Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii.

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S-transferases. RNA-seq-based genome-wide transcriptional analyses supported by quantitative PCR identified glutathione S-transferase, GSTe2 (RNA-seqFC = 2.93 and qRT-PCRFC = 8.4, p < 0.0043) and CYP450, CYP6Z2 (RNA-seqFC = 2.39 and qRT-PCRFC = 11.7, p < 0.0177) as the most overexpressed detoxification genes in the pyrethroid-resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p < 0.0114), CYP4G16 (FC = 2.02, p < 0.0005), and CYP4G17 (FC = 1.86, p < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace-1 mutation was detected in bendiocarb-resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon.

Clothianidin-resistant Anopheles gambiae adult mosquitoes from Yaoundé, Cameroon, display reduced susceptibility to SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying.

BACKGROUND: Chronic exposure of mosquito larvae to pesticide residues and cross-resistance mechanisms are major drivers of tolerance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid prequalified for Indoor Residual Spraying (IRS). METHODS: Using standard bioassays, we tested if reduced susceptibility to clothianidin can affect the efficacy of SumiShield® 50WG, one of four new IRS formulations containing clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp sampled from urban, suburban and agricultural areas of Yaoundé, Cameroon. RESULTS: We found that in this geographic area, the level of susceptibility to the active ingredient predicted the efficacy of SumiShield 50WG. This formulation was very potent against populations that reached 100% mortality within 72 h of exposure to a discriminating concentration of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield 50WG in An. gambiae adults collected from a farm where the spraying of the two neonicotinoids acetamiprid and imidacloprid for crop protection is likely driving resistance to clothianidin. CONCLUSIONS: Despite the relatively small geographic extend of the study, the findings suggest that cross-resistance may impact the efficacy of some new IRS formulations and that alternative compounds could be prioritized in areas where neonicotinoid resistance is emerging.

Anopheles funestus Populations across Africa Are Broadly Susceptible to Neonicotinoids but with Signals of Possible Cross-Resistance from the GSTe2 Gene.

Evaluating the susceptibility of malaria vectors to the new WHO-recommended products is a key step before large-scale deployment. We mapped the susceptibility profile of Anopheles funestus to neonicotinoids across Africa and established the diagnostic doses of acetamiprid and imidacloprid with acetone + MERO as solvent. Indoor resting An. funestus were collected in 2021 in Cameroon, Malawi, Ghana and Uganda. Susceptibility to clothianidin, imidacloprid and acetamiprid was evaluated using CDC bottle assays and offsprings of the field-caught adults. The L119F-GSTe2 marker was genotyped to assess the potential cross-resistance between clothianidin and this DDT/pyrethroid-resistant marker. Mosquitoes were susceptible to the three neonicotinoids diluted in acetone + MERO, whereas low mortality was noticed with ethanol or acetone alone. The doses of 6 µg/mL and 4 µg/mL were established as diagnostic concentrations of imidacloprid and acetamiprid, respectively, with acetone + MERO. Pre-exposure to synergists significantly restored the susceptibility to clothianidin. A positive correlation was observed between L119F-GSTe2 mutation and clothianidin resistance with the homozygote resistant mosquitoes being more able to survive than heterozygote or susceptible. This study revealed that An. funestus populations across Africa are susceptible to neonicotinoids, and as such, this insecticide class could be effectively implemented to control this species using IRS. However, potential cross-resistance conferred by GSTe2 calls for regular resistance monitoring in the field.

Resistance to clothianidin reduces the efficacy of SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying, against Anopheles gambiae.

Chronic exposure of mosquito larvae to pesticide residues in agricultural areas is often associated with evolution of resistance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid qualified for Indoor Residual Spraying (IRS). Using standard bioassays, we tested if reduced susceptibility to clothianidin affects the efficacy of SumiShield® 50WG, one of the two newly approved formulations, which contains 50% clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield® 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp collected from urban, suburban and agricultural areas of Yaoundé. We found that the level of susceptibility to the active ingredient predicted the efficacy of SumiShield® 50WG. This formulation was very potent against populations that achieved 100% mortality within 72 h of exposure to a discriminating dose of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield® 50WG in An. gambiae adults collected from a farm where spraying of acetamiprid and imidacloprid is driving cross-resistance to clothianidin. These findings indicate that more potent formulations of clothianidin or different insecticides should be prioritized in areas where resistance is emerging.

Marked aggravation of pyrethroid resistance in major malaria vectors in Malawi between 2014 and 2021 is partly linked with increased expression of P450 alleles.

BACKGROUND: Increased intensity of pyrethroid resistance is threatening the effectiveness of insecticide-based interventions to control malaria in Africa. Assessing the extent of this aggravation and its impact on the efficacy of these tools is vital to ensure the continued control of major vectors. Here we took advantage of 2009 and 2014 data from Malawi to establish the extent of the resistance escalation in 2021 and assessed its impact on various bed nets performance. METHODS: Indoor blood-fed and wild female Anopheles (An) mosquitoes were collected with an electric aspirator in Chikwawa. Cocktail and SINE PCR were used to identify sibling species belonging to An. funestus group and An. gambiae complex. The susceptibility profile to the four classes of insecticides was assessed using the WHO tubes bioassays. Data were saved in an Excel file. Analysis was done using Vassarstats and figures by Graph Pad. RESULTS: In this study, a high level of resistance was observed with pyrethroids (permethrin, deltamethrin and alpha-cypermethrin with mortality rate at 5x discriminating concentration (DC) < 50% and Mortality rate at 10x DC < 70%). A high level of resistance was also observed to carbamate (bendiocarb) with mortality rate at 5x DC < 25%). Aggravation of resistance was also noticed between 2009 and 2021. For pyrethroids, the mortality rate for permethrin reduced from 47.2% in 2009 to 13% in 2014 and 6.7% in 2021. For deltamethrin, the mortality rate reduced from 42.3% in 2009 to 1.75% in 2014 and 5.2% in 2021. For Bendiocarb, the mortality rate reduced from 60% in 2009 to 30.1% in 2014 and 12.2% in 2021. The high resistance observed is consistent with a drastic loss of pyrethroid-only bed nets efficacy although Piperonyl butoxide (PBO)-based nets remain effective. The resistance pattern observed was linked with high up-regulation of the P450 genes CYP6P9a, CYP6P9b and CYP6M7 in An. funestus s.s. mosquitoes surviving exposure to deltamethrin at 1x, 5x and 10x DC. A significant association was observed between the 6.5 kb structural variant and resistance escalation with homozygote resistant (SV+/SV+) more likely to survive exposure to 5x and 10x (OR = 4.1; P < 0.001) deltamethrin than heterozygotes. However, a significant proportion of mosquitoes survived the synergist assays with PBO suggesting that other mechanisms than P450s are present. CONCLUSIONS: This resistance aggravation in An. funestus s.s. Malawian population highlights an urgent need to deploy novel control tools not relying on pyrethroids to improve the effectiveness of vector control.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa.

BACKGROUND: New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa. METHODS: Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin. RESULTS: Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin (11.4‒51.9% mortality in Nkolondom, 31.7‒48.2% in Mangoum, 34.6‒56.1% in Mayuge, 39.4‒45.6% in Obuasi, 83.7‒89.3% in Congo and 71.1‒95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Similar observations were done for imidacloprid and acetamiprid. Synergist assays (PBO, DEM and DEF) with clothianidin revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR = 0.5; P = 0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR = 2.10; P = 0.013). CONCLUSIONS: This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone + MERO (4 µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Pyrethroid Resistance Aggravation in Ugandan Malaria Vectors Is Reducing Bednet Efficacy.

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.

Genome-Wide Transcriptional Analysis and Functional Validation Linked a Cluster of Epsilon Glutathione S-Transferases with Insecticide Resistance in the Major Malaria Vector Anopheles funestus across Africa.

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus, with a significant recovery of susceptibility observed when GSTe2, 3, 4, 5 and GSTe6 were silenced. These findings established that GSTe3, 4, 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTes in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon.

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (<40% mortality). An. gambiae s.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae s.s. corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the high Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

Correction: Cytochrome P450 metabolic resistance (CYP6P9a) to pyrethroids imposes a fitness cost in the major African malaria vector Anopheles funestus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exploring the Mechanisms of Multiple Insecticide Resistance in a Highly Plasmodium-Infected Malaria Vector Anopheles funestus Sensu Stricto from Sahel of Northern Nigeria.

The Nigerian Government is scaling up the distribution of insecticide-treated bed nets for malaria control, but the lack of surveillance data, especially in the Sudan/Sahel region of the country, may hinder targeting priority populations. Here, the vectorial role and insecticide resistance profile of a population of a major malaria vector Anopheles funestus sensu stricto from Sahel of Nigeria was characterised. An. funestus s.s. was the only vector found, with a high human blood index (100%) and a biting rate of 5.3/person/night. High Plasmodium falciparum infection was discovered (sporozoite rate = 54.55%). The population is resistant to permethrin (mortality = 48.30%, LT50 = 65.76 min), deltamethrin, DDT (dichlorodiphenyltrichloroethane) and bendiocarb, with mortalities of 29.44%, 56.34% and 54.05%, respectively. Cone-bioassays established loss of efficacy of the pyrethroid-only long-lasting insecticidal nets (LLINs); but 100% recovery of susceptibility was obtained for piperonylbutoxide (PBO)-containing PermaNet®3.0. Synergist bioassays with PBO and diethyl maleate recovered susceptibility, implicating CYP450s (permethrin mortality = 78.73%, χ2 = 22.33, P < 0.0001) and GSTs (DDT mortality = 81.44%, χ2 = 19.12, P < 0.0001). A high frequency of 119F GSTe2 mutation (0.84) was observed (OR = 16, χ2 = 3.40, P = 0.05), suggesting the preeminent role of metabolic resistance. These findings highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in Nigeria.

Cytochrome P450 metabolic resistance (CYP6P9a) to pyrethroids imposes a fitness cost in the major African malaria vector Anopheles funestus.

Metabolic resistance threatens the sustainability of pyrethroid-based malaria control interventions. Elucidating the fitness cost and potential reversal of metabolic resistance is crucial to design suitable resistance management strategies. Here, we deciphered the fitness cost associated with the CYP6P9a (P450-mediated metabolic resistance) in the major African malaria vector Anopheles funestus. Reciprocal crosses were performed between a pyrethroid susceptible (FANG) and resistant (FUMOZ-R) laboratory strains and the hybrid strains showed intermediate resistance. Genotyping the CYP6P9a-R resistance allele in oviposited females revealed that CYP6P9a negatively impacts the fecundity as homozygote susceptible mosquitoes (CYP6P9a-SS) lay more eggs than heterozygote (OR = 2.04: P = 0.01) and homozygote resistant mosquitoes. CYP6P9a also imposes a significant fitness cost on the larval development as homozygote resistant larvae (CYP6P9a-RR) developed significantly slower than heterozygote and homozygote susceptible mosquitoes (χ2 = 11.2; P = 0.0008). This fitness cost was further supported by the late pupation of homozygote resistant than susceptible mosquitoes (OR = 2.50; P < 0.01). However, CYP6P9a does not impact the longevity as no difference was observed in the life span of mosquitoes with different genotypes (χ2 = 1.6; P = 0.9). In this hybrid strain, a significant decrease of the resistant CYP6P9a-RR genotype was observed after ten generations (χ2 = 6.6; P = 0.01) suggesting a reversal of P450-based resistance in the absence of selection. This study shows that the P450-mediated metabolic resistance imposes a high fitness cost in malaria vectors supporting that a resistance management strategy based on rotation could help mitigate the impact of such resistance.

An Experimental Hut Evaluation of PBO-Based and Pyrethroid-Only Nets against the Malaria Vector Anopheles funestus Reveals a Loss of Bed Nets Efficacy Associated with GSTe2 Metabolic Resistance.

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7; p < 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.

Temporal escalation of Pyrethroid Resistance in the major malaria vector Anopheles coluzzii from Sahelo-Sudanian Region of northern Nigeria.

Despite the highest global burden of malaria, information on bionomics and insecticide resistance status of malaria vectors is grossly lacking in the densely populated Sahelo-Sudanian region of Nigeria. To support evidence-based vector control we characterised transmission and resistance profiles of Anopheles coluzzii populations from three sites in northern Nigeria. High sporozoite infection (~19.51%) was found in the An. coluzzii populations. A high pyrethroid resistance was observed with only 1% mortality against deltamethrin, a high LD50 (96.57 µg/ml), and a high LT50 (170.27 min, resistance ratio of ~51 compared with the fully susceptible Ngoussou colony). Moderate carbamate resistance was observed. Synergist bioassays significantly recovered deltamethrin susceptibility implicating CYP450s (mortality = 85%, χ2 = 134.04, p < 0.0001) and esterases (mortality = 56%, χ2 = 47.31, p < 0.0001). Reduced bed net efficacy was also observed, with mortalities on exposure to the roof of PermaNet3.0 (PBO + deltamethrin) more than 22 times compared to the side panel (deltamethrin). TaqMan genotyping revealed a high frequency of 1014F kdr mutation (82%) with significant difference in genotype distribution associated with permethrin resistance [OR = 4.69 (CI:1.53-14.35, χ2 = 8.22 p = 0.004]. Sequencing of exons 18-21 of the VGSC led to detection of two additional nonsynonymous mutations, Ile10148Asn and Ser1156Gly. These findings highlight the threats posed by the highly resistant An. coluzzii to malaria control in Nigeria.

Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, Anopheles funestus.

Background: Metabolic resistance is a serious challenge to current insecticide-based interventions. The extent to which it affects natural populations of mosquitoes including their reproduction ability remains uncharacterised. Here, we investigated the potential impact of the glutathione S-transferase L119F-GSTe2 resistance on the mating competitiveness of male Anopheles funestus, in Cameroon. Methods: Swarms and indoor resting collections took place in March, 2018 in Tibati, Cameroon. WHO tube and cone assays were performed on F 1 mosquitoes from indoor collected females to assess the susceptibility profile of malaria vectors. Mosquitoes mated and unmated males collected in the swarms were genotyped for the L119F metabolic marker to assess its association with mating male competitiveness. Results: Susceptibility and synergist assays, showed that this population was multiple resistant to pyrethroids, DDT and carbamates, likely driven by metabolic resistance mechanisms. Cone assays revealed a reduced efficacy of standard pyrethroid-nets (Olyset and PermaNet 2.0) with low mortality (<25%) whereas synergist PBO-Nets (Olyset Plus and PermaNet 3.0) retained greater efficacy with higher mortality (>80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in this An.funestus population at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21 An. funestus couples were isolated from those swarms.  A comparative genotyping of the L119F-GSTe2 mutation between mated and unmated males revealed that heterozygote males 119L/F-RS were less able to mate than homozygote susceptible (OR=7.2, P<0.0001). Surprisingly, heterozygote mosquitoes were also less able to mate than homozygote resistant (OR=4.2, P=0.010) suggesting the presence of a heterozygote disadvantage effect. Overall, mosquitoes bearing the L119-S susceptible allele were significantly more able to mate than those with 119F-R resistant allele (OR=2.1, P=0.03). Conclusion: This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.

Exposure to the insecticide-treated bednet PermaNet 2.0 reduces the longevity of the wild African malaria vector Anopheles funestus but GSTe2-resistant mosquitoes live longer.

BACKGROUND: Despite the increased report of insecticide resistance in malaria vectors, its impact on mosquito's life-traits after exposure to insecticide-treated nets remains under investigated. Here, we assessed the effects of exposure to PermaNet 2.0 on several life traits of An. gambiae s.l. and An. funestus s.l. field mosquitoes in Cameroon. METHODOLOGY: Female Anopheles mosquitoes were collected indoor using electric aspirators in southern Cameroon (Obout) in 2016. After assessing the resistance status of F1 from the field collected-mosquitoes, progeny of the first generation (An. funestus s.l.) and seventh generation (An. gambiae s.l.) were used to assess the long-term effect of exposure to PermaNet 2.0 on several life-traits of these vectors (longevity, blood feeding ability, fecundity and fertility) in comparison to untreated net. In addition, the L119F-GSTe2 mutation associated with DDT/pyrethroids resistance in An. funestus was genotyped to assess its association with increased life-span post-exposure. PRINCIPAL FINDINGS: Both An. funestus and An. gambiae were resistant to pyrethroids and DDT with a greater level in the latter. Pyrethroid-only nets PermaNet 2.0 (17.5% mortality) and Olyset (0% mortality) exhibited a significantly reduced efficacy against An. funestus in contrast to a greater efficacy for PBO-based Nets Olyset Plus (65% mortality), PermaNet 3.0 top (100% mortality). In both species, mosquitoes that survived exposure to PermaNet 2.0 exhibited a significantly reduced longevity than those non-exposed (6.95 days vs 12.46 for An. funestus P<0.001; 8.87 vs 11.25 days for An. gambiae; P<0.001). However, no significant difference was observed for blood feeding and fecundity in both species. In addition, molecular analysis of the L119F-GSTe2 mutation revealed that this mutation is associated with an increase in the chance of surviving after exposure to this net in An. funestus. CONCLUSIONS: These results show that although the PermaNet 2.0 presents a reduced efficacy against resistant populations, it remains efficient after exposure by reducing the life expectancy of the vectors which could contribute in the reduction of malaria incidence.

Escalation of Pyrethroid Resistance in the Malaria Vector Anopheles funestus Induces a Loss of Efficacy of Piperonyl Butoxide-Based Insecticide-Treated Nets in Mozambique.

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.

Escalation of Pyrethroid Resistance in the Malaria Vector Anopheles funestus Induces a Loss of Efficacy of Piperonyl Butoxide­Based Insecticide-Treated Nets in Mozambique

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. 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. 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. 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

Fitness Costs of the Glutathione S-Transferase Epsilon 2 (L119F-GSTe2) Mediated Metabolic Resistance to Insecticides in the Major African Malaria Vector Anopheles Funestus.

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.

Exploring insecticide resistance mechanisms in three major malaria vectors from Bangui in Central African Republic.

Malaria remains the main cause of mortality and morbidity in the Central African Republic. However, the main malaria vectors remain poorly characterised, preventing the design of suitable control strategies. Here, we characterised the patterns and mechanisms of insecticide resistance in three important vectors from Bangui. Mosquitoes were collected indoors, using electrical aspirators in July 2016 in two neighborhoods at Bangui. WHO bioassays performed, using F2 An. gambiae sensu lato (s.l.), revealed a high level of resistance to type I (permethrin) and II (deltamethrin) pyrethroids and dichlorodiphenyltrichloroethane (< 3% mortality). Molecular analysis revealed the co-occurrence of Anopheles coluzzii (56.8 %) and An. gambiae s.s. (43.2%) within the An. gambiae complex. Anopheles funestus s.s. was the sole species belonging to An. funestus group. Both kdr-w (40% of homozygotes and 60% of heterozygotes/kdr-w/wild type) and kdr-e (37.5% of heterozygotes) mutations were found in An. gambiae. Contrariwise, only the kdr-w (9.5% homozygotes and 85.7% of heterozygotes) was detected in An. coluzzii. Quantitative RT-PCR showed that CYP6M2 and CYP6P3 are not upregulated in An. coluzzii from Bangui. Analysis of the sodium channel gene revealed a reduced diversity in An. coluzzii and An. gambiae s.s. In An. funestus s.s., the pyrethroid/DDT GSTe2 L119F resistance allele was detected at high frequency (54.7%) whereas a very low frequency for Rdl was observed. Polymorphism analysis of GSTe2 and GABA receptor gene in An. funestus revealed the presence of one resistant haplotype for each gene. This study provides baseline information to help guide current and future malaria vector control interventions in CAR.