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

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 [3]. 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 [4]. 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 [5]. 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 [14]. 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) [15] 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 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 [22]. 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 [15]. 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 [23]. 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 [26], 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 [19]. 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 [27] and can confer very high resistance in combination with P450 metabolic enzymes [28]. 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 [29]. 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 [30] and bendiocarb to a lesser extent [28]. CYP6P3 has been reported as over-expressed in pyrethroid-resistant field populations of An. gambiae s.l. from Dodowa in Ghana [31], Akron and Gbejromede in Benin, Orugun in Nigeria [32], and Tiassale in Côte d’Ivoire [28] 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.