Metabolic resistance to insecticides such as pyrethroids in mosquito vectors threatens control of malaria in Africa. Unless it is managed, recent gains in reducing malaria transmission could be lost. To improve monitoring and assess the impact of insecticide resistance on malaria control interventions, we elucidated the molecular basis of pyrethroid resistance in the major African malaria vector, Anopheles funestus. We showed that a single cytochrome P450 allele (CYP6P9a_R) in A. funestus reduced the efficacy of insecticide-treated bednets for preventing transmission of malaria in southern Africa. Expression of key insecticide resistance genes was detected in populations of this mosquito vector throughout Africa but varied according to the region. Signatures of selection and adaptive evolutionary traits including structural polymorphisms and cis-regulatory transcription factor binding sites were detected with evidence of selection due to the scale-up of insecticide-treated bednet use. A cis-regulatory polymorphism driving the overexpression of the major resistance gene CYP6P9a allowed us to design a DNA-based assay for cytochrome P450–mediated resistance to pyrethroid insecticides. Using this assay, we tracked the spread of pyrethroid resistance and found that it was almost fixed in mosquitoes from southern Africa but was absent from mosquitoes collected elsewhere in Africa. Furthermore, a field study in experimental huts in Cameroon demonstrated that mosquitoes carrying the resistance CYP6P9a_R allele survived and succeeded in blood feeding more often than did mosquitoes that lacked this allele. Our findings highlight the need to introduce a new generation of insecticide-treated bednets for malaria control that do not rely on pyrethroid insecticides.

Background Insecticide resistance poses a serious threat to insecticide-based interventions in Africa. There is a fear that resistance escalation could jeopardize malaria control efforts. Monitoring of cases of aggravation of resistance intensity and its impact on the efficacy of control tools is crucial to predict consequences of resistance. Methods The resistance levels of an Anopheles funestus population from Palmeira, southern Mozambique, were characterized and their impact on the efficacy of various insecticide-treated nets established. Results A dramatic loss of efficacy of all long-lasting insecticidal nets (LLINs), including piperonyl butoxide (PBO)–based nets (Olyset Plus), was observed. This An. funestus population consistently (2016, 2017, and 2018) exhibited a high degree of pyrethroid resistance. Molecular analyses revealed that this resistance escalation was associated with a massive overexpression of the duplicated cytochrome P450 genes CYP6P9a and CYP6P9b, and also the fixation of the resistance CYP6P9a_R allele in this population in 2016 (100%) in contrast to 2002 (5%). However, the low recovery of susceptibility after PBO synergist assay suggests that other resistance mechanisms could be involved. Conclusions The loss of efficacy of pyrethroid-based LLINs with and without PBO is a concern for the effectiveness of insecticide-based interventions, and action should be taken to prevent the spread of such super-resistance.

Elucidating the genetic basis of metabolic resistance to insecticides in malaria vectors is crucial to prolonging the effectiveness of insecticide-based control tools including long lasting insecticidal nets (LLINs). Here, we show that cis-regulatory variants of the cytochrome P450 gene, CYP6P9b, are associated with pyrethroid resistance in the African malaria vector Anopheles funestus. A DNA-based assay is designed to track this resistance that occurs near fixation in southern Africa but not in West/Central Africa. Applying this assay we demonstrate, using semi-field experimental huts, that CYP6P9b-mediated resistance associates with reduced effectiveness of LLINs. Furthermore, we establish that CYP6P9b combines with another P450, CYP6P9a, to additively exacerbate the reduced efficacy of insecticide-treated nets. Double homozygote resistant mosquitoes (RR/RR) significantly survive exposure to insecticide-treated nets and successfully blood feed more than other genotypes. This study provides tools to track and assess the impact of multi-gene driven metabolic resistance to pyrethroids, helping improve resistance management.

Malaria remains the deadliest vector-borne disease in the world. With nearly half of the world's population at risk, 216 million people suffered from malaria in 2016, with over 400,000 deaths, mainly in sub-Saharan Africa. Important global efforts have been made to eliminate malaria leading to significant reduction in malaria cases and mortality in Africa by 42% and 66%, respectively. Early diagnosis, improved drug therapies and better health infrastructure are key components, but this extraordinary success is mainly due the use of long-lasting insecticidal nets (LLINs) and indoor residual sprayings (IRS) of insecticide. Unfortunately, the emergence and spread of resistance in mosquito populations against insecticides is jeopardising the effectiveness of the most efficient malaria control interventions. To help establish suitable resistance management strategies, it is vital to better understand the distribution of resistance, its mechanisms and impact on effectiveness of control interventions and malaria transmission. In this chapter, we present the current status of insecticide resistance worldwide in main malaria vectors as well as its impact on malaria transmission, and discuss the molecular mechanisms and future perspectives.

Metabolic resistance to insecticides threatens malaria control. However, little is known about its fitness cost in field populations of malaria vectors, thus limiting the design of suitable resistance management strategies. Here, we assessed the association between the glutathione S-transferase GSTe2-mediated metabolic resistance and life-traits of natural populations of Anopheles funestus. A total of 1200 indoor resting blood-fed female An. funestus (F0) were collected in Mibellon, Cameroon (2016/2017), and allowed to lay eggs individually. Genotyping of F1 mosquitoes for the L119F-GSTE2 mutation revealed that L/L119-homozygote susceptible (SS) mosquitoes significantly laid more eggs than heterozygotes L119F-RS (odds ratio (OR) = 2.06; p < 0.0001) and homozygote resistant 119F/F-RR (OR = 2.93; p < 0.0001). L/L119-SS susceptible mosquitoes also showed the higher ability for oviposition than 119F/F-RR resistant (OR = 2.68; p = 0.0002) indicating a reduced fecundity in resistant mosquitoes. Furthermore, L119F-RS larvae developed faster (nine days) than L119F-RR and L119F-SS (11 days) (X2 = 11.052; degree of freedom (df) = 4; p = 0.02) suggesting a heterozygote advantage effect for larval development. Interestingly, L/L119-SS developed faster than 119F/F-RR (OR = 5.3; p < 0.0001) revealing an increased developmental time in resistant mosquitoes. However, genotyping and sequencing revealed that L119F-RR mosquitoes exhibited a higher adult longevity compared to RS (OR > 2.2; p < 0.05) and SS (OR > 2.1; p < 0.05) with an increased frequency of GSTe2-resistant haplotypes in mosquitoes of D30 after adult emergence. Additionally, comparison of the expression of GSTe2 revealed a significantly increased expression from D1-D30 after emergence of adults (Anova test (F) = 8; df= 3; p = 0.008). The negative association between GSTe2 and some life traits of An. funestus could facilitate new resistance management strategies. However, the increased longevity of GSTe2-resistant mosquitoes suggests that an increase in resistance could exacerbate malaria transmission.

Background The scale-up in the distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying has significantly reduced malaria burden and mortality. However, insecticide resistance, among other factors, is responsible for a recent rebound in malaria transmission in 2015–2016, threatening the progress so far made. As a contribution towards understanding patterns of resistance and its mechanism in the field we characterized a population of Anopheles gambiae ( s.l .) from Gounougou, a Guinea savanna of north/central Cameroon. Results Indoor collection conducted in September 2017 identified Anopheles coluzzii and Anopheles arabiensis as the unique Anopheles vector species, with abundances of 83 and 17%, respectively. Analysis of infection with TaqMan assays using heads/thoraces of indoor collected females of An. coluzzii revealed a low Plasmodium falciparum parasite rate of 4.7%. Bioassays conducted with female An. coluzzii revealed extreme resistance, with low mortalities of only 3.75 ± 1.25%, 3.03 ± 1.59% and 1.45 ± 1.45%, respectively, for permethrin, deltamethrin and DDT. In contrast, high susceptibility was obtained with the organophosphates and carbamates, with mortalities in the range of 98–100%. Synergist assays with piperonyl butoxide (PBO) recovered some susceptibility with increased mortality for permethrin to 14.88 ± 8.74%, and for deltamethrin to 32.50 ± 10.51% (~27-fold increase compared to mortalities with deltamethrin alone, χ 2 = 29, df = 1, P < 0.0001). These correlated with the results of cone bioassays which revealed complete loss of efficacy of Olyset®Net (0% mortality) and PermaNet®2.0 (0% mortality), and the considerable loss of efficacy of Olyset®Plus (mortality of 2 ± 2%), PermaNet®3.0 side panel (mortality of 2 ± 2%) and PermaNet3.0® roof (mortality of 16 ± 5.1%). Time-course bioassays conducted with deltamethrin established a high intensity of resistance, with LT 50 of 309.09 (95% CI 253.07–393.71, Fiducial), and a resistance ratio of 93.09 compared with the fully susceptible Ngoussou laboratory colony. TaqMan genotyping revealed a high frequency of the 1014F allele (65.25%) in the An. coluzzii populations. Sequencing of a fragment of the voltage-gated sodium channel identified a single An. arabiensis female harbouring the 1014S kdr mutation. Conclusions This finding of high pyrethroid and DDT resistance in An. coluzzii from north-central Cameroon is a major obstacle to malaria control using pyrethroid bednets and indoor residual spraying with DDT.

Metabolic resistance to insecticides is threatening malaria control in Africa. However, the extent to which it impacts malaria transmission remains unclear. Here, we investigated the association between a marker of glutathione S-transferase mediated metabolic resistance and Plasmodium infection in field population of Anopheles funestus s.s. in comparison to the A296S-RDL target site mutation. The 119F-GSTe2 resistant allele was present in southern (Obout) (56%) and central (Mibellon) (25%) regions of Cameroon whereas the 296S-RDL resistant allele was detected at 98.5% and 15% respectively. The whole mosquito Plasmodium and sporozoite infection rates were 57% and 14.8% respectively in Obout (n = 508) and 19.7% and 5% in Mibellon (n = 360). No association was found between L119F-GSTe2 genotypes and whole mosquito infection status. However, when analyzing oocyst and sporozoite infection rates separately, the resistant homozygote 119F/F genotype was significantly more associated with Plasmodium infection in Obout than both heterozygote (OR = 2.5; P = 0.012) and homozygote susceptible (L/L119) genotypes (OR = 2.10; P = 0.013). In contrast, homozygote RDL susceptible mosquitoes (A/A296) were associated more frequently with Plasmodium infection than other genotypes (OR = 4; P = 0.03). No additive interaction was found between L119F and A296S. Sequencing of the GSTe2 gene showed no association between the polymorphism of this gene and Plasmodium infection. Glutathione S-transferase metabolic resistance is potentially increasing the vectorial capacity of resistant An . funestus mosquitoes. This could result in a possible exacerbation of malaria transmission in areas of high GSTe2-based metabolic resistance to insecticides.

Monitoring cases of insecticide resistance aggravation and the effect on the efficacy of control tools is crucial for successful malaria control. In this study, the resistance intensity of major malaria vectors from Uganda was characterised and its impact on the performance of various insecticide-treated nets elucidated. High intensity of resistance to the discriminating concentration (DC), 5× DC, and 10× DC of pyrethroids was observed in both Anopheles funestus and Anopheles gambiae in Mayuge and Busia leading to significant reduced performance of long-lasting insecticidal nets (LLINs) including the piperonyl butoxide (PBO)-based nets (Olyset Plus). Molecular analysis revealed significant over-expression of cytochrome P450 genes (CYP9K1 and CYP6P9a/b). However, the expression of these genes was not associated with resistance escalation as no difference was observed in the level of expression in mosquitoes resistant to 5× DC and 10× DC compared to 1× DC suggesting that other resistance mechanisms are involved. Such high intensity of pyrethroid resistance in Uganda could have terrible consequences on the effectiveness of insecticide-based interventions and urgent action should be taken to prevent the spread of super-resistance in malaria vectors.