Comparative assessment of insecticide resistance phenotypes in two major malaria vectors, Anopheles funestus and Anopheles arabiensis in south-eastern Tanzania

Effective use of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) has tremendously reduced malaria transmission in sub-Saharan Africa [1, 2]. Despite this reduction, malaria transmission continues in several areas, driven by mosquitoes that are either physiologically [3‐5] or behaviourally resistant [6‐10] to current insecticide-based interventions. Resistance to commonly used pyrethroids has also necessitated a change of insecticide classes for IRS to either carbamates, organophosphates or more recently neonicotinoids approved by the World Health Organization (WHO) [11]. Similarly new insecticide-treated nets (ITNs) are being developed that contain either multiple insecticide classes [12] or pyrethroids and synergists [13, 14], and are expected to improve the control of resistant mosquitoes.

Mosquito resistance involves different mechanisms, through which they can withstand exposures to insecticides. These include metabolic resistance, target-site resistance, behavioural resistance, and cuticular resistance [15‐17]. The discriminating concentration is used to evaluate the phenotypic resistance which when detected, the level of resistance can be subsequentlly determined by using intensity assays. The intensity assays uses the 5× and 10× discriminating concentration in a stepwise manner aiming at providing the information on the range of resistance present in a target vector [18]. Mosquitoes that express metabolic forms of resistance produce large quantities of enzymes or alternate the enzyme catalytic centre to efficiently detoxify the insecticide. The specific enzymes include monooxygenases (i.e. cytochrome P450s), which detoxify pyrethroids and carbamates, glutathione-S-transferases (GSTs), which detoxify organochlorides like DDT [17], and esterases, which detoxify pyrethroids and organophosphates [19, 20]. The degree to which the enzymatic proteins are expressed, and the level of resistance can be assessed using quantitative polymerase chain reaction (qPCR). Phenotypic assays use synergists, such as piperonyl butoxide (PBO), which enhance the potency of an insecticide by inhibiting the enzymes responsible for the insecticide metabolism [21]. On the other hand, some mosquitoes may have one or multiple target-site mutations due to modification of protein receptors usually targeted by insecticides (e.g. the voltage-gated sodium channels targeted by pyrethroids and organochlorides), thereby blocking or reducing the effectiveness of the insecticides [22‐24]. Recently, scientists have also demonstrated that a sensory appendage protein (SAP2) enriched in the legs of malaria-carrying mosquitoes can also confer resistance to insecticides, thus allowing these mosquitoes to survive contact with ITNs [25].

Different Anopheles species have diverse levels of competencies in pathogen transmission, and also respond different to interventions based on their behaviour and physiology [26, 27]. With the rise of insecticide resistance in the vector populations, the choice of interventions will depend on characteristics of the local vectors. Comprehensive understanding of the distribution and underlying mechanisms of insecticide resistance is, therefore, important for planning and implementing vector control interventions.

In the villages of south-eastern Tanzania, where Anopheles funestus have been reported to be implicated in most of the ongoing malaria transmission [28‐30], signs of resistance to most public health pesticides have been observed. Anopheles funestus also appears to survive longer than other co-existing vector species (parity rates are higher than Anopheles arabiensis) [28]. However, since the species is highly anthropophagic (prefers to blood-feed on humans over that of other vertebrates) [31, 32] and endophilic (prefers to bite indoors) [33], one would expect its populations and transmission activity to have been significantly reduced by ITNs now widely used in Tanzania for more than one decade [34‐37]. Indeed, historical evidence from both East and Southern Africa suggests that effective insecticide-based indoor interventions can eliminate An. funestus on a local scale [38, 39]. Anopheles gambiae sensu stricto (s.s.), which is generally considered the most competent malaria vector, shares similar behaviours with An. funestus, i.e. high degree of anthropophily and endophilly [31, 32, 40]. However, An. gambiae s.s. unlike An. funestus has been highly impacted by ITNs in Kenya and Tanzania [41‐43].

An important question, therefore, is why and how An. funestus, despite being highly anthropophagic and endophilic, survived the ITN onslaught, and why it continues to mediate most malaria transmission in rural south-eastern Tanzania despite co-occurrence with a different malaria vector species, An. arabiensis. One hypothesis has been that An. funestus expresses higher intensities of resistance to most of the commonly used insecticides in comparison to other malaria vectors and is, therefore, far less impacted by insecticidal interventions. A meta-analysis of various datasets has verified this phenomenon at global scale [44], but specific field tests comparing resistance intensities in the different vector species are limited. As shown by Kaindoa et al. in a study conducted in south-eastern Tanzania, An. funestus was resistant to pyrethroids, organochlorides, and carbamates [28]. Another study from the same study area demonstrated that resistance of An. arabiensis to diagnostic insecticide concentrations varied between nearby locations and seasons [45]. However, the intensity and mechanisms of these resistance phenotypes were not compared between species.

This study, therefore, compared the intensities and knockdown time of insecticide-resistance between the two main malaria vectors, An. funestus and An. arabiensis, in rural south-eastern Tanzania. Potential involvement of metabolic resistance and insecticide potency enhancement by Piperonyl Butoxide (PBO) synergists was also assessed.

In this study, both An. arabiensis and An. funestus were resistant to pyrethroids and DDT. However, An. funestus exhibited far lower mortalities when subjected to pyrethroids at either the baseline concentration, five times concentration or the ten times concentration in the intensity bioassays. This suggests that while An. funestus is strongly resistant to the pyrethroids, the level of resistance in An. arabiensis was either low or moderate. This is the first study to directly compare resistance intensities of these two vectors in the area, and therefore provides important information on potential performance of current or future interventions against malaria. Given the differential contribution of the two vectors to overall transmission, their responsiveness to insecticidal interventions is an important factor for consideration in the elimination efforts.

Initial findings from standard WHO susceptibility assays by Kaindoa et al. [28] and Matowo et al. [45] on the two malaria vectors in the same study area observed that the baseline mortalities were higher in An. arabiensis than An. funestus. This was the initial indication that the intensity of resistance would be different between the two species, and necessitated additional tests according to standard WHO assays [18]. The duration at which either 50% or 95% of mosquitoes would be knocked-down varied between species, insecticides and study villages (Table 1).

Knock-down time was high when mosquitoes were exposed to standard concentrations of pyrethroids, but slowed as the concentrations increased. Since no kdr mutation tests were done, it is not possible to determine whether these observations were associated with the voltage-gated sodium channel protein mutation [57]. The slowest knockdown time was observed when the mosquito species were exposed to bendiocarb, these findings still support the potency of the insecticide (Table 1). The new findings clearly demonstrate that An. funestus populations, despite being the more dominant vector of malaria in the area, would be much more difficult to control using current pyrethroid-based interventions, in particular the LLINs.

As demonstrated by Matowo et al. for both An. arabiensis and Culex mosquitoes [45, 58], there were signs of fine-scale spatial variations in insecticide resistance. For example, An. funestus populations from Tulizamoyo were resistant to bendiocarb but populations of the same species from the other two villages were susceptible to the same chemical (Fig. 2). Similarly, the mortality percentages observed at 5× and 10× doses varied between the villages (Figs. 2, 3). This might be attributed to differences in the use of agricultural pesticides for crop protection in these villages [59]. Surprisingly, An. arabiensis from Ikwambi were 100% susceptible to DDT, against which both species from the other study villages were resistant (Fig. 2), which further suggests fine-scale spatial differences in resistance profiles.

The currently observed dominance of An. funestus is likely to be contributed by their well-documented resistance to commonly used insecticides [28, 60‐64], their high survival probabilities in the wild [28, 30] and high levels of anthropophily [28, 31, 33]. Its dominance in areas where insecticidal interventions such as ITNs are widely implemented is particularly surprising given that scale-up of ITNs has coincided with significant declines in populations of other anthropophilic vectors such as An. gambiae s.s. [41‐43]. Today, in rural south-eastern Tanzania, An. funestus co-exists with other Anopheles species, namely An. arabiensis, An. leesoni, Anopheles coustani, Anopheles squamosus, Anopheles rivulorum and Anopheles pharoensis [28]. However, it is known to carry most of the ongoing malaria transmission, sometime implicating to nearly nine in every ten new cases, even in areas where it occurs in lower densities than An. arabiensis [28, 29]. It was thus hypothesized that its dominance may at least be partly driven by stronger insecticide resistance levels to insecticides commonly used for public health, notably the pyrethroids used on bed nets.

As insecticide resistance increases across Africa, some populations have been observed to withstand up to 1000 times the standard concentrations [65], making it an urgent need to find new classes or combinations of insecticides [3‐5, 17]. In areas where An. funestus is dominant, such as in south-eastern Tanzania, the decisions on which insecticides to be implemented in vector control measures should reflect intensity of resistance in this species, even if it is difficult to find its larvae. Anopheles funestus were resistant up to ten times the WHO-recommended concentration of pyrethroids, clearly indicating that this class of insecticides can no longer be useful in the area and must be urgently replaced by other classes such as organophosphates, against which resistance is not yet detected.

The synergist tests in this study showed complete or almost complete restoration of susceptibility in the malaria vector mosquitoes nearly from all study areas. This full restoration is a likely indicator of metabolic resistance [58, 66] and suggests that ITNs which have both PBO and pyrethroids, such as PermaNet 3.0 [14] and Olyset Plus [13], may be suitable for malaria prevention in these areas, and could potentially provide better protection than standard LLINs [67]. Synergist pre-exposure combined with deltamethrin had a greater restoration in An. funestus than when the synergist was combined with permethrin (Fig. 4), but in both cases there was still substantial restoration. This could be likely due to different resistance levels against the two pyrethroid classes as observed by Rakotoson et al. [68] when An. arabiensis were pre-exposed to PBO. Partial restoration of susceptibility observed in An. funestus mosquitoes might be a sign of multiple metabolic resistance forms or other resistance mechanisms including the target-site mutation [69]. This could also be a manifestation of the demonstrated high intensities of pyrethroid resistance (Fig. 3). These findings are in line with the previous studies on the resistance of malaria vectors to pyrethroids and organochlorides and incomplete susceptibility restoration after the synergist pre-exposure to pyrethroids [68]. Nonetheless, further exploration is needed to identify the specific metabolic enzymes responsible for the observed resistance under biochemical tests. Additionally, the level of these resistant enzymes needs to be assessed using quantitative PCR assays in both An. arabiensis and An. funestus.

Despite largely achieving the stated aims, the findings of this study should be considered only as indicative and not in any way conclusive. This is due to the various methodological limitations faced during the study. First, the overall collection of the specimen was distributed over several months, and may bave been influenced by seasonal variations in resistance, as previously demonstrated [45]. Besides, given the scarcity of An. funestus specimen in some of the villages, the tests used just 120 mosquitoes. Third, this study was the use of wild mosquitoes which may have varying ages, which is an important factor long-demonstrated to impact resistance [70‐72].

While this way of testing gives a true representation of the natural mosquito population in communities and their ability to withstand insecticidal interventions, it makes it difficult to compare the tests in a conclusive manner. The WHO guidelines recommend the use of age-synchronized F₁ generation, 3–5 days old [18]. In this study the challenge was minimized by: (a) collecting the adult female mosquitoes at the edges of the village near potential aquatic habitats, thus maximizing the chances of getting young nulliparous mosquitoes [49], (b) adding an acclimatization period of mosquitoes for 24 h between the actual mosquito collection and the resistance tests, and (c) using the CDC light trap for mosquito collection, thereby capitalizing collection of nulliparous host-seeking mosquitoes [73‐75]. In addition, the tests did not combine collections from multiple days, but instead used synchronized days for each replicate, thus ensuring that the mosquito ages were approximately similar. It is recognized however that these improvements slightly improved the tests but are not adequate to enable conclusive determination or comparison of resistance levels.

Another limitation was the non-amplification of the samples where 8% (n = 12) of An.arabiensis and 24% (n = 62) of An. funestus complex were unidentifiable. It is possible that either there were polymorphisms in the ITS2 region of rDNA amplified in these assays, which might have been the main contributor of the observed non-amplification (Mapua et al., unpublished data), or there were a few other sibling species for which no primers were available in the assay. Future studies should involve a larger sample size, and possibly individual analysis of specimen to distinguish between species.

Overall, this study has demonstrated that other than the differential importance of malaria vector species and the multiplicity of malaria transmission in different settings, the responsiveness of these vectors towards different insecticides may also vary. In rural south-eastern Tanzania, An. funestus, which now dominates malaria transmission, also indicate stronger resistance to pyrethroids commonly used on ITNs than its counterpart, An. arabiensis. Despite its rarity at aquatic stage, collection methods must endeavour to find this vector with synchronized age and study its resistance profile so that effective interventions can be mounted. Lastly, the study also emphasizes that decisions on which insecticidal interventions to apply should be informed by geographical and species-specific studies rather than generalized studies. In this cases, it appears that PBO-based LLINs and IRS with non pyrethroids, such as organophosphates may be appropriate for now, as the main vectors are still susceptible to these treatments.