The currently observed dominance of An. funestus is likely to be contributed by their well-documented resistance to commonly used insecticides [27, 57–61], their high survival probabilities in the wild [27, 29] and high levels of anthropophily [27, 30, 32]. Its dominance in areas where insecticidal interventions such as ITNs are widely implemented is particularly suprising given that scale-up of ITNs has coincided with significant declines in populations of other anthropophilic vectors such as An. gambiae s.s. [40–42]. Today, in rural south-eastern Tanzania, An. funestus co-exists with other Anopheles species, namely An. arabiensis, Anopheles coustani, Anopheles squamosus, Anopheles leesoni, Anopheles rivulorum and Anopheles pharoensis . However, it is known to carry most of the ongoing malaria transmission, sometime mediating nearly nine in every ten new cases, even in areas where it occurs in lower densities than An. arabiensis [27, 28]. 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.
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  and Matowo et al  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 necessaitated additional tests according to standard WHO assays . 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 [44, 62], 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 . 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.
As insecticide resistance increases across Africa, some populations have been observed to withstand up to 1000 times the standard concentrations , 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. An. 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 [62, 65] and suggests that ITNs which have both PBO and pyrethroids such as PermaNet 3.0  and Olyset Plus  may be suitable for malaria prevention in these areas, and could potentially provide better protection than standard LLINs . 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  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 . 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 . 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.
One limitation of this study was the use of wild mosquitoes which may have varying ages, which is an important factor long-demonstrated to impact resistance [69–71]. However, this way of testing gives a true representation of the natural mosquito population in communities, and their ability to withstand insecticidal interventions. The WHO guidelines recommend the use of F1 generation, 3–5 days old . In this study therefore, this limitation 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 , b) adding an acclimatization period of mosquitoes for 24 hours 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 [72–74]. 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.
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 unidentifyiable. 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.
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, is also much more resistant 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 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 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.