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.

Nationwide surveillance identifies yellow fever and chikungunya viruses transmitted by various species of Aedes mosquitoes in Nigeria.

Background: Since its reemergence in 2017, yellow fever (YF) has been active in Nigeria. The Nigeria Centre for Disease Control (NCDC) has coordinated responses to the outbreaks with the support of the World Health Organization (WHO). The National Arbovirus and Vectors Research Centre (NAVRC) handles the vector component of these responses. This study sought to identify the vectors driving YF transmission and any of the targeted arboviruses and their distribution across states. Methods: Eggs, larvae and pupae as well as adult mosquitoes were collected in observational, analytical, and cross-sectional surveys conducted in sixteen YF outbreak states between 2017 and 2020. Adult mosquitoes (field-collected or reared from immature stages) were morphologically identified, and arboviruses were detected using RT-qPCR at the African Centre of Excellence for Genomics of Infectious Diseases (ACEGID). Results: Aedes mosquitoes were collected in eleven of the sixteen states surveyed and the mosquitoes in nine states were found infected with arboviruses. A total of seven Aedes species were collected from different parts of the country. Aedes aegypti was the most dominant (51%) species, whereas Aedes africanus was the least (0.2%). Yellow fever virus (YFV) was discovered in 33 (~26%) out of the 127 Aedes mosquito pools. In addition to YFV, the Chikungunya virus (CHIKV) was found in nine pools. Except for Ae. africanus, all the Aedes species tested positive for at least one arbovirus. YFV-positive pools were found in six (6) Aedes species while CHIKV-positive pools were only recorded in two Aedes species. Edo State had the most positive pools (16), while Nasarawa, Imo, and Anambra states had the least (1 positive pool). Breteau and house indices were higher than normal transmission thresholds in all but one state. Conclusion: In Nigeria, there is a substantial risk of arbovirus transmission by Aedes mosquitoes, with YFV posing the largest threat at the moment. This risk is heightened by the fact that YFV and CHIKV have been detected in vectors across outbreak locations. Hence, there is an urgent need to step up arbovirus surveillance and control activities in the country.

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector Anopheles coluzzii.

BACKGROUND: Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. RESULTS: Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. CONCLUSIONS: These findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa.

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field-applicable markers to better track resistance Africa-wide.

Pyrethroid resistance in the New World malaria vector Anopheles albimanus is mediated by cytochrome P450 CYP6P5.

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α-cypermethrin with comparable affinities (KM of 3.3 µM ± 0.4 and 3.6 µM ± 0.5, respectively), but exhibited a 2.7-fold higher catalytic rate for α-cypermethrin (kcat of 6.02 min-1 ± 0.2) versus deltamethrin (2.68 min-1 ± 0.09). Time-course assays revealed progressive depletion of the above pyrethroids with production of four HPLC-detectable metabolites. Low depletion was obtained with type I pyrethroid, permethrin. Transgenic expression in Drosophila melanogaster demonstrated that overexpression of CYP6P5 alone conferred type II pyrethroid resistance, with only 16% and 55.3% mortalities in flies exposed to 0.25% α-cypermethrin and 0.15% deltamethrin, respectively. Synergist bioassays using P450 inhibitor piperonylbutoxide significantly recovered susceptibility (mortality = 73.6%, p < 0.001) in synergized flies exposed to 4% piperonylbutoxide, plus 0.25% α-cypermethrin, compared to non-synergized flies (mortality = 4.9%). Moderate resistance was also observed towards 4% DDT. These findings established the preeminent role of CYP6P5 in metabolic resistance in An. albimanus, highlighting challenges associated with deployment of insecticide-based control tools in the Americas.

Temporal Evaluation of Insecticide Resistance in Populations of the Major Arboviral Vector Aedes Aegypti from Northern Nigeria.

To support evidence-based control measures, two Nigerian Aedes populations (BUK and Pantami) were characterised. Larval bioassay using temephos and deltamethrin revealed a significant increase in deltamethrin resistance, with LC50 of 0.018mg/L (resistance ratio compared to New Orleans, RR = 2.250) in 2018 increasing ~6-fold, by 2019 (LC50 = 0.100mg/L, RR = 12.5), and ~11-fold in 2020 (LC50 = 0.198mg/L, RR = 24.750). For the median deltamethrin concentration (0.05mg/L), a gradual decrease in mortality was observed, from 50.6% in 2018, to 44.9% in 2019, and 34.2% in 2020. Extremely high DDT resistance was observed, with <3% mortalities and LT50s of 352.87 min, 369.19 min and 406.94 min in 2018, 2019 and 2020, respectively. Significant temporal increase in resistance was observed towards ƛ-cyhalothrin (a type II pyrethroid) over three years. Synergist bioassays with diethylmaleate and piperonylbutoxide significantly recovered DDT and ƛ-cyhalothrin susceptibility respectively, implicating glutathione S-transferases and CYP450s. Cone bioassays revealed increased resistance to the PermaNet® 3.0, side panels (mortalities of 94% in 2018, 66.4% in 2019, and 73.6% in 2020), while full susceptibility was obtained with the roof of PermaNet® 3.0. The F1534C kdr mutation occurred in low frequency, with significant correlation between heterozygote genotypes and DDT resistance. This temporal increase in resistance is a major challenge for control of this vector of public health importance.

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.

2La Paracentric Chromosomal Inversion and Overexpressed Metabolic Genes Enhance Thermotolerance and Pyrethroid Resistance in the Major Malaria Vector Anopheles gambiae.

Changes in global temperature are impacting the spread/intensity of vector-borne diseases, including malaria, and accelerating evolutionary/adaptive changes in vector species. These changes, including chromosomal inversions and overexpression and/or changes in allele frequencies of thermotolerance-associated genes, may facilitate insecticide resistance through pleiotropy. This study investigated the impact of thermotolerance on pyrethroid resistance in four populations of the malaria vector An. gambiae s.l., from the savanna/sub-Sahel of northern Nigeria. Anopheles coluzzii and An. gambiae s.s. were the only malaria vectors found, sympatric in all the sites, with the former species predominant. High thermotolerance was observed, with no mortality at 38 °C, and LT50 of ~44 °C. Significantly high permethrin resistance was observed (mortality < 50%) in 44 °C heat-hardened (exposure to an intermediately high temperature provides protection to a more severe temperature or insecticide) larvae from two sites, BUK and Pantami, compared with the control, and heat-hardened adult females from Auyo (mortality = 3.00% ± 1.20, χ2 = 5.83, p < 0.01) compared with the control (12.00% ± 4.65). The 2La chromosomal inversion was detected at ~50% in subset of larvae and 58% in subset of adult females genotyped. A significant association was observed (OR = 7.2, p < 0.03) between permethrin resistance and the 2La/+a rearrangement compared with 2L+a/+a, in BUK larvae. For all sites, permethrin resistance correlated with 2La/a homozygosity in adult females (R = 5.02, p = 0.01). qRT-PCR identified six genes commonly induced/overexpressed, including the heat shock protein 70 (AGAP004581) which was 2468× and 5× overexpressed in heat-hardened and permethrin-resistant females, respectively; trehalose-6-phosphate synthase (AGAP008227); and the ionotropic glutamate receptor genes, IR25a (AGAP010272) and IR21a (AGAP008511). This study highlights challenges associated with insecticide-based malaria vector control, and the epidemiological significance of taking climate variables into account for the design/choice of control measures.

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.

High pyrethroid/DDT resistance in major malaria vector Anopheles coluzzii from Niger-Delta of Nigeria is probably driven by metabolic resistance mechanisms.

Entomological surveillance of local malaria vector populations is an important component of vector control and resistance management. In this study, the resistance profile and its possible mechanisms was characterised in a field population of the major malaria vector Anopheles coluzzii from Port Harcourt, the capital of Rivers state, in the Niger-Delta Region of Nigeria. Larvae collected in Port-Harcourt, were reared to adulthood and used for WHO bioassays. The population exhibited high resistance to permethrin, deltamethrin and DDT with mortalities of 6.7% ± 2.4, 37.5% ± 3.2 and 6.3% ± 4.1, respectively, but were fully susceptible to bendiocarb and malathion. Synergist bioassays with piperonylbutoxide (PBO) partially recovered susceptibility, with mortalities increasing to 53% ± 4, indicating probable role of CYP450s in permethrin resistance (χ2 = 29.48, P < 0.0001). Transcriptional profiling revealed five major resistance-associated genes overexpressed in the field samples compared to the fully susceptible laboratory colony, Ngoussou. Highest fold change (FC) was observed with GSTe2 (FC = 3.3 in permethrin exposed and 6.2 in unexposed) and CYP6Z3 (FC = 1.4 in exposed and 4.6 in unexposed). TaqMan genotyping of 32 F0 females detected the 1014F and 1575Y knockdown resistance (kdr) mutations with frequencies of 0.84 and 0.1, respectively, while 1014S mutation was not detected. Sequencing of a fragment of the voltage-gated sodium channel, spanning exon 20 from 13 deltamethrin-resistant and 9 susceptible females revealed only 2 distinct haplotypes with a low haplotype diversity of 0.33. The findings of high pyrethroid resistance but with a significant degree of recovery after PBO synergist assay suggests the need to move to PBO-based nets. This could be complemented with carbamate- or organophosphate-based indoor residual spraying in this area.

Polymorphism Analysis of pfmdr1 and pfcrt from Plasmodium falciparum Isolates in Northwestern Nigeria Revealed the Major Markers Associated with Antimalarial Resistance.

Suspicion of failure in the effectiveness of artemisinin-based combination therapies (currently the first-line treatment of malaria, worldwide) is leading to the unofficial use of alternative antimalarials, including chloroquine and sulfadoxine/pyrimethamine, across northern Nigeria. To facilitate evidence-based resistance management, antimalarial resistance mutations were investigated in Plasmodium falciparum multidrug resistance-1 (pfmdr1) and chloroquine resistance transporter (pfcrt), in isolates from Kano, northwestern Nigeria. Out of the 88 samples genotyped for pfmdr1N86Y mutation using PCR/restriction fragment length polymorphism, one sample contained the 86Y mutation (86Yfrequency = 1.14%). The analysis of 610 bp fragments of pfmdr1 from 16 isolates revealed two polymorphic sites and low haplotype diversity (Hd = 0.492), with only 86 Y mutations in one isolate, and 184 F replacements in five isolates (184Ffrequency = 31.25%). The analysis of 267 bp fragments of pfcrt isolates revealed high polymorphism (Hd = 0.719), with six haplotypes and seven non-synonymous polymorphic sites. Eleven isolates (61.11%) were chloroquine-resistant, CQR (C72V73I74E75T76 haplotype), two of which had an additional mutation, D57E. An additional sequence was CQR, but of the C72V73M74E75T76 haplotype, while the rest of the sequences (33.33%) were chloroquine susceptible (C72V73M74N75K76 haplotype). The findings of these well characterized resistance markers should be considered when designing resistance management strategies in the northwestern Nigeria.

Identification of Mutations in Antimalarial Resistance Gene Kelch13 from plasmodium falciparum Isolates in Kano, Nigeria.

Malaria control relies on first-line treatments that use artemisinin-combination therapies (ACT). Unfortunately, mutations in the plasmodium falciparum kelch13 gene result in delayed parasite clearance. Research on what is causing ACT failure is non-existent in northwestern Nigeria. Thus, the presence of mutations in kelch13 in P. falciparum isolates from Kano, Nigeria was investigated in this study. Microscopic examination of 154 blood samples obtained from patients revealed a high prevalence of P. falciparum infection (114 positive individuals, slide positivity rate = 74.03%). The 114 patients were administered Cartef® (ACT) and out of the 50 patients that returned for the 14-day follow up, 11 were positive for P. falciparum (slide positivity rate = 22%). On day 0, 80 samples out of 114 and 11 samples on day 14 (91 out of 125 microscopy-positive samples) were positive with Plasmodium according to the PCR of cytochrome oxidase I, which corresponds to 72.8%. A fragment of the kelch13 gene encompassing the propeller domains was sequenced in 49 samples, alongside samples of the susceptible strain pf_3D7. Low polymorphism was observed, suggesting a lack of selection on this gene, and only six mutations (Glu433Gly, Phe434Ile, Phe434Ser, Ile684Asn, Ile684Thr and Glu688Lys) were found. The epidemiologic impact of these mutations and their potential role in ACT resistance needs to be investigated further.

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.

High insecticide resistance in the major malaria vector Anopheles coluzzii in Chad Republic.

BACKGROUND: The Sahel region of Chad Republic is a prime candidate for malaria pre-elimination. To facilitate pre-elimination efforts in this region, two populations of Anopheles coluzzii from Central Chad Republic were characterized, their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated. METHODS: Bloodfed female Anopheles gambiae s.l. resting indoor, were collected at N'djamena and Massakory, Chad in 2018 and characterized for species composition, and infection rate was determined using the TaqMan assay. Susceptibility to various insecticides was assessed using WHO tube bioassays. Cone bioassays were conducted using various long-lasting insecticidal nets (LLINs). Results were analysed using Chi Square test. Knockdown resistance (kdr) and ace-1 markers were investigated by TaqMan genotyping. RESULTS: Anopheles coluzzii was the major vector found in N'djamena (100%) and Massakory (~ 94%). No Plasmodium was found in 147 bloodfed F0 An. coluzzii (82 from N'djamena and 65 from Massakory). High intensity pyrethroid resistance was observed with mortalities of < 2% for permethrin, deltamethrin and etofenprox, and with < 50% and < 60% dead following exposure to 10× diagnostic doses of deltamethrin and permethrin, respectively. For both sites, < 10% mortalities were observed with DDT. Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations, implicating CYP450s (mortality = 13.6% for permethrin, χ2 = 22.8, df = 1, P = 0.0006; mortality = 13.0% for deltamethrin, χ2 = 8.8, df = 1, P < 0.00031). Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs; and a 100% recovery of susceptibility following exposure to the roof of PermaNet®3.0, containing piperonylbutoxide. Both populations were susceptible to malathion, but high bendiocarb resistance was observed in Massakory population. The absence of ace-1 mutation points to the role of metabolic resistance in the bendiocarb resistance. Both 1014F and 1014S mutations were found in both populations at around 60% and < 20% respectively. Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism. CONCLUSIONS: Multiple resistance in An. coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in the Sahel of this country. The pyrethroid-synergists LLINs (e.g. PermaNet®3.0) and organophosphate-based IRS maybe the alternatives for malaria control in this region.

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.

High Plasmodium infection and multiple insecticide resistance in a major malaria vector Anopheles coluzzii from Sahel of Niger Republic.

BACKGROUND: Information on insecticide resistance and the mechanisms driving it in the major malaria vectors is grossly lacking in Niger Republic, thus hindering control efforts. To facilitate evidence-based malaria control, the role of Anopheles coluzzii population from southern Niger, in malaria transmission, its insecticides resistance profile and the molecular mechanisms driving the resistance were characterized. METHODS: Blood fed female Anopheles gambiae sensu lato resting indoor were collected at Tessaoua, Niger. Source of blood was established using PCR and infection with Plasmodium determined using TaqMan assay. Resistance profile was established with the major public health insecticides, and resistance intensity determined with deltamethrin. Synergist assays were conducted with piperonyl butoxide and diethyl maleate. Presence of L1014F and L1014S knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) was investigated using TaqMan genotyping, and strength of selection pressure acting on the Anopheles populations determined by assessing the genetic diversity of a fragment spanning exon-20 of the VGSC from alive and dead females. RESULTS: High human blood index (96%) and high Plasmodium falciparum infection (~ 13%) was observed in the An. coluzzii population. Also, a single mosquito was found infected with Plasmodium vivax. High pyrethroid and organochloride resistance was observed with mortalities of less than 20% for deltamethrin, permethrin, α-cypermethrin, and DDT. A high LD50 (156.65 min) was obtained for deltamethrin, with a resistance ratio of ~ 47.18 compared to the susceptible Ngoussou colony. Moderate carbamate resistance was observed, and a full susceptibility to organophosphates recorded. Synergist bioassays with piperonyl butoxide and diethyl maleate significantly recovered deltamethrin and DDT susceptibility, respectively implicating CYP450 s (mortality = 82%, χ2 = 84.51, p < 0.0001) and glutathione S-transferases (mortality = 58%, χ2 = 33.96, p < 0.001) in resistance. A high frequency of 1014F kdr mutation (82%) was established, with significant difference in genotype distribution associated with permethrin resistance [odds ratio = 7.71 (95% CI 2.43-14.53, χ2 = 13.67, p = 0.001]. Sequencing of intron-1 of the voltage-gated sodium channel (VGSC) revealed a low genetic diversity. CONCLUSION: High pyrethroid resistance highlight the challenges to the effectiveness of the pyrethroids-based ITNs and indoor residual spraying (IRS) against An. coluzzii in Niger. The pyrethroids-synergists LLINs and organophosphate-based IRS maybe the alternatives for malaria control in southern Niger.

A combination of metabolic resistance and high frequency of the 1014F kdr mutation is driving pyrethroid resistance in Anopheles coluzzii population from Guinea savanna of Cameroon.

BACKGROUND: The scale-up in the distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying has significantly reduced malaria burden and mortality. However, insecticide resistance, among other factors, is responsible for a recent rebound in malaria transmission in 2015-2016, threatening the progress so far made. As a contribution towards understanding patterns of resistance and its mechanism in the field we characterized a population of Anopheles gambiae (s.l.) from Gounougou, a Guinea savanna of north/central Cameroon. RESULTS: Indoor collection conducted in September 2017 identified Anopheles coluzzii and Anopheles arabiensis as the unique Anopheles vector species, with abundances of 83 and 17%, respectively. Analysis of infection with TaqMan assays using heads/thoraces of indoor collected females of An. coluzzii revealed a low Plasmodium falciparum parasite rate of 4.7%. Bioassays conducted with female An. coluzzii revealed extreme resistance, with low mortalities of only 3.75 ± 1.25%, 3.03 ± 1.59% and 1.45 ± 1.45%, respectively, for permethrin, deltamethrin and DDT. In contrast, high susceptibility was obtained with the organophosphates and carbamates, with mortalities in the range of 98-100%. Synergist assays with piperonyl butoxide (PBO) recovered some susceptibility with increased mortality for permethrin to 14.88 ± 8.74%, and for deltamethrin to 32.50 ± 10.51% (~27-fold increase compared to mortalities with deltamethrin alone, χ2 = 29, df = 1, P < 0.0001). These correlated with the results of cone bioassays which revealed complete loss of efficacy of Olyset®Net (0% mortality) and PermaNet®2.0 (0% mortality), and the considerable loss of efficacy of Olyset®Plus (mortality of 2 ± 2%), PermaNet®3.0 side panel (mortality of 2 ± 2%) and PermaNet3.0® roof (mortality of 16 ± 5.1%). Time-course bioassays conducted with deltamethrin established a high intensity of resistance, with LT50 of 309.09 (95% CI 253.07-393.71, Fiducial), and a resistance ratio of 93.09 compared with the fully susceptible Ngoussou laboratory colony. TaqMan genotyping revealed a high frequency of the 1014F allele (65.25%) in the An. coluzzii populations. Sequencing of a fragment of the voltage-gated sodium channel identified a single An. arabiensis female harbouring the 1014S kdr mutation. CONCLUSIONS: This finding of high pyrethroid and DDT resistance in An. coluzzii from north-central Cameroon is a major obstacle to malaria control using pyrethroid bednets and indoor residual spraying with DDT.

A cytochrome P450 allele confers pyrethroid resistance on a major African malaria vector, reducing insecticide-treated bednet efficacy.

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

Pyrethroid Resistance in the Major Malaria Vector Anopheles funestus is Exacerbated by Overexpression and Overactivity of the P450 CYP6AA1 Across Africa.

Resistance to pyrethroids (the ingredients in bed net insecticides) in the major malaria vector Anopheles funestus is threatening recent gains in the fight against malaria. Here, we established the role of an over-expressed P450, A. funestus CYP6AA1 in insecticides resistance. Transcription profiling of CYP6AA1 across Africa using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that it is significantly more over-expressed in southern African populations compared to West (Benin) and East African (Uganda). Heterologous expression in Escherichia coli coupled with metabolism assays demonstrated that CYP6AA1 metabolises type I (permethrin) and type II (deltamethrin) pyrethroids, as well as bendiocarb (a carbamate). Transgenic Drosophila melanogaster flies over-expressing CYP6AA1 were significantly more resistant to pyrethroid insecticides, permethrin and deltamethrin compared with control flies not expressing the gene, validating the role of this gene in pyrethroid resistance. In silico modelling and docking simulations predicted the intermolecular receptor-ligand interactions which allow this P450 to metabolise the pyrethroids and bendiocarb. Validation of CYP6AA1 as a pyrethroid resistance gene makes it possible to monitor the spread of resistance in the field where this P450 is over-expressed. Its potential cross-resistance role makes it necessary to monitor the gene closely to inform control programs on molecular basis of multiple resistance in the field.

Genome-Wide Transcription and Functional Analyses Reveal Heterogeneous Molecular Mechanisms Driving Pyrethroids Resistance in the Major Malaria Vector Anopheles funestus Across Africa.

Pyrethroid resistance in malaria vector, An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern (Malawi), East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes (CYP6P9a and CYP6P9b) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies.