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.

Knowing the extent and spread of insecticide resistance in malaria vectors is vital to successfully manage insecticide resistance in Africa. This information in the main malaria vector, Anopheles funestus sensu stricto, is completely lacking in the most populous country in Africa, Nigeria. This study reports the insecticide susceptibility status and the molecular basis of resistance of An. funestus as well as its involvement in malaria transmission in Akaka-Remo, a farm settlement village in southwest Nigeria. Plasmodium infection analysis using TaqMan protocol coupled with a nested PCR revealed an infection rate of 8% in An. funestus s.s. from Akaka-Remo. WHO susceptibility tests showed this species has developed multiple resistance to insecticides in the study area. Anopheles funestus s.s. population in Akaka-Remo is highly resistant to organochlorines: dieldrin (8%) and DDT (10%). Resistance was also observed against pyrethroids: permethrin (68%) and deltamethrin (87%), and the carbamate bendiocarb (84%). Mortality rate with DDT slightly increased (from 10 to 30%, n = 45) after PBO pre-exposure indicating that cytochrome P450s play little role in DDT resistance while high mortalities were recorded after PBO pre-exposure with permethrin (from 68 to 100%, n = 70) and dieldrin (from 8 to 100%, n = 48) suggesting the implication of P450s in the observed permethrin and dieldrin resistance. High frequencies of resistant allele, 119F in F0 (77%) and F1 (80% in resistant and 72% in susceptible) populations with an odd ratio of 1.56 (P = 0.1859) show that L119F-GSTe2 mutation is almost fixed in the population. Genotyping of the A296S-RDL mutation in both F0 and F1 samples shows an association with dieldrin resistance with an odd ratio of 81 (P < 0.0001) (allelic frequency (R) = 76% for F0; for F1, 90 and 10% were observed in resistant and susceptible populations, respectively) as this mutation is not yet fixed in the population. The study reports multiple insecticide resistance in An. funestus from Akaka Remo. It is, therefore, necessary to pay more attention to this major malaria vector for effective malaria control in Nigeria.

Background:The arbovirus vector,Aedes albopictus,originating from Asia, has recently invaded African countries, including the Republic of the Congo, where it was associated with a chikungunya outbreak. Up until now, little was known about its distribution in relation to the nativeAedes aegyptiand how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution ofAe. albopictusandAe. aegyptiin the Republic of the Congo and explored the genetic diversity of the invading species,Ae. albopictus. Methods:Immature stages ofAedeswere collected in nine locations in the Republic of the Congo in 2017 following a north-south transect and reared to adult stage. Adults were morphologically identified, counted and grouped according to species and location. Genetic diversity ofAe. albopictuswas assessed by analyzing the cytochrome oxidase I (COI) gene. Results: Ae. albopictusandAe. aegyptiwere found together across the country in all the locations investigated. The invasive species is predominant over the native species in all locations except Brazzaville, suggesting thatAe. albopictusis displacingAe. aegyptiacross Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire,Ae. albopictuswas more prevalent thanAe. aegyptiin the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity ofAe. albopictuswith only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed thatAe. albopictusfrom Congo originated from other tropical Asian countries such as China, likely as a result of increasing trade links. Conclusion:These findings are important for the implementation of vector control strategies and can serve as a foundation for further research on these vectors in the country.

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.

Lambda-cyhalothrin (λ-cyhalothrin) is the most commonly used pyrethroid insecticide for vegetable farming in Benin. This insecticide is misused and overused by farmers, and hence may pose health hazards to consumers. We monitored λ-cyhalothrin residues in lettuce and cabbage from farms at the market gates in Cotonou and Parakou using high performance liquid chromatography (HPLC) analysis techniques. These residues were also monitored on samples directly from farms (on-farm sampling) for 14 days post-treatment. Potential factors such as photolysis and hydrolysis involved in λ-cyhalothrin degradation were also screened. Results revealed that the level of λ-cyhalothrin residue concentrations in lettuce from Houeyiho decreased from 4.2 mg/kg on Day 1 to about 0.2 mg/kg on Day 7. On Day 9, analyzed lettuces were all λ-cyhalothrin free. In contrast, even 14 days after treatment of cabbage from Bawera (Parakou), we still recorded the presence of λ-cyhalothrin residues in analyzed samples. For samples from market gates, λ-cyhalothrin residues were found in lettuce from two markets out of the nine surveyed in Cotonou. Interestingly, none of these contaminated samples had residues above the maximum residue limit for lettuce (MRL = 0.5 mg/kg). Similarly, in Parakou, samples from all five surveyed vegetable markets were contaminated with λ-cyhalothrin residues at concentrations below the MRL for cabbage (MRL = 0.2 mg/kg). We conclude that λ-cyhalothrin residues in lettuce and cabbage from farms and markets in Parakou and Cotonou are within the MRL, and hence are relatively safe for consumption.