Mosquito populations in West African countries display a high level of resistance and multiple resistance profiles [34]. Country-wide surveys over the last decade in Benin have reported that pyrethroid resistance is widespread in malaria vector populations [35–40]. Investigating tools that can complement or replace existing ones is therefore necessary to strengthen resistance management plans (27). This study provides key information on the performance of dual active ingredient ITNs, IG2, P3, and RG, against a pyrethroid-resistant population of An. gambiae s.s. from Za-Kpota in Southern Benin using experimental hut trials (EHTs).
Following the WHO susceptibility test, a low mortality rate was recorded for pyrethroid insecticides (permethrin and deltamethrin) in An. gambiae s.s. from Za-Kpota. This confirms resistance to those insecticide, which has also been reported in the neighbouring localities of Za-Kpota in Southern Benin (28, 29). Regarding the mortality rates recorded against deltamethrin in the population from Za-Kpota, a synergist assay with 4 % PBO as carried out, which revealed a potential involvement of cytochrome P450 genes in observed phenotypic resistance. This indicates that the pyrethroid resistance observed might also be driven by metabolic resistance mechanisms in Za-Kpota’s population of An. gambiae s.s.
For our experimental hut trial, the 72-hour mortality rates were considered. The pyrethroid-PBO based net (P3) has also displayed higher toxicity against field free-flying pyrethroid resistant An. gambiae s.s. when compared to the pyrethroid-only based net (P2). P3 is treated with a high concentration of deltamethrin on the side panels (2.1g/kg) and (4.0g/kg plus PBO (25g/kg) on the roof panel). Thus, the high toxicity observed with P3 could be attributed to the presence of an amount of active ingredients on this net. Given the well-known synergistic effect of the PBO on pyrethroid resistance and the data from the WHO susceptibility test, the difference in killing effect recorded can be due to the partial or complete restoration of susceptibility to deltamethrin, thus increasing P3 net efficacy over P2 net. The same trend was observed between Olyset and Olyset Plus nets (30) confirming the improved protective role of PBO nets on malaria prevalence in area where the resistance phenotype is mainly driven by P450 genes [8–13].
Nevertheless, P3 doesn't provide total personal protection (the mortality induced below the 80%). These results highlight the possible presence of resistance mechanisms other than metabolic resistance mediated by monooxygenases in the vector population. This pointed out a worry concerning the efficacy of P3 nets, which have already been reported in several other studies in Southern Benin (31, 32). Since studies have questioned its real performance against resistant malaria vectors where mechanisms such as GST-based metabolic resistance occurred (33), much attention is being paid to the new dual-active ingredient insecticide-treated nets (IG2 and RG) containing each, two products with different modes of action. Interestingly in this study, higher mortality rate was recorded with IG2 and RG nets against pyrethroid-resistant An. gambiae s.s. compared to the standard pyrethroid-only net P2.
The highest mortality observed with IG2 nets could be due to the combined toxic effects of alpha-cypermethrin and chlorfenapyr. Chlorfenapyr is activated when the N-ethoxymethyl group is removed by oxidation mediated by some cytochrome P450 enzymes, producing the toxic metabolite tralopyril (34). The later disturb the proton gradient across mitochondrial membranes and impair ATP production (oxidative phosphorylation), leading to cell death (35, 36). As demonstrated in this study, cytochrome P450 enzymes are involved in pyrethroid resistance in in Za-Kpota’s population of An. gambiae s.s. Thus, overexpression of these enzymes may enhance chlorfenapyr activation (37–39). This could increase the toxicity of nets with insecticide combinations of pyrethroids and chlorfenapyr. The similar observation has been pointed out in several experiments in the huts (40–42).
This study also showed that while inducing high mortality compared to P2, IG2 aged induced significantly low mortality compared to new IG2. This could be due to a decrease in the insecticide content over time of ITNs in operational use, as demonstrated in several studies (43, 44). In contrast, Martin et al.(45) have demonstrated that after 20 washes (supposed to mimic a 36 months field net) IG2 washed did not induce a significantly different mortality from IG2 unwashed, confirming the wash resistance of this net. This contrast reveals that the decrease in insecticide content of insecticide-treated nets over time would not be proportional to its reduction after washing. Future new nets durability studies should consequently focus more on aged nets, whose aging in the field considers their environmental use conditions (temperature, humidity).
In addition to the high toxicity displayed by IG2, RG and P3 nets, they were able to provide irritancy properties, allowing them to inhibit blood feeding in pyrethroid resistant An. gambiae s.s. from Za-Kpota. However, only RG net significantly reduced the blood feeding compared to standard P2 net. Of the ITNs evaluated in this study, the RG ITN was treated with the highest concentration of alpha-cypermethrin (216mg/m2). This significant reduction in blood feeding induced by RG could be due to the high alphacypermethrin concentration. The latter could trigger an avoidance behaviour in the vectors(46) and therefore affect their ability to take blood meal. As a key parameter influencing malaria transmission potential(47), blood feeding behaviour observed with new nets could be beneficial for malaria vector control (40–42). This confirms that high-dose pyrethroids would continue to have a valuable role concerning blood-feeding inhibition and personal protection (48).
In addition to the standard parameters such as mortality and blood feeding, sublethal exposure effect could provide complementary valuable information to better appreciate the performance of vector control strategies. It may be regarded as reductions in longevity, development rates, feeding, oviposition, fertility, fecundity, changes in sex ratio or changes in behaviour (49). Several studies have shown that insecticide resistance mechanisms can negatively affect the reproductive fitness and longevity of Anopheles mosquitoes (50–52). In this study, we analysed sublethal ITN exposure in collected An. gambiae s.s. through ovipositing, fecundity, fertility, and longevity in which a risk of death has been estimated. No significant difference in ovipositing rate in collected blood-fed An. gambiae s.s. with standard P2 net and the other ITNs was observed.
Exposure to dual-ingredient nets (IG2, IG2 aged, P3 and P3 aged) induced a reduction in fecundity compared to the standard P2 net. This may be due to the synergistic action of alphacypermethrin, PBO, or chlorfenapyr on fecundity in resistant mosquitoes. This could be explained by a resource-based trade-off between fecundity and survival (53). It is a physiological reaction flow that could be activated when mosquitoes are exposed to dual-ingredient nets. It causes an overuse of energy which impacts resource availability for fecundity. Such regulation could indicate a high cost of adaptation linked to insecticide resistance (54) which has been described in previous studies (55, 56). The reduction in fecundity would logically translate, from an epidemiological point of view, into a reduction in vector density and, hence, a reduction in transmission (57).
Further, reducing vector longevity is the objective of current insecticide-based malaria control methods and insecticide resistance management strategies. In this study, the risk of death was very high with the two-ingredient nets, with IG2 inducing the highest mortality. This shows once more the efficacy of these nets, and pre-eminently the combined toxic effects of alpha-cypermethrin and chlorfenapyr. Chlorfenapyr has a delayed impact (58) due to its mode of activation, so it could persist for several days after exposure, thus disrupting several life-history traits of the mosquito, including longevity. Together, this indicates that new net in the community is likely to impede the development cycle of vectors and thus contribute to reducing malaria transmission. These generated data add to the data available to better parameterize malaria transmission models in order to better predict the long-term efficacy of new ITNs.