cytochrome P450 monooxygenase superfamilies are prominent candidates to confer metabolic resistance to pyrethroid insecticides. Both carboxylesterases and P450 enzymes have been shown to be involved in pyrethroid resistance in Australian Helicoverpa armigera, the noctuid species possessing by far the most reported resistance cases worldwide. However, specific enzymes responsible for pyrethroid resistance in field populations of this species have not yet been identified. Here, we show that the resistance toward fenvalerate in an Australian strain of H. armigera is due to a unique P450 enzyme, CYP337B3, which arose from unequal crossing-over between two parental P450 genes, resulting in a chimeric enzyme. CYP337B3 is capable of metabolizing fenvalerate into 4′-hydroxyfenvalerate, which exhibits no toxic effect on susceptible larvae; enzymes from the parental P450 genes showed no detectable fenvalerate metabolism. Furthermore, a polymorphic H. armigera strain could be bred into a susceptible line possessing the parental genes CYP337B1 and CYP337B2 and a resistant line possessing only CYP337B3. The exclusive presence of CYP337B3 in resistant insects of this strain confers a 42-fold resistance to fenvalerate. Thus, in addition to previously documented genetic mechanisms of resistance, recombination can also generate selectively advantageous variants, such as this chimeric P450 enzyme with an altered substrate specificity leading to a potent resistance mechanism.     

Selection of permethrin resistance in the malaria vector Anopheles stephensi.

The laboratory strain of Anopheles stephensi, a well-known urban malaria vector, was selected with permethrin, a synthetic pyrethroid at LD90 level up to five generations. The selection resulted in the development of resistance in F5 generation to the tune of 13-fold to permethrin and cross-resistance to the tune of 7-fold to cypermethrin, 9-fold to alphamethrin, and 10-fold to deltamethrin. The development of cross-resistance to 4% DDT was also noticed. The susceptibility status against 5% malathion was maintained throughout the five generations. The synergistic effect of piperonyl butoxide with permethrin did not overcome the development of resistance. The development of resistance showed a significant relationship between hatchability and different generations.     

江苏省传疟按蚊对菊酯类杀虫剂抗药性的监测

目的了解连续多年采用菊酯类杀虫剂处理蚊帐灭蚊后媒介按蚊的抗性情况.方法采用WHO成蚊滤纸接触法,以全国蚊类抗性监测网提供的区分剂量法来判定抗性级别.结果连续采用菊酯类杀虫剂处理蚊帐5年以上地区的中华按蚊对溴氰菊酯和二氯苯醚菊酯均已产生初级抗性,5年以下地区中华按蚊对这两种杀虫剂尚未产生抗性;连续灭蚊5年以上地区未捕获嗜人按蚊,5年以下地区嗜人按蚊对菊酯类杀虫剂仍较敏感.结论嗜人按蚊对溴氰菊酯和二氯苯醚菊酯尚未产生抗性,中华按蚊虽已产生抗性,但抗性水平仍较低,在今后的疟疾防治工作中应注意加强监测.     

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid‐resistant Aedes aegypti?

Background Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non‐specific esterases, and to enhance cuticular penetration of the insecticide. Objective To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. Methods Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti‘detox chip’. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM‐resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM‐treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. Conclusion Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.     

Monitoring of insecticides resistance in main malaria vectors in a malarious area of Kahnooj district, Kerman province, southeastern Iran

BACKGROUND AND OBJECTIVES: Kahnooj district in south of Iran is an endemic area for malaria where Anopheles stephensi (Liston) is a main malaria vector and An. dthali (Patton) a secondary vector. According to the national strategy plan on monitoring of insecticides resistance, this study was performed on susceptibility and irritability levels of An. stephensi and An. dthali to different insecticides in the district. METHODS: The susceptibility and irritability levels of field strains of An. stephensi and An. dthali at the adult and larval stages to discriminative dose of different imagicides was determined as recommended by WHO. RESULTS: Using discriminative dose and WHO criteria it was found that An. stephensi is resistant to DDT and dieldrin with 36.1 +/- 2.3 and 62.2 +/- 1.95 mortality rates, respectively; but susceptible to other insecticides. An. dthali was found to susceptible to all tested insecticides. The larvae of An. stephensi, exhibited 100% mortality for temephos and malathion, but 44 +/- 4.32 for discriminative dose of fenitrothion. The results of irritability level for DDT and pyrethroids showed that permethrin had the most irritancy effect on An. stephensi and An. dthali. DDT and deltamethrin showed the least irritancy effect against An. stephensi with 0.42 +/- 0.08 and 0.77 +/- 0.12 take-offs/min/adult, respectively, however, lambdacyhalothrin had the least irritancy effect against An. dthali with 0.096 +/- 0.02 take-offs/min/ adult. The mean number of take-offs/min/adult with permethrin showed significant difference to DDT, lambdacyhalothrin, cyfluthrin and deltamethrin. INTERPRETATION AND CONCLUSION: Pyrethroid insecticides are being used as indoor residual sprays in Iran. Based on our results, the main malaria vectors in the region are still susceptible to pyrethroid insecticides. Therefore, we propose the use of pyrethroids with low irritancy effect in rotation with carbamate insecticides in two interval seasonal peaks of malaria transmission. Biological control including larvivorous fishes, using of local made Bacillus thuringiensis and larvicides such as chlorpyrifos-methyl are the main larval control in the region. Result on larval test exhibited the susceptibility of main vectors to some larvicides, although the 100% mortality was not obtained using fenitrothion and this is postulated the use of this insecticide in agriculture pest control. Monitoring and evaluation of insecticides resistance in malaria vectors in the region could provide an essential clue for judicious use of insecticides.