Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypti

Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and β esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of β-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.     

Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana

Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south-western Ghana and from urban and rural localities in Ogun State, south-west Nigeria. Adult mosquitoes, reared from field-collected larvae, were exposed to the WHO-recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post-exposure. Susceptibility of An. gambiae s.l. to DDT was 94-100% in Ghana and 72-100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97-100% in Ghana, 95-100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr-type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid-impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S-form, whereas the M-form predominated in urban samples. It is suggested that 'urban island' populations of An. arabiensis and of An. gambiae s.s. M-form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.     

Structure of an insect epsilon class glutathione S-transferase from the malaria vector Anopheles gambiae provides an explanation for the high DDT-detoxifying activity

Glutathione S-transferases (GSTs), a major family of detoxifying enzymes, play a pivotal role in insecticide resistance in insects. In the malaria vector Anopheles gambiae, insect-specific epsilon class GSTs are associated with resistance to the organochlorine insecticide DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane]. Five of the eight class members have elevated expression levels in a DDT resistant strain. agGSTe2 is considered the most important GST in conferring DDT resistance in A. gambiae, and is the only member of the epsilon class with confirmed DDT-metabolizing activity. A delta class GST from the same species shows marginal DDT-metabolizing activity but the activity of agGSTe2 is approximately 350x higher than the delta class agGST1-6. To investigate its catalytic mechanism and the molecular basis of its unusually high DDT-metabolizing ability, three agGSTe2 crystal structures including one apo form and two binary complex forms with the co-factor glutathione (GSH) or the inhibitor S-hexylglutathione (GTX) have been solved with a resolution up to 1.4A. The structure of agGSTe2 shows the canonical GST fold with a highly conserved N-domain and a less conserved C-domain. The binding of GSH or GTX does not induce significant conformational changes in the protein. The modeling of DDT into the putative DDT-binding pocket suggests that DDT is likely to be converted to DDE [1,1-dichloro-2,2-bis-(p-chlorophenyl)ethylene] through an elimination reaction triggered by the nucleophilic attack of the thiolate group of GS(-) on the beta-hydrogen of DDT. The comparison with the less active agGST1-6 provides the structural evidence for its high DDT-detoxifying activity. In short, this is achieved through the inclination of the upper part of H4 helix (H4' helix), which brings residues Arg112, Glu116, and Phe120 closer to the GSH-binding site resulting in a more efficient GS(-)-stabilizing hydrogen-bond-network and higher DDT-binding affinity.     

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.     

Insecticide resistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, Culex quinquefasciatus, in northeastern India

The insecticide resistance status of Culex quinquefasciatus Say (Diptera: Culicidae) to DDT and deltamethrin across army cantonments and neighbouring villages in northeastern India was investigated. In India, DDT is still the insecticide of choice for public health programmes. In military stations, pyrethroids, especially deltamethrins, are used for insecticide‐treated nets (ITNs). Recent information on the levels of resistance to DDT and deltamethrin in mosquito populations of northeastern India is scare. Continued monitoring of insecticide resistance status, identification of the underlying mechanisms of resistance in local mosquito populations and the establishment of a baseline data bank of this information are of prime importance. Insecticide susceptibility assays were performed on wild‐caught adult female Cx. quinquefasciatus mosquitoes to the discriminating doses recommended by the World Health Organisation (WHO) to DDT (4%) and deltamethrin (0.05%). Across all study sites, mortality as a result of DDT varied from 11.9 to 50.0%, as compared with 91.2% in the susceptible laboratory strain (S‐Lab), indicating that Cx. quinquefasciatus is resistant to DDT. The species was found to be 100% susceptible to deltamethrin in all study sites except Benganajuli and Rikamari. Knock‐down times (KDT) in response to deltamethrin varied significantly between study sites (P < 0.01) from 8.3 to 17.8 min for KDT₅₀ and 37.4 to 69.5 min for KDT₉₀. All populations exceeded the threshold level of alpha‐esterase, beta‐esterase and glutathion S‐transferase (GST) established for the S‐Lab susceptible strain, and all populations had 100% elevated esterase and GST activity, except Missamari and Solmara. Beta‐esterase activity in Field Unit II (96.9%) was less than in any of the other populations. Benganajuli had the highest activity level for all the enzymes tested. There was a significant correlation between all enzyme activity levels and insecticide resistance phenotype by populations (P < 0.05). The results presented here provide the first report and baseline information of the insecticide resistance status of Cx. quinquefasciatus in northeastern India, and associated information about biochemical mechanisms that are essential for monitoring the development of insecticide resistance in the area.     

Characterization of mosquito larval habitats and assessment of insecticide-resistance status of Anopheles gambiae senso lato in urban areas in southwestern Ghana

The study was carried out to characterize potential larval habitats in the city of Sekondi with the aim of assessing the relative importance of anthropogenic and natural water bodies as larval habitats. Insecticide-resistance status of Anopheles gambiae senso lato in the southwestern part of the coastal savannah zone in Ghana was also assessed against four different classes of insecticides. Larval surveys were carried out in two communities that are separated by a lagoon. Although the lagoon was a potential mosquito larval habitat, we showed that it was not an important mosquito breeding site. The major larval habitats were anthropogenic, resulting from human behavior. Some of the organically polluted breeding sites were inhabited by both An. gambiae s.l. and Culex quinquefasciatus larvae. The data also showed that An. gambiae s.l. has currently developed a strong resistance to DDT and pyrethroid insecticides in southwestern Ghana, where the species was reported to be susceptible about a decade ago. The use of insecticides in households was implicated as a possible cause of the development of resistance among An. gambiae s.l. populations in the area. The management of insecticide resistance among malaria vectors needs urgent attention if insecticide-treated materials can continue to be used for malaria control.     

Age-dependent response to insecticides and enzymatic variation in susceptible and resistant codling moth larvae

Insecticide resistance in the codling moth, Cydia pomonella, partly results from increased metabolic detoxification. The aim of this study was to follow the age variations in larval susceptibility to deltamethrin and teflubenzuron in one susceptible (S) strain, and two resistant (Rv and Rt) ones selected for resistance to deltamethrin and diflubenzuron, respectively. The age variation of the activities of cytochrome P450-dependent monooxygenase (MFO), glutathione S-transferases (GST), and esterases in S and both resistant strains were simultaneously investigated. The highest levels of insecticide resistance were recorded in late instars in both resistant strains, although Rv neonates exhibited enhanced resistance to deltamethrin. The involvement of an additional deltamethrin-specific mechanism of resistance, which could be mainly expressed in early instars, was supported by previous demonstration of a kdr point mutation in the Rv strain. The cross-resistance between deltamethrin and teflubenzuron indicated the involvement of non-specific metabolic pathways in resistance to teflubenzuron, rather than target site modification. A positive correlation between enhanced GST activities and deltamethrin resistance suggested that this mechanism might take place into the adaptive response of C. pomonella to pyrethroids treatments. Enhanced MFO activity was recorded in each instar of the two resistant strains compared to the susceptible one. But these activities were not correlated to the responses to deltamethrin nor to teflubenzuron. In the light of these findings, studying age-dependence of responses to selection is central to the implementation of monitoring tests of resistances, especially if the target instars are difficult to collect in the field.     

Establishing the role of detoxifying enzymes in field‐evolved resistance to various insecticides in the brown planthopper (Nilaparvata lugens) in South India

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the major pests of rice throughout Asia. Extensive use of insecticides for suppressing N. lugens has resulted in the development of insecticide resistance leading to frequent control failures in the field. The aim of the present study was to evaluate resistance in the field populations of N. lugens from major rice growing states of South India to various insecticides. We also determined the activity of detoxifying enzymes (esterases [ESTs], glutathione S‐transferases [GSTs], and mixed‐function oxidases [MFOs]). Moderate levels of resistance were detected in the field populations to acephate, thiamethoxam and buprofezin (resistance factors 1.05–20.92 fold, 4.52–14.99 fold, and 1.00–18.09 fold, respectively) as compared with susceptible strain while there were low levels of resistance to imidacloprid (resistance factor 1.23–6.70 fold) and complete sensitivity to etofenoprox (resistance factor 1.05–1.66 fold). EST activities in the field populations were 1.06 to 3.09 times higher than the susceptible strain while for GST and MFO the ratios varied from 1.29 to 3.41 and 1.03 to 1.76, respectively. The EST activity was found to be correlated to acephate resistance (r = 0.999, P ≥ 0.001). The high selection pressure of organophosphate, neonicotinoid, and insect growth regulator (IGR) in the field is likely to be contributing for resistance in BPH to multiple insecticides, leading to control failures. The results obtained will be beneficial to IPM recommendations for the use of effective insecticides against BPH.     

Pyrethroid resistance mechanisms in the head louse Pediculus capitis from Israel: implications for control

In Israel, the head louse, Pediculus capitis, developed resistance to DDT through the extensive use of this insecticide until the 1980s. In 1991, permethrin was introduced for control of DDT resistant P. capitis in Israel, leading to control failure of this pyrethroid insecticide by 1994. Pyrethroid resistance of P. capitis in Israel extends to phenothrin, which has not been used for louse control. We identified a glutathione S-transferase(GST)-based mechanism of DDT resistance in the Israeli head lice. This GST mechanism occurred before 1989, while permethrin resistance in P. capitis developed after 1994, suggesting that the main GST resistance mechanism selected by DDT use does not confer any pyrethroid cross-resistance. Esterase activity levels were equivalent in pyrethroid resistant and susceptible P. capitis field-collected in Israel, and in a susceptible strain of P. humanus, the body louse, indicating no involvement of any esterase-based mechanism in resistance. A weak monooxygenase-based permethrin metabolism resistance mechanism was the only factor identified which could account for any of the observed pyrethroid resistance in P. capitis. However, the lack of synergism of phenothrin resistance by piperonyl butoxide suggests that a non-oxidative mechanism is also present in the resistant lice. Therefore it seems probable that pyrethroid resistance in Israeli P. capitis is due to a combination of nerve insensitivity (knockdown resistance or 'kdr') and monooxygenase resistance mechanisms.     

Comparative evaluation of carbosulfan- and permethrin-impregnated curtains for preventing house-entry by the malaria vector Anopheles gambiae in Burkina Faso

Pyrethroid-impregnated bednets and curtains are widely employed to reduce the risk of malaria transmission, but pyrethroid-resistance is becoming more prevalent among malaria vector Anopheles mosquitoes (Diptera: Culicidae). As an alternative treatment for curtains, we assessed carbosulfan (a carbamate insecticide) in comparison with permethrin as the standard pyrethroid, against endophilic female mosquitoes of the Anopheles gambiae Giles complex in a village near Ouagadougou, Burkina Faso. The main criterion evaluated was the impact of curtains (hung inside windows, eaves and doorways) on the number of An. gambiae s.l. females active indoors at night. Light-traps were operated overnight (21.00-06.00 hours beside occupied untreated bednets) to sample mosquitoes in houses fitted with net curtains treated with carbosulfan 0.2 g ai/m2 or permethrin 1 g ai/m2 or untreated, compared with houses without curtains. The treated and untreated curtains significantly reduced the numbers of mosquitoes collected indoors, compared with houses without curtains. Carbosulfan-treated curtains had a highly significantly greater effect than permethrin-treated or untreated curtains, the scale of the difference being estimated as three-fold. However, there was no significant difference between the impact of untreated and permethrin-treated curtains on densities of An. gambiae s.l. trapped indoors. Samples of the An. gambiae complex comprised An. arabiensis Patton and both the S- and M-forms of An. gambiae Giles s.s. Susceptibility tests revealed some resistance to DDT and low frequencies of permethrin-resistance, insufficient to explain the poor performance of permethrin on curtains. Among survivors from the diagnostic dosage of permethrin were some specimens of all three members of the An. gambiae complex, but the kdr resistance mechanism was detected only in the S-form of An. gambiae s.s. Questions arising for further investigation include clarification of resistance mechanisms in, and foraging behaviour of, each member of the An. gambiae complex in this situation and the need to decide whether carbosulfan-treated curtains are acceptably safe for use to reduce risks of malaria transmission.     

Resistance management strategies in malaria vector mosquito control. Baseline data for a large-scale field trial against Anopheles albimanus in Mexico

Abstract. A high level of DDT resistance and low levels of resistance to organophosphorus, carbamate and pyrethroid insecticides were detected by discriminating dose assays in field populations of Anopheles albimanus in Chiapas, southern Mexico, prior to a large-scale resistance management project described by Hemingway et al. (1997) . Biochemical assays showed that the DDT resistance was caused by elevated levels of glutathione S-transferase (GST) activity leading to increased rates of metabolism of DDT to DDE. The numbers of individuals with elevated GST and DDT resistance were well correlated, suggesting that this is the only major DDT resistance mechanism in this population. The carbamate resistance in this population is conferred by an altered acetylcholinesterase (AChE) -based resistance mechanism. The level of resistance observed in the bioassays correlates with the frequency of individuals homozygous for the altered AChE allele. This suggests that the level of resistance conferred by this mechanism in its heterozygous state is below the level of detection by the WHO carbamate discriminating dosage bioassay. The low levels of organophosphate (OP) and pyrethroid resistance could be conferred by either the elevated esterase or monooxygenase enzymes. The esterases were elevated only with the substrate pNPA, and are unlikely to be causing broad spectrum OP resistance. The altered AChE mechanism may also be contributing to the OP but not the pyrethroid resistance. Significant differences in resistance gene frequencies were obtained from the F₁ mosquitoes resulting from adults obtained by different collection methods. This may be caused by different insecticide selection pressures on the insects immediately prior to collection, or may be an indication that the indoor- and outdoor-resting A. albimanus collections are not from a randomly mating single population. The underlying genetic variability of the populations is currently being investigated by molecular methods.     

The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides

The Epsilon glutathione transferase (GST) class in the dengue vector Aedes aegypti consists of eight sequentially arranged genes spanning 53,645 bp on super contig 1.291, which maps to chromosome 2. One Epsilon GST, GSTE2, has previously been implicated in conferring resistance to DDT. The amino acid sequence of GSTE2 in an insecticide susceptible and a DDT resistant strain differs at five residues two of which occur in the putative DDT binding site. Characterization of the respective recombinant enzymes revealed that both variants have comparable DDT dehydrochlorinase activity although the isoform from the resistant strain has higher affinity for the insecticide. GSTe2 and two additional Epsilon GST genes, GSTe5 and GSTe7, are expressed at elevated levels in the resistant population and the recombinant homodimer GSTE5-5 also exhibits low levels of DDT dehydrochlorinase activity. Partial silencing of either GSTe7 or GSTe2 by RNA interference resulted in an increased susceptibility to the pyrethroid, deltamethrin suggesting that these GST enzymes may also play a role in resistance to pyrethroid insecticides.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.     

Characterization of deltamethrin resistance in field populations of Aedes aegypti in Thailand.

Five field collections of adult Aedes aegypti mosquitoes from different areas in Bangkok and Pathum Thani provinces were subjected to susceptibility tests against deltamethrin. Low levels of resistance were detected among all populations tested (RR50 = 8-17.2) compared to the susceptible strain, Bora (French Polynesia). Among the five populations tested, the BKH (Bang Khen, Bangkok) and PSC (Phasicharoen, Bangkok) populations showed a higher level of deltamethrin resistance than the other three populations (RR50 of BKH = 17.2, and of PSC= 13.6) and cross-resistance to DDT was observed in these strains. Biochemical analysis showed a significant elevation of mixed function oxidases enzyme activity in all populations. There was an elevation of non-specific esterases in all populations except BKL, and there was no consistent association of glutathione S-transferases with deltamethrin and DDT resistance, although not all populations were bioassayed for DDT. The partial cDNA sequence of the para-type voltage-dependent sodium channel (IIS4-IIS6) was determined for BKH and PSC populations. Common amino acid substitution, leucine to phenylalanine in the IIS6 region, found for insects including Anopheles gambiae was not found in either the BKH or the PSC populations. However, two other amino acid substitutions (proline substituted with serine at position 64 in the PSC population and leucine with phenylalanine at position 69 in the BKH population) were found in the IIS5-IIS6 inter-segment region sequenced. The role these substitutions play in target site resistance is uncertain at this time.     

Glutathione S-transferase and insecticide resistance in laboratory strains and field populations of Musca domestica

Glutathione S-transferase (GST) activity of 10 house fly laboratory strains and 21 field populations are described with two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), commonly associated with resistance. Three laboratory strains selected by tetrachlorvinphos, lindane (gamma-HCH) and dimethoate, respectively, had significantly elevated CDNB- and DCNB-GST activities. The multiresistant field population 791a had significantly elevated CDNB- and DCNB-GST activities. Many strains recorded several individuals with high CDNB- and/or DCNB-GST activity and to evaluate these differences, phenotypes were defined by cluster analysis. A phenotype, GST-R, indicating high CDNB- and DCNB-GST activities, was found in 23 of 31 laboratory strains or field populations. GST-R was likely to be involved in gamma-HCH resistance in strain 17e, tetrachlorvinphos resistance in strain 39m2b, and dimethoate resistance in strain 49r2b. The frequency of GST-R in selected laboratory strains and field population correlated with the frequency of house flies surviving the organophosphate azamethiphos either topically applied or by ingestion. There was no significant correlation between GST activity and the toxicity of the organophosphate dimethoate or the pyrethroids bioresmethrin and pyrethrin.     

Trends in the selection of insecticide resistance in Anopheles gambiae s.l. mosquitoes in northwest Tanzania during a community randomized trial of longlasting insecticidal nets and indoor residual spraying

Anopheles gambiae s.l. (Diptera: Culicidae) in Muleba, Tanzania has developed high levels of resistance to most insecticides currently advocated for malaria control. The kdr mutation has almost reached fixation in An. gambiae s.s. in Muleba. This change has the potential to jeopardize malaria control interventions carried out in the region. Trends in insecticide resistance were monitored in two intervention villages using World Health Organization (WHO) susceptibility test kits. Additional mechanisms contributing to observed phenotypic resistance were investigated using Centers for Disease Control (CDC) bottle bioassays with piperonylbutoxide (PBO) and S,S,S‐tributyl phosphorotrithioate (DEF) synergists. Resistance genotyping for kdr and Ace‐1 alleles was conducted using quantitative polymerase chain reaction (qPCR). In both study villages, high phenotypic resistance to several pyrethroids and DDT was observed, with mortality in the range of 12–23%. There was a sharp decrease in mortality in An. gambiae s.l. exposed to bendiocarb (carbamate) from 84% in November 2011 to 31% in December 2012 after two rounds of bendiocarb‐based indoor residual spraying (IRS). Anopheles gambiae s.l. remained susceptible to pirimiphos‐methyl (organophosphate). Bendiocarb‐based IRS did not lead to the reversion of pyrethroid resistance. There was no evidence for selection for Ace‐1 resistance alleles. The need to investigate the operational impact of the observed resistance selection on the effectiveness of longlasting insecticidal nets and IRS for malaria control is urgent.     

Three years of insecticide resistance monitoring in Anopheles gambiae in Burkina Faso: resistance on the rise?

ABSTRACT: Background and methods A longitudinal Anopheles gambiae s.l. insecticide-resistance monitoring programme was established in four sentinel sites in Burkina Faso. For three years, between 2008 and 2010, WHO diagnostic dose assays were used to measure the prevalence of resistance to all the major classes of insecticides at the beginning and end of the malaria transmission season. Species identification and genotyping for target site mutations was also performed and the sporozoite rate in adults determined. RESULTS: At the onset of the study, resistance to DDT and pyrethroids was already prevalent in An. gambiae s.l. from the south-west of the country but mosquitoes from the two sites in central Burkina Faso were largely susceptible. Within three years, DDT and permethrin resistance was established in all four sites. Carbamate and organophosphate resistance remains relatively rare and largely confined to the south-western areas although a small number of bendiocarb survivors were found in all sites by the final round of monitoring. The ace-1R target site resistance allele was present in all localities and its frequency exceeded 20% in 2010 in two of the sites. The frequency of the 1014 F kdr mutation increased throughout the three years and by 2010, the frequency of 1014 F in all sites combined was 0.02 in Anopheles arabiensis, 0.56 in An. gambiae M form and 0.96 in An. gambiae S form. This frequency did not differ significantly between the sites. The 1014 S kdr allele was only found in An. arabiensis but its frequency increased significantly throughout the study (P = 0.0003) and in 2010 the 1014 S allele frequency was 0.08 in An. arabiensis. Maximum sporozoite rates (12%) were observed in Soumousso in 2009 and the difference between sites is significant for each year. CONCLUSION: Pyrethroid and DDT resistance is now established in An. gambiae s.l. throughout Burkina Faso. Results from diagnostic dose assays are highly variable within and between rounds of testing, and hence it is important that resistance monitoring is carried out on more than one occasion before decisions on insecticide procurement for vector control are made. The presence of 1014 S in An. gambiae s.l., in addition to 1014 F, is not unexpected given the recent report of 1014 S in Benin but highlights the importance of monitoring for both mutations throughout the continent. Future research must now focus on the impact that this resistance is having on malaria control in Burkina Faso.