Insecticide resistance profile of Anopheles gambiae from a phase II field station in Cové, southern Benin: implications for the evaluation of novel vector control products
© Ngufor et al. 2015
Received: 14 August 2015
Accepted: 2 November 2015
Published: 18 November 2015
Novel indoor residual spraying (IRS) and long-lasting insecticidal net (LLIN) products aimed at improving the control of pyrethroid-resistant malaria vectors have to be evaluated in Phase II semi-field experimental studies against highly pyrethroid-resistant mosquitoes. To better understand their performance it is necessary to fully characterize the species composition, resistance status and resistance mechanisms of the vector populations in the experimental hut sites.
Bioassays were performed to assess phenotypic insecticide resistance in the malaria vector population at a newly constructed experimental hut site in Cové, a rice growing area in southern Benin, being used for WHOPES Phase II evaluation of newly developed LLIN and IRS products. The efficacy of standard WHOPES-approved pyrethroid LLIN and IRS products was also assessed in the experimental huts. Diagnostic genotyping techniques and microarray studies were performed to investigate the genetic basis of pyrethroid resistance in the Cové Anopheles gambiae population.
The vector population at the Cové experimental hut site consisted of a mixture of Anopheles coluzzii and An. gambiae s.s. with the latter occurring at lower frequencies (23 %) and only in samples collected in the dry season. There was a high prevalence of resistance to pyrethroids and DDT (>90 % bioassay survival) with pyrethroid resistance intensity reaching 200-fold compared to the laboratory susceptible An. gambiae Kisumu strain. Standard WHOPES-approved pyrethroid IRS and LLIN products were ineffective in the experimental huts against this vector population (8–29 % mortality). The L1014F allele frequency was 89 %. CYP6P3, a cytochrome P450 validated as an efficient metabolizer of pyrethroids, was over-expressed.
Characterizing pyrethroid resistance at Phase II field sites is crucial to the accurate interpretation of the performance of novel vector control products. The strong levels of pyrethroid resistance at the Cové experimental hut station make it a suitable site for Phase II experimental hut evaluations of novel vector control products, which aim for improved efficacy against pyrethroid-resistant malaria vectors to WHOPES standards. The resistance genes identified can be used as markers for further studies investigating the resistance management potential of novel mixture LLIN and IRS products tested at the site.
Malaria vector control and prevention today largely depends on the use of insecticides applied as indoor residual spray (IRS) or long-lasting insecticidal nets (LLINs). Both interventions are effective [1, 2] and have contributed immensely to reducing the burden of malaria in recent years . Nevertheless, the rapid development and spread of insecticide resistance to the limited classes of insecticides approved for vector control poses a major threat to the gains so far achieved in malaria control . The need for new insecticide products that can circumvent existing mechanisms of resistance to current insecticides has become critical.
There is currently concerted effort from the international vector control community towards the development of new public health insecticides. The present portfolio of the Innovative Vector Control Consortium (IVCC) for example, is designed to produce three entirely new classes of insecticides by 2023 with no cross-resistance to existing classes of insecticides . The IVCC has set up several projects and some novel IRS and LLIN products have been taken to evaluation phase. The World Health Organization (WHO) through its pesticide evaluation scheme (WHOPES) has set criteria that such products must meet to obtain recommendation for large-scale use against malaria vectors. WHOPES guidelines require that these products be evaluated in Phase I laboratory, Phase II semi-field experimental hut studies and in Phase III randomized controlled trials in households or village clusters [6, 7].
Experimental huts are a good simulation of human-occupied houses which allow the performance of indoor vector control interventions/products to be measured in terms of their ability to kill mosquitoes, prevent feeding and deter mosquitoes from entering a home. Several experimental hut stations have been constructed across sub-Saharan Africa and used for evaluating various indoor vector control products/tools following WHOPES guidelines [8–13]. However, the resistance profile of the local vector populations in some of these studies was not fully characterized at the time of evaluation, making interpretation of results complex . Considering the movement towards the evaluation of a new generation of vector control products that are expected to show improved efficacy against insecticide-resistant mosquitoes in order to attain WHO recommendation for large-scale use in pyrethroid resistant areas, it becomes imperative that the vector populations at experimental hut stations used for such evaluations are highly resistant and the profile of resistance and vector species is fully characterized and understood at the time of evaluation. In addition, because insecticide resistance tends to vary from one locality to another usually over very short distances, the local vector resistance profile reported for such Phase II evaluations has to be specific for the vicinity of the experimental hut station where the study was conducted. Furthermore, the new guidelines for substantiating efficacy claims of novel LLINs in areas of high resistance recently published by the Vector Control Advisory Group (VCAG)  specifies criteria that the vector populations in hut sites used for Phase II evaluations of such novel products should meet; the vector population should be well characterized and should have a pyrethroid resistance ratio of at least ten-fold compared to a susceptible laboratory strain.
The current study was designed to fully characterize insecticide resistance in the Anopheles gambiae sl vector population from an experimental hut station in Cové, Benin, a newly constructed site belonging to the Pan African Malaria Vector Research Consortium (PAMVERC) collaborative site between the London School of Hygiene and Tropical Medicine (LSHTM) and Centre de Recherches Entomologique de Cotonou (CREC) in parallel with a series of Phase II evaluations of novel IRS and LLIN products to WHOPES standards. Studies were also performed to assess year-round hut-entry rates in the Cové site. The efficacy of current WHOPES-approved pyrethroid IRS and LLINs was assessed in the experimental huts. The implications of the findings for the evaluation of the novel vector control tools aimed at improving the control of insecticide-resistant vector populations is discussed.
Cové site and experimental huts
To assess year-round variation in mosquito density and hut-entry rates in the experimental huts at Cové, consenting human volunteers slept under an untreated net in the huts and mosquitoes were collected daily over 12 months and brought to the laboratory for identification.
WHO susceptibility bioassays
WHO susceptibility tests to assess the prevalence of resistance were performed using papers obtained from Universiti Sains Malaysia, impregnated with a range of insecticides from the four approved classes for vector control: namely, 0.75 % permethrin, 4 % DDT, 0.05 % deltamethrin, 0.1 % bendiocarb, 5 % malathion, and 4 % fenithrothion. Adult female mosquitoes 2–3 days old, which emerged from larvae collected from breeding sites next to the experimental huts, were exposed for 1 h to the insecticide-treated papers and mortality was recorded 24 h later. Approximately 100 mosquitoes (four replicates of 25 mosquitoes) were used per test and the average mortality was calculated. Control mosquitoes were exposed to untreated papers. Resistant females which remained alive after 24 h were kept for one more day until they were 3–4 days old, after which they were preserved submerged in RNA later for genotyping, target site resistance and microarray studies.
Resistance intensity dose–response bioassay
To determine the intensity of resistance to pyrethroids in the wild An. gambiae Cové strain, mosquitoes that emerged from larvae collected from breeding sites at the experimental hut station were tested in CDC bottle bioassays treated with a range of doses of alphacypermethrin from 0.05 to 5 µg. Comparison was made with the laboratory-susceptible An. gambiae Kisumu strain tested with a dose range of 0.0005–0.05 µg. Alphacypermethrin was chosen for this study because it was one of the main pyrethroid insecticides used as a positive control in the parallel hut evaluations. A total of 100 mosquitoes were exposed for 1 h at each concentration and deaths were scored 24 h later. Log-dosage mortality curves were generated using probit analysis and estimates of the dose required to kill 50 % (LD50) of each strain and the resistance ratios relative to the susceptible laboratory strain were generated (PoloPlus version 1.0).
The effect of the insecticide synergist piperonyl butoxide (PBO), the primary action of which is to inhibit P450 mono-oxygenase enzymes, was evaluated in CDC bottle bioassays. One-hundred and fifty-two to five day old adult females were exposed (in batches of 25 mosquitoes) for 30 min in bottles treated with permethrin (21.5 µg/bottle) either alone or after 1 h pre-exposure to PBO (400 µg/bottle). Mosquitoes were also exposed to PBO alone. Control bottles were treated with acetone alone. The laboratory-susceptible Kisumu strain was also tested in bottles treated with permethrin alone for comparison. Mortality was recorded after 24 h.
Efficacy of standard pyrethroid LLIN and IRS in experimental huts in Cové, Benin
Untreated control net
Olyset Net (permethrin-incorporated LLIN)
Interceptor 1 (alphacypermethrin-coated LLIN)
Deltamethrin IRS applied at 25 mg/sq m
Alphacypermethrin IRS applied at 25 mg/sq m
During the trials, consenting human volunteers slept in the huts from dusk to dawn to attract mosquitoes. Mosquitoes were collected in the morning and brought to the laboratory for identification and scoring of mortality, blood feeding rate and exophily. The nets were rotated between huts to account for hut attractiveness to mosquitoes while the sleepers were rotated on successive nights to reduce any bias due to individual attractiveness to mosquitoes in accordance with a Latin square design.
Chemoprophylaxis was provided to volunteer sleepers prior to the hut studies. Approval was obtained from the ethics review boards of the London School of Hygiene and Tropical Medicine and the Ministry of Health in Benin.
Species identification and target site resistance
To identify vector species and target site resistance in Cové, DNA was extracted from a pair of legs taken from each female mosquito tested in the WHO susceptibility bioassays, and also from a set of unexposed mosquitoes (N = 89) collected from the Cové hut site at the beginning of the previous dry season (September–October 2014) for comparison between seasons. The legs were transferred to 96-well plates and extraction was done using a buffer-based boiling method. Species identification was conducted on each DNA sample using standard PCR techniques [16, 17]. TaqMan assays were used to characterize each sample for genotypes at three loci in the voltage-gated, sodium channel, target site (L1014F, L1015S and N1575Y) [18, 19] which confer resistance to DDT and pyrethroids, and also for the ace-1 G119S resistance mutation in acetylcholinesterase, the target site of organophosphates and carbamates .
Monthly hut-entry rates
WHO susceptibility bioassays
Resistance intensity bioassays
Intensity of pyrethroid (alpha-cypermethrin) resistance in Anopheles gambiae sl from experimental hut station in Cové, Benin
LD50 (95 % CI)
LD95 (95 % CI)
RR 50 (95 % CI)
PBO synergist bioassays
Efficacy of standard pyrethroid LLIN and IRS in experimental huts in Cové, Benin
Species identification PCR results revealed that the vector population was 100 % An. gambiae (N = 187). SINE PCR results showed that the population consisted of a mixture of the Anopheles coluzzii and An. gambiae s.s., the proportions of which showed seasonal variation. Twenty-three per cent of 89 samples collected in the dry season were An. gambiae s.s. while no An. gambiae s.s. was found in samples collected during the rainy season (N = 169); Chi-sq = 29.2, 1 df, P = 6 × 10−8.
Target site resistance alleles
Frequency of target site resistance alleles in Anopheles gambiae sl from experimental hut station in Cové, Benin
Resistant allele freq (%)
The current study was designed to provide an in-depth characterization of the genotype and resistance profile of An. gambiae s.l. at a newly constructed experimental hut station in Cové, Benin, being used for WHOPES Phase II evaluation of novel IRS and LLIN products that are expected to show improved performance against pyrethroid-resistant mosquito vectors and potentially manage resistance. Characterizing the vector population in an experimental hut site is crucial to the accurate interpretation of results obtained in the evaluation of such products.
For any given experimental hut study, a minimum average hut-entry rate is usually required for the study to have enough statistical power to demonstrate the expected impact . As expected of most rice growing areas in West Africa, the Cové experimental hut site is characterized by very high mosquito vector densities, which resulted in high hut-entry rates, making the site suitable for most Phase II product evaluation studies. Nevertheless, the results also showed seasonality in vector density linked to the rice cropping season with the lowest hut entry recorded in the months of June–September, a period during which no rice growing activity is ongoing and rice paddies are dry. Assuming that the rice cropping seasons in Cové remain unchanged, plans have been made to exclude this period where possible when planning hut evaluation studies in this site.
The resistance bioassays demonstrated very high levels of phenotypic resistance to pyrethroids and DDT in the Cové wild An. gambiae s.l. population, revealing a resistance ratio of 20-fold (compared to a susceptible laboratory strain) which far exceeds the minimum of ten-fold set by the VCAG for the evaluation of new LLINs with efficacy claims against highly pyrethroid-resistant mosquitoes . This was confirmed by the low mortality rates (<30 %) achieved with standard WHOPES-approved pyrethroid LLINs and IRS in the experimental huts in Cové. Mortality rates of >80 % with pyrethroid IRS and insecticide-treated nets have been reported in earlier experimental hut studies in a previously pyrethroid-susceptible area in Benin . The findings here constitute further evidence of the poor performance of current pyrethroid IRS and LLIN products when confronted with highly pyrethroid-resistant vector populations [9, 14, 23, 24]. The novel IRS and LLIN products being tested in the experimental huts in Cové involve non-pyrethroid classes of insecticide with new modes of action to which there are currently no records of resistance in malaria vectors; hence, improved mortality rates are expected.
The genotyping results revealed that the vector population in the Cové experimental hut station is composed of a mixture of An. coluzzii and An. gambiae s.s. with their relative abundance varying through the course of the year. While all samples genotyped from the March cropping season (start of the rainy season in 2014) were An. coluzzi, An. gambiae s.s. was found in a smaller proportion (23 %) only in samples that were collected at the start of the October annual rice cropping season in the Cové site (start of the dry season in 2013). Seasonal variation in these two sub-species of An. gambiae has been reported in several areas in West Africa [25, 26]. Unlike the An. coluzzi which is well adapted to rice paddies in West Africa, An. gambiae s.s. tends to occupy transitory man-made breeding sites, such as pits, ponds and puddles which are more likely to exist in the dry season in Cové. Nevertheless, because both species contribute similarly to disease transmission and are anthropophilic and endophilic , they can be equally targeted by the various novel IRS and LLIN products being evaluated in the experimental huts in Cové. Further studies are underway to investigate the extent of the variation in both vector sub-species in Cové through the course of the year, in relation to other human activities at the site and to assess the impact it may have on phenotypic resistance to pyrethroids.
The resistance genotyping studies suggests that the pyrethroid resistance encountered in the Cové vector population could be partly attributable to high frequencies of the 1014F kdr allele (0.89 allele frequency), with the 1014S mutation absent and the 1575Y allele very rare. The latter should be monitored however because it can effectively double the resistance conferred by 1014F alone . Ace-1 119S was also very rare, which likely explains the lack of any carbamate or organophosphate resistance. However, again this requires monitoring because it is strongly diagnostic of resistance to each insecticide class  and can confer very high resistance in combination with P450 metabolic enzymes . Indeed, some vector populations in West Africa with high frequencies of pyrethroid resistance mutations and the ace-1 119S mutation are resistant to all current classes of insecticides . The PBO synergism data suggested a moderate impact of P450 enzymes in resistance, which was confirmed by the microarray study that identified two P450 genes as over-expressed in pyrethroid-selected An. coluzzi in Cové. Of these, CYP6P3 has been previously validated as a metabolizer of class I and II pyrethroids  and bendiocarb to a lesser extent . CYP6P3 has been reported as over-expressed in pyrethroid-resistant field populations of An. gambiae s.l. from Dodowa in Ghana , Akron and Gbejromede in Benin, Orugun in Nigeria , and Tiassale in Côte d’Ivoire  and is thus operationally considered a key diagnostic marker of pyrethroid resistance. Further studies will be needed to investigate the other genes which were over-expressed in the microarray study for their roles in conferring pyrethroid resistance.
In addition to improving the control of pyrethroid-resistant malaria vectors, the novel LLIN and IRS products evaluated at the Cové site are expected to also manage pyrethroid resistance since they are treated with insecticide mixtures [4, 33]. To investigate this, surviving mosquitoes from the different experimental hut treatments have been preserved in RNAlater for further studies comparing their capacity to prevent selection on insecticide resistance genes. Based on the microarray results obtained in the present study, CYP6P3 can be considered a suitable candidate gene for investigating selection of metabolic resistance to pyrethroids in follow-up quantitative real time PCR studies.
CN, RN, DW, and MR designed the study. CN JF, KS, AF, and AO performed the experiments, CN and DW analysed the data. CN wrote the paper and DW, KS and MR made important contributions to the manuscript. The fieldwork was supervised by CN, RN and MA. All authors read and approved the final manuscript.
We thank Prof Hilary Ranson (LSTM) for coordinating molecular analysis and Damien Todjinou and Achille Oumbouke (CREC) for field assistance. The study was funded by the Bill and Melinda Gates Foundation through the Innovative Vector Control Consortium (IVCC).
The authors declare they have no competing interests.
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