The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo.

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control.

Epidemiology of malaria, schistosomiasis, and geohelminthiasis amongst children 3-15 years of age during the dry season in Northern Cameroon.

BACKGROUND: The double burden of malaria and helminthiasis in children poses an obvious public health challenge, particularly in terms of anemia morbidity. While both diseases frequently geographically overlap, most studies focus on mono-infection and general prevalence surveys without molecular analysis. The current study investigated the epidemiological determinants of malaria, schistosomiasis, and geohelminthiasis transmission among children in the North Region of Cameroon. METHODOLOGY: School and pre-school children aged 3-15 year-of-age were enrolled from three communities in March 2021 using a community cross-sectional design. Capillary-blood samples were obtained, and each was examined for malaria parasites using rapid-diagnostic-test (RDT), microscopy, and PCR while hemoglobin level was measured using a hemoglobinometer. Stool samples were analyzed for Schistosoma mansoni, S. guineensis, and soil-transmitted-helminthiasis (STH) infections using the Kato Katz method, and urine samples were assessed for the presence of S. haematobium eggs (including hybrids) using the standard urine filtration technique. RESULT: A malaria prevalence of 56% (277/495) was recorded by PCR as opposed to 31.5% (156/495) by microscopy and 37.8% (186/495) by RDT. Similarly, schistosomiasis was observed at prevalence levels of up to 13.3% (66/495) overall [S. haematobium (8.7%); S. mansoni (3.8%); mixed Sh/Sm (0.6%); mixed Sh/Sm/Sg (0.2%). Both infections were higher in males and the 3-9 year-of-age groups. A high frequency of PCR reported P. falciparum mono-infection of 81.9% (227/277) and mixed P. falciparum/P. malariae infection of 17.3% (48/277) was observed. Malaria-helminths co-infections were observed at 13.1% (65/495) with marked variation between P. falciparum/S. haematobium (50.8%, 33/65); P. falciparum/S. mansoni (16.9%, 11/65) and P. falciparum/Ascaris (9.2%, 6/65) (χ2 = 17.5, p = 0.00003). Anemia prevalence was 32.9% (163/495), categorically associated with P. falciparum (45.8%, 104/227), Pf/Sh (11.5%, 26/227), and Pf/Sm (3.9%, 9/227) polyparasitism. CONCLUSION: Polyparasitism with malaria and helminth infections is common in school-aged children despite periodic long-lasting insecticide-treated nets (LLINs) distribution and regular school-based praziquantel (for schistosomiasis) and albendazole (for STH) campaigns. Co-existence of Plasmodium parasites and helminths infections notably Schistosoma species among children may concurrently lead to an increase in Plasmodium infection with an enhanced risk of anemia, highlighting the necessity of an integrated approach for disease control interventions.

The duplicated P450s CYP6P9a/b drive carbamates and pyrethroids cross-resistance in the major African malaria vector Anopheles funestus.

Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC<7). Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant An. funestus, this locus exhibits a reduced nucleotide diversity, significant p-values when comparing allele frequencies, and the most non-synonymous substitutions. Recombinant enzyme metabolism assays showed that both CYP6P9a/b metabolize carbamates. Transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between carbamate resistance and CYP6P9a genotypes with homozygote resistant An. funestus (CYP6P9a and the 6.5kb enhancer structural variant) exhibiting a greater ability to withstand bendiocarb/propoxur exposure than homozygote CYP6P9a_susceptible (e.g Odds ratio = 20.8, P<0.0001 for bendiocarb) and heterozygotes (OR = 9.7, P<0.0001). Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that pyrethroid resistance escalation poses to the efficacy of other classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions.

Escalating pyrethroid resistance in two major malaria vectors Anopheles funestus and Anopheles gambiae (s.l.) in Atatam, Southern Ghana.

BACKGROUND: Aggravation of insecticide resistance in malaria vectors is threatening the efforts to control malaria by reducing the efficacy of insecticide-based interventions hence needs to be closely monitored. This study investigated the intensity of insecticide resistance of two major malaria vectors An. funestus sensu stricto (s.s.) and An. gambiae sensu lato (s.l.) collected in southern Ghana and assessed the bio-efficacy of several long-lasting insecticidal nets (LLINs) against these mosquito populations. METHODS: The insecticide susceptibility profiles of Anopheles funestus s.s. and Anopheles gambiae s.l. populations from Obuasi region (Atatam), southern Ghana were characterized and the bio-efficacy of some LLINs was assessed to determine the impact of insecticide resistance on the effectiveness of these tools. Furthermore, molecular markers associated with insecticide resistance in both species were characterized in the F0 and F1 populations using PCR and qPCR methods. RESULTS: Anopheles funestus s.s. was the predominant species and was resistant to pyrethroids, organochlorine and carbamate insecticides, but fully susceptible to organophosphates. An. gambiae s.l. was resistant to all four insecticide classes. High intensity of resistance to 5 × and 10 × the discriminating concentration (DC) of pyrethroids was observed in both species inducing a considerable loss of efficacy of long-lasting insecticidal nets (LLINs). Temporal expression analysis revealed a massive 12-fold increase in expression of the CYP6P4a cytochrome P450 gene in An. funestus s.s., initially from a fold change of 41 (2014) to 500 (2021). For both species, the expression of candidate genes did not vary according to discriminating doses. An. gambiae s.l. exhibited high frequencies of target-site resistance including Vgsc-1014F (90%) and Ace-1 (50%) while these mutations were absent in An. funestus s.s. CONCLUSIONS: The multiple and high intensity of resistance observed in both malaria vectors highlights the need to implement resistance management strategies and the introduction of new insecticide chemistries.

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.

Experimental Hut Trials Reveal That CYP6P9a/b P450 Alleles Are Reducing the Efficacy of Pyrethroid-Only Olyset Net against the Malaria Vector Anopheles funestus but PBO-Based Olyset Plus Net Remains Effective.

Malaria remains a major public health concern in Africa. Metabolic resistance in major malaria vectors such as An. funestus is jeopardizing the effectiveness of long-lasting insecticidal nets (LLINs) to control malaria. Here, we used experimental hut trials (EHTs) to investigate the impact of cytochrome P450-based resistance on the efficacy of PBO-based net (Olyset Plus) compared to a permethrin-only net (Olyset), revealing a greater loss of efficacy for the latter. EHT performed with progenies of F5 crossing between the An. funestus pyrethroid-resistant strain FUMOZ and the pyrethroid-susceptible strain FANG revealed that PBO-based nets (Olyset Plus) induced a significantly higher mortality rate (99.1%) than pyrethroid-only nets (Olyset) (56.7%) (p < 0.0001). The blood-feeding rate was higher in Olyset compared to Olyset Plus (11.6% vs. 5.6%; p = 0.013). Genotyping the CYP6P9a/b and the intergenic 6.5 kb structural variant (SV) resistance alleles showed that, for both nets, homozygote-resistant mosquitoes have a greater ability to blood-feed than the susceptible mosquitoes. Homozygote-resistant genotypes significantly survived more with Olyset after cone assays (e.g., CYP6P9a OR = 34.6; p < 0.0001) than homozygote-susceptible mosquitoes. A similar but lower correlation was seen with Olyset Plus (OR = 6.4; p < 0.001). Genotyping EHT samples confirmed that CYP6P9a/b and 6.5 kb_SV homozygote-resistant mosquitoes survive and blood-feed significantly better than homozygote-susceptible mosquitoes when exposed to Olyset. Our findings highlight the negative impact of P450-based resistance on pyrethroid-only nets, further supporting that PBO nets, such as Olyset Plus, are a better solution in areas of P450-mediated resistance to pyrethroids.

A 6.5kb Intergenic Structural Variation Exacerbates the Fitness Cost of P450-Based Metabolic Resistance in the Major African Malaria Vector Anopheles funestus.

Metabolic-based resistance to insecticides limit the control of medically important pests, and it is extremely detrimental in the ongoing struggle to control disease vectors. Elucidating the fitness cost of metabolic resistance in major malaria vectors is vital for successful resistance management. We established the fitness cost of the 6.5kb structural variant (6.5kb-sv) between the duplicated CYP6P9a/b P450s using the hybrid strain generated from the crossing between two An. funestus laboratory strains. Furthermore, we assessed the cumulative impact of this marker with the duplicated P450 genes. We established that individuals that were homozygote for the resistant structural variant (SV) presented reduced fecundity and slow development relative to those that were homozygote for the susceptible SV. Furthermore, we observed that 6.5kb act additively with CYP6P9a and CYP6P9b to exacerbate the reduced fecundity and the increased development time of resistant mosquitoes since double/triple homozygote susceptible (SS/SS/SS) significantly laid more eggs and developed faster than other genotypes. Moreover, a restoration of susceptibility was noted over 10 generations in the insecticide-free environment with an increased proportion of susceptible individuals. This study highlights the negative impact of multiple P450-based resistance on the key physiological traits of malaria vectors. Such high fitness costs suggest that in the absence of selection pressure, the resistant individuals will be outcompeted in the field. Therefore, this should encourage future strategies based on the rotation of insecticides to reduce selection pressure and to slow the spread of pyrethroid resistance.

Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon.

BACKGROUND: Monitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors. METHODS: Anopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile. RESULTS: The study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.0%) and oocyst (14.4%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.9%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.4%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations. CONCLUSION: The emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

RNAseq-based gene expression profiling of the Anopheles funestus pyrethroid-resistant strain FUMOZ highlights the predominant role of the duplicated CYP6P9a/b cytochrome P450s.

Insecticide-based interventions, notably long-lasting insecticidal nets, against mosquito vectors of malaria are currently threatened by pyrethroid resistance. Here, we contrasted RNAseq-based gene expression profiling of laboratory-resistant (FUMOZ) and susceptible (FANG) strains of the major malaria vector Anopheles funestus. Cytochrome P450 genes were the predominant over-expressed detoxification genes in FUMOZ, with high expression of the duplicated CYP6P9a (fold-change of 82.23 vs FANG) and CYP6P9b (FC 11.15). Other over-expressed P450s belonged to the same cluster of P450s corresponding to the resistance to pyrethroid 1 (rp1) quantitative trait loci (QTL) on chromosome 2R. Several Epsilon class glutathione S-transferases were also over-expressed in FUMOZ, as was the ATP-binding cassette transporter AFUN019220 (ABCA) which also exhibited between-strain alternative splicing events at exon 7. Significant differences in single-nucleotide polymorphism frequencies between strains occurred in resistance QTLs rp1 (CYP6P9a/b and CYP6AA1), rp2 on chromosome 2L (CYP6Z1, CYP6M7, and CYP6Z3), and rp3 on chromosome 3R (CYP9J5, CYP9J4, and CYP9J3). Differences were also detected in CYP4G17 and CYP4G16 genes on the X chromosome, both of which are associated with cuticular resistance in Anopheles gambiae. A close analysis of nonsynonymous diversity at the CYP6P9a/b loci revealed a drastic loss of diversity in FUMOZ with only a single polymorphism and 2 haplotypes vs 18 substitutions and 8 haplotypes in FANG. By contrast, a lowly expressed cytochrome P450 (CYP4C36) did not exhibit diversity differences between strains. We also detected the known pyrethroid resistance conferring amino acid change N384S in CYP6P9b. This study further elucidates the molecular bases of resistance in An. funestus, informing strategies to better manage widespread resistance across Africa.

Genetic Diversity of Cytochrome P450s CYP6M2 and CYP6P4 Associated with Pyrethroid Resistance in the Major Malaria Vectors Anopheles coluzzii and Anopheles gambiae from Yaoundé, Cameroon.

Assessing the genetic diversity of metabolic resistance genes, such as cytochrome P450s, helps to understand the dynamics and evolution of resistance in the field. Here, we analyzed the polymorphisms of CYP6M2 and CYP6P4, associated with pyrethroid resistance in Anopheles coluzzii and Anopheles gambiae, to detect potential resistance markers. Field-caught resistant mosquitos and susceptible lab strains were crossed, and F4 was exposed to permethrin for 15 min and 90 min to discriminate highly susceptible (HS) and highly resistant (HR) mosquitos, respectively. Significant permethrin mortality reduction was observed after pre-exposure to PBO, suggesting the gene involvement of P450s. qPCR analysis revealed significant overexpression of CYP6M2 (FC = 19.57 [95% CI 13.96-25.18] for An. coluzzii; 10.16 [7.86-12.46] for An. gambiae) and CYP6P4 (FC = 6.73 [6.15-7.30] An. coluzzii; 23.62 [26.48-20.76] An. gambiae). Full-gene and ≈1 kb upstream were sequenced. For CYP6M2, the upstream region shows low diversity in HR and HS (overall Hd = 0.49, π = 0.018), whereas the full-gene shows allelic-variation but without evidence of ongoing selection. CYP6P4 upstream region showed a lower diversity in HR (Hd = 0.48) than HS (Hd = 0.86) of An. gambiae. These results highlighted that CYP6P4-associated resistance is potentially driven by modification in upstream region. However, further work is needed to determine the real causative variants that will help design rapid detection tools.

The cytochrome P450 CYP325A is a major driver of pyrethroid resistance in the major malaria vector Anopheles funestus in Central Africa.

The overexpression and overactivity of key cytochrome P450s (CYP450) genes are major drivers of metabolic resistance to insecticides in African malaria vectors such as Anopheles funestus s.s. Previous RNAseq-based transcription analyses revealed elevated expression of CYP325A specific to Central African populations but its role in conferring resistance has not previously been demonstrated. In this study, RT-qPCR consistently confirmed that CYP325A is highly over-expressed in pyrethroid-resistant An. funestus from Cameroon, compared with a control strain and insecticide-unexposed mosquitoes. A synergist bioassay with PBO significantly recovered susceptibility for permethrin and deltamethrin indicating P450-based metabolic resistance. Analyses of the coding sequence of CYP325A Africa-wide detected high-levels of polymorphism, but with no predominant alleles selected by pyrethroid resistance. Geographical amino acid changes were detected notably in Cameroon. In silico homology modelling and molecular docking simulations predicted that CYP325A binds and metabolises type I and type II pyrethroids. Heterologous expression of recombinant CYP325A and metabolic assays confirmed that the most-common Cameroonian haplotype metabolises both type I and type II pyrethroids with depletion rate twice that the of the DR Congo haplotype. Analysis of the 1 kb putative promoter of CYP325A revealed reduced diversity in resistant mosquitoes compared to susceptible ones, suggesting a potential selective sweep in this region. The establishment of CYP325A as a pyrethroid resistance metabolising gene further explains pyrethroid resistance in Central African populations of An. funestus. Our work will facilitate future efforts to detect the causative resistance markers in the promoter region of CYP325A to design field applicable DNA-based diagnostic tools.

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.

Combined over-expression of two cytochrome P450 genes exacerbates the fitness cost of pyrethroid resistance in the major African malaria vector Anopheles funestus.

Metabolic resistance driven by multiple P450 genes is worsening insecticide resistance in malaria vectors. However, it remains unclear whether such multiple over-expression imposes an additive fitness cost in the vectors. Here, we showed that two highly over-expressed P450 genes (CYP6P9a and CYP6P9b) combine to impose additive fitness costs in pyrethroid-resistant Anopheles funestus. Genotyping of the CYP6P9b resistance allele in hybrid mosquitoes from a pyrethroid-resistant FUMOZ-R and the susceptible FANG strains revealed that this gene imposes a fitness cost in resistant mosquitoes similar to CYP6P9a. Homozygote susceptible CYP6P9b_S (SS) significantly lay more eggs than the resistant (OR = 2.2, P = 0.04) and with greater hatching rate (p < 0.04). Homozygote resistant larvae CYP6P9b_R (RR) developed significantly slower than homozygote susceptible from L1-L4 (χ2 = 7.2; P = 0.007) with a late pupation observed for RR compared to both heterozygotes and homozygotes susceptible (χ2 = 11.17; P = 0.0008). No difference was observed between genotypes for adult longevity with no change in allele frequency and gene expression across the lifespan. Furthermore, we established that CYP6P9b combines with CYP6P9a to additively exacerbate the fitness cost of pyrethroid resistance with a greater reduction in fecundity/fertility and increased developmental time of double homozygote resistant mosquitoes. Moreover, an increased proportion of double homozygote susceptible individuals was noted over 10 generations in the insecticide-free environment (χ2 = 6.3; P = 0.01) suggesting a reversal to susceptibility in the absence of selection. Such greater fitness cost imposed by multiple P450 genes shows that resistance management strategy based on rotation could help slow the spread of resistance.

Insecticide Resistance Profiling of Anopheles coluzzii and Anopheles gambiae Populations in the Southern Senegal: Role of Target Sites and Metabolic Resistance Mechanisms.

The emergence and spread of insecticide resistance among the main malaria vectors is threatening the effectiveness of vector control interventions in Senegal. The main drivers of this resistance in the Anopheles gambiae complex (e.g., An. gambiae and Anopheles coluzzii) remains poorly characterized in Senegal. Here we characterized the main target site and metabolic resistances mechanisms among the An. gambiae and An. coluzzii populations from their sympatric and allopatric or predominance area in Senegal. Larvae and pupae of An. gambiae s.l. were collected, reared to adulthood, and then used for insecticides susceptibility and synergist assays using the WHO (World Health Organisation) test kits for adult mosquitoes. The TaqMan method was used for the molecular characterization of the main target site insecticide resistance mechanisms (Vgsc-1014F, Vgsc-1014S, N1575Y and G119S). A RT-qPCR (Reverse Transcriptase-quantitative Polymerase Chaine Reaction) was performed to estimate the level of genes expression belonging to the CYP450 (Cytochrome P450) family. Plasmodium infection rate was investigated using TaqMan method. High levels of resistance to pyrethroids and DDT and full susceptibility to organophosphates and carbamates where observed in all three sites, excepted a probable resistance to bendiocarb in Kedougou. The L1014F, L1014S, and N1575Y mutations were found in both species. Pre-exposure to the PBO (Piperonyl butoxide) synergist induced a partial recovery of susceptibility to permethrin and full recovery to deltamethrin. Subsequent analysis of the level of genes expression, revealed that the CYP6Z1 and CYP6Z2 genes were over-expressed in wild-resistant mosquitoes compared to the reference susceptible strain (Kisumu), suggesting that both the metabolic resistance and target site mutation involving kdr mutations are likely implicated in this pyrethroid resistance. The presence of both target-site and metabolic resistance mechanisms in highly pyrethroid-resistant populations of An. gambiae s.l. from Senegal threatens the effectiveness and the sustainability of the pyrethroid-based tools and interventions currently deployed in the country. The Kdr-west mutation is widely widespread in An. coluzzii sympatric population. PBO or Duo nets and IRS (Indoor Residual Spraying) with organophosphates could be used as an alternative measure to sustain malaria control in the study area.

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.

CYP6P9-Driven Signatures of Selective Sweep of Metabolic Resistance to Pyrethroids in the Malaria Vector Anopheles funestus Reveal Contemporary Barriers to Gene Flow.

Pyrethroid resistance in major malaria vectors such as Anopheles funestus threatens malaria control efforts in Africa. Cytochrome P450-mediated metabolic resistance is best understood for CYP6P9 genes in southern Africa in An. funestus. However, we do not know if this resistance mechanism is spreading across Africa and how it relates to broader patterns of gene flow across the continent. Nucleotide diversity of the CYP6P9a gene and the diversity pattern of five gene fragments spanning a region of 120 kb around the CYP6P9a gene were surveyed in mosquitoes from southern, eastern and central Africa. These analyses revealed that a Cyp6P9a resistance-associated allele has swept through southern and eastern Africa and is now fixed in these regions. A similar diversity profile was observed when analysing genomic regions located 34 kb upstream to 86 kb downstream of the CYP6P9a locus, concordant with a selective sweep throughout the rp1 locus. We identify reduced gene flow between southern/eastern Africa and central Africa, which we hypothesise is due to the Great Rift Valley. These potential barriers to gene flow are likely to prevent or slow the spread of CYP6P9-based resistance mechanism to other parts of Africa and would to be considered in future vector control interventions such as gene drive.

A 6.5-kb intergenic structural variation enhances P450-mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5-kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole-genome pooled sequencing detected an intergenic 6.5-kb SV between duplicated CYP6P9a/b P450s in pyrethroid-resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (p < .0001) for the SV- carrying fragment than the SV- free one. Quantitative real-time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV- (1.7- to 2-fold) and homozygotes SV-/SV- (4-to 5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV-/SV-; odds ratio [OR] = 2,079.4, p < .001). The 6.5-kb SV is present at high frequency in southern Africa (80%-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid-treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote-resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets.

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.