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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.

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) 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 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.

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.

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. Graphical Abstract

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.

Background New active ingredients, mostly repurposed from the agricultural sector, are gradually being introduced into malaria vector control programs. Prior to scaling up novel insecticides, it is vital to assess if their use in agriculture has already selected mosquito populations with reduced susceptibility in some areas. The aim of the present study was to determine if the use of neonicotinoids in agricultural pest management could drive resistance to clothianidin—a neonicotinoid recently prequalified by WHO for indoor residual spraying. Methods We carried out intensive sampling of Anopheles gambiae, An. coluzzii and Culex sp. larvae from a large suburban farm and from 4 surrounding locations belonging to the rural and urban ecosystems of Yaounde, Cameroon. Using WHO tube tests and CDC bottle bioassays, we tested the recommended diagnostic doses of clothianidin against field-collected populations using two laboratory strains as susceptible controls. We exposed adult mosquitoes to the insecticide for 1 h and monitored mortality rates for seven consecutive days. Results An. coluzzii and Culex sp. mosquitoes were fully susceptible to clothianidin reaching 100% mortality by day 3. By contrast, An. gambiae populations revealed heterogeneous profiles ranging from susceptibility to strong resistance. Populations from the suburban farm, where neonicotinoids are routinely used, displayed the lowest mortality rate (46.5 % ± 5.65) and were capable of surviving 5X and 10X the diagnostic dose of clothianidin. When clothianidin was tested in combination with the synergist piperonyl butoxide, mortality of resistant populations increased from 46.5 % to 92.7 % implying that Cytochrome P450 enzymes play an integral role in the genetic mechanism underlying clothianidin resistance in An. gambiae. Conclusions These findings show that small clusters of resistance to clothianidin may exist around farmlands where agricultural neonicotinoids are being used. Patches of intensive agriculture should be particularly monitored in indoor residual spraying target areas.

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.

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 international airport of Yaoundé, the capital city of 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 2km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adult progeny. WHO bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was fixed above 98% mortality rate and mortality rates below 90% were considered indicative of confirmed insecticide resistance according to the WHO test procedures for insecticide resistance monitoring. 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 infection rate. Both species exhibited high levels of resistance to pyrethroids (Plasmodium infection rate highlights the challenges that public health vector control programs encounter in sustaining the regular effectiveness of contemporary insecticide-based control interventions in forested areas. More particularly, the resistance observed against the carbamates and possible resistance against the organophosphates constitutes a major concern for IRS.

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.

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,Plasmodiuminfection 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 locality situated 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F1adults. Bioassays were performed to assess resistance profile to the four insecticides classes. 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 andPlasmodiuminfection rates were investigated.Results:Anopheles funestuss.s. was the most abundant species in Elende (85%) followed byAnopheles gambiaes.s. (15%) with both having similar sporozoite rate. Both species exhibited high levels of resistance to the pyrethroids, permethrin and deltamethrin (An. gambiaes.s. was resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate.An. funestuss.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119Sace-1 resistance allele (15%) were detected inAn. funestuss.s. andAn. gambiaes.s., respectively.Furthermore, the high pyrethroid/DDT resistances inAn. gambiaecorresponded with an increase frequency of 1014Fkdrallele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression ofCYP6P5,CYP6P9aandCYP6P9b.Conclusion:The resistance to multiple insecticide classes observed in these vector populations alongside the significantPlasmodiumsporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

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

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 F2An. gambiae sensu lato (s.l.), revealed a high level of resistance to type I (permethrin) and II (deltamethrin) pyrethroids and dichlorodiphenyltrichloroethane (

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., An extensive loss of efficacy of all pyrethroid-based Insecticide-treated bednets, including PBO-based bednets is recorded in a highly pyrethroid resistant population of the malaria vector An. funestus (Mozambique) exhibiting high expression of cytochrome P450 genes with fixation of CYP6P9a_R allele.

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 maleAnopheles 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 F1mosquitoes 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 (80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in thisAn. funestuspopulation at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21An. funestuscouples 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, PConclusion:This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.

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 maleAnopheles 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 F1mosquitoes 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 (80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in thisAn.funestuspopulation at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21An. funestuscouples 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, PConclusion:This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.